A cell electroporation device and method

Abstract

This invention relates to the field of cell electroporation technology, and more particularly to a cell electroporation device and method. The device includes: a controller configured to acquire excitation parameters; a flexible electrode array module configured to apply an electric field to target cells; the flexible electrode array module includes a flexible substrate and multiple electrodes; the region where the electrodes are located has a micro-via array; a multi-channel phased-array excitation module configured to generate corresponding high-voltage pulses and introduce a phase delay for each channel; and electric fields are applied to the electrodes according to the phase delay, thereby causing the electric fields applied by each electrode to interfere and superimpose with each other under the action of the micro-vias, forming a fused electric field of the target radius at the target perforation depth for electroporation of the target cells. This invention achieves precise electroporation of target cells, such as adherent cells, suspended cell populations, three-dimensional cell clusters, or organoids, by superimposing electric field interference at the target depth to form a fused electric field of the target radius.

Description

Technical Field

[0001] This invention belongs to the field of cell electroporation technology, and particularly relates to a cell electroporation device and method. Background Technology

[0002] Tumor cells exhibit widespread cellular heterogeneity, a major cause of drug-resistant cell subtypes during tumor treatment and closely related to tumor metastasis. Single-cell analysis is an effective method for studying cellular heterogeneity. Currently, traditional biotechnologies, such as PCR and Western blotting, often require the extraction and analysis of markers from large numbers of cells, failing to accurately reflect heterogeneity at the single-cell level and easily leading to incomplete analytical results. Recent advancements in single-cell analysis techniques, combining traditional methods with micro / nanochips, enable genomic and proteomic analyses at the single-cell level, but these methods still cannot guarantee cell viability and involve complex procedures with poor reproducibility.

[0003] In situ cell analysis can provide information on cellular markers and allow for real-time observation of cell state, thus yielding rich information on cellular behavior. In particular, it enables direct comparison of cellular heterogeneity at the single-cell level. Single-cell in situ analysis often requires the delivery of exogenous functional molecules into the cell or the extraction of intracellular substances into the extracellular space for analysis. Current delivery technologies can be categorized into viral vectors, chemical methods, and physical methods.

[0004] Viral vectors, such as adenoviruses and lentiviruses, have a high advantage in delivery efficiency. However, their application in gene detection is limited because viral gene fragments can also be introduced into the host chromosome.

[0005] Chemical methods primarily utilize artificially synthesized micro / nanomaterials as carriers to deliver biomolecules via endocytosis and efflux within cells. For smaller biomolecules, such as siRNAs, chemical methods offer high delivery efficiency. However, for larger biomolecules, such as CRISPR-Cas9 plasmids (>9 kb), the delivery efficiency is very limited.

[0006] The main principle of physical delivery is to apply a physical field, such as an electric field or a magnetic field, to the cell membrane, causing the cell membrane to instantly create a reversible channel. At the same time, exogenous molecules in the surrounding solution, such as DNA, RNA, and drugs, are transferred under the influence of the physical field, thereby enabling the delivery of functional molecules.

[0007] Electroporation, as a simple and direct physical delivery method, has been widely used in biomedical research, including regenerative medicine, adoptive immunotherapy, and in vivo gene editing. Compared with other physical methods such as microinjection, sonar perforation, and osmosis, electroporation is easier to perform and more universally applicable.

[0008] Electroporation, also known as electrotransfection, is a commonly used method in cell transfection. Because cell membranes are selectively permeable to external substances, controlling eukaryotic gene experiments requires introducing specific biological DNA or RNA fragments into eukaryotic cells. Applying a certain potential difference across the cell membrane and maintaining it for a period of time creates micropores, enhancing the cell membrane's permeability. When electroporation occurs, the permeability and membrane conductivity increase instantaneously, allowing molecules that normally cannot pass through the cell membrane, such as hydrophilic molecules, DNA, proteins, viral particles, and drug particles, to enter the cell. After the potential difference is removed within a short time, the cell membrane recovers and becomes a selective permeability barrier again. Compared to traditional chemical and viral transfection, electroporation has wider applicability and advantages: it is suitable for plasmids and genomic fragments of tens of kilobytes, avoids chemical and viral contamination, causes no permanent cell damage, and allows for transient transfection. Therefore, electroporation technology has broad application prospects in biophysics, molecular biology, and clinical medicine.

[0009] Current electroporation devices can be mainly divided into two types: one uses standard electroporation cuvettes with an electrode spacing of 1-4 mm, which is on the millimeter scale, while cell size is on the micrometer scale. Therefore, a large voltage (generally from several hundred volts to thousands of volts) needs to be applied, and the electric field is uneven. Each cell is in a different electric field environment, which makes cells near the electrodes prone to death, while cells located in the weaker electric field cannot be perforated and transfected. The survival rate and transfection efficiency are relatively low. Moreover, each operation can only put a small number of cells and plasmids according to the volume of the standard electroporation cuvette, resulting in low experimental efficiency. The other type is micro-spacing (generally about 1-100 μm) cell electroporation devices (generally made using micro-nano fabrication technology). Because the electrode spacing is small, the voltage required by this type of device is greatly reduced compared to the first type of device, making it safer to use and often resulting in higher electroporation efficiency.

[0010] For example, patent application CN106085845A discloses a cell electroporation chip device and its fabrication method based on a U-shaped groove microelectrode array. This cell electroporation chip integrates a large number of U-shaped groove microelectrodes on the chip, focusing the electric field within the microchannels into the grooves, enabling efficient cell electroporation under low-voltage conditions. The integrated electrode array allows for the simultaneous perforation of a large number of cells. Simultaneously, the groove structure facilitates cell capture, and single-cell capture can be achieved through fluid control within the chip, thus completing single-cell perforation. The grooves are isolated by silicon dioxide and polysilicon, and this part is not electrified, preventing cell aggregation and perforation in this area. An aluminum lead layer ensures good electrical conductivity; the silicon dioxide passivation layer and chip material ensure good biocompatibility and corrosion resistance. This device can be widely used in research on cell lysis, cell electrotransfection, and cell electrofusion.

[0011] For example, patent application CN115684309A discloses a nanobranched microelectrode array device, its fabrication method, and its applications. By combining hydrothermal growth and standard microfabrication processes, a unique nanobranched microelectrode array was developed. The three-dimensional nanobranched structure, with its high aspect ratio, can form a tight coupling with cardiomyocytes, reducing signal leakage. Simultaneously, it allows for cell electroporation at low voltage, obtaining high-quality intracellular signals. This nanobranched microelectrode array device enables long-term intracellular recording. The high aspect ratio nanobranched structure creates a tight cell-electrode interface, allowing electroporation at multiple sites and delaying cell membrane closure after electroporation, thereby extending the time for electrode intracellular access and achieving long-term intracellular recording.

[0012] For example, utility model patent CN210176871U provides an electroporation chip and an electroporation system. The electroporation chip includes: a substrate; a cell culture chamber located on the substrate for containing cell samples; a microelectrode array formed on the substrate, the microelectrode array including multiple sets of microelectrodes located in the cell culture chamber; and electrode leads, including an anode lead corresponding to each set of microelectrodes and a common cathode lead, the electrode leads being used to transmit electrical signals to one or more sets of microelectrodes in the microelectrode array. This device can perform in-situ electroporation on adherent or suspended cells.

[0013] The aforementioned prior art has the following drawbacks: (1) Uneven electric field distribution leads to excessive damage to some cells in the cell population and failure to effectively transfect some cells, resulting in large differences in treatment effects; (2) In a three-dimensional cell cluster or tissue environment, the electric field is mainly concentrated on the surface, and it is difficult for deep cells to be effectively perforated; (3) To compensate for insufficient efficiency, it is often necessary to increase the voltage, but high voltage can easily cause heat accumulation and irreversible damage, thereby reducing cell survival rate and reproducibility. Summary of the Invention

[0014] The purpose of this invention is to provide a cell electroporation device and method that partially solves or alleviates at least one of the above-mentioned deficiencies in the prior art, and can form a fused electric field of target radius by superimposing electric field interference at the target depth to perform electroporation on target cells more accurately.

[0015] To solve the aforementioned technical problems, the present invention specifically adopts the following technical solution: A first aspect of the present invention is to provide a cell electroporation device, comprising: The controller is configured to acquire excitation parameters, including voltage amplitude, pulse width, frequency, and phase delay. A flexible electrode array module is configured to apply an electric field to target cells; wherein the flexible electrode array module includes a flexible substrate and a plurality of electrodes disposed on the flexible substrate; the region where the electrodes are located has a micro-via array, the micro-via array being used to converge the electric field applied by the electrodes and guide the direction of the electric field; A multi-channel phased excitation module, connected to the controller and the flexible electrode array module, is configured to generate corresponding high-voltage pulses based on the voltage amplitude, pulse width, and frequency in the excitation parameters, and to introduce a phase delay for each channel. Electric fields are applied to the electrodes corresponding to the channels according to the phase delay, so that the electric fields applied by each electrode interfere with and superimpose with each other, forming a fused electric field of the target radius at the target perforation depth, so as to electroperforate the target cells.

[0016] Furthermore, the micro-vias in the micro-via array penetrate the flexible substrate and are arranged in a ring, a rectangle, or a radial pattern.

[0017] Furthermore, the aperture of the micro-vias in the micro-via array is 50nm-500nm, and the spacing between the micro-vias is 1um-5um.

[0018] Furthermore, the inner wall or orifice region of the micro-hole is provided with a stimuli-responsive sealing layer; such that in the non-working state, the sealing layer expands to block the micro-hole, while in the working state, the sealing layer contracts in response to external stimuli to open the micro-hole; the external stimuli include one or more of pH, electric field or temperature.

[0019] Furthermore, the plurality of electrodes are symmetrically distributed in a ring array, and the spacing between two adjacent electrodes is 0.5mm-2mm; the maximum phase delay between the electrodes is 360°, and the phase delay between adjacent electrodes is equal.

[0020] Furthermore, there are an even number of electrodes, and the electrodes arranged at intervals are connected together to form a group.

[0021] Furthermore, the flexible substrate is made of polyimide film, PDMS or TPU, with a thickness of 80um-120um.

[0022] Furthermore, the voltage amplitude output by the multi-channel phase-controlled excitation module is 10V-100V, the pulse width is 10us-1000us, and the frequency is 1Hz-2Hz.

[0023] A second aspect of the present invention is to provide a method for performing cell electroporation using the above-described cell electroporation device, comprising: S101, attach the flexible electrode array module of the cell electroporation device to the target object; S102, using the cell electroporation device to apply an electric field to the target object to electroporate the target cells on the target object; Specifically, step S102 includes the following steps: S1021, the controller of the cell electroporation device acquires the cell parameters configured by the user; the cell parameters include cell type and target perforation depth; S1022, the controller matches the corresponding excitation parameters in a pre-built cell-excitation parameter correspondence table according to the cell parameters; the excitation parameters include voltage amplitude, pulse width, frequency, and phase delay; S1023, the multi-channel phased excitation module of the cell electroporation device generates a corresponding high-voltage pulse based on the matched excitation parameters voltage amplitude, pulse width and frequency, and introduces a phase delay for each channel; the electrodes corresponding to the channels apply electric fields simultaneously according to the phase delay, so that the electric fields applied by each electrode interfere with each other and are superimposed, forming a fused electric field of the target radius at the target perforation depth, so as to perform electroporation on the target cells.

[0024] Furthermore, in step S01, when processing adherent cells, the flexible electrode array module is attached to the bottom / top surface of the cell culture dish or chip, so that the electric field is uniformly applied to the cell monolayer. When processing a population of suspended cells, the flexible electrode array module is attached to the inner wall of the microfluidic chip channel to achieve controlled perforation of cells during the flow process; When processing three-dimensional cell clumps or organoid models, the flexible electrode array module is arranged around the supporting substrate and achieves focused electroporation at the internal target depth through phased excitation.

[0025] Beneficial effects: In response to the problem that the electric field distribution of traditional electroporation is relatively diffuse and the depth of action is difficult to control, this invention achieves three-dimensional fine control of the electric field distribution through the synergistic effect of "micropore guidance" and "phase control excitation". It can not only focus, but also control the depth of focus, which is crucial for targeting cells at different depths.

[0026] Furthermore, multiple micropore arrangement patterns (annular, square, and radial) are provided. The annular arrangement ensures a symmetrical and uniform electric field that converges in the central region, enabling precise, non-invasive electroporation of individual cells. The square arrangement creates a regular and uniform electric field with a large coverage area, allowing for electroporation of large areas of monolayer cells. The radial arrangement concentrates the electric field lines towards the central axis, enabling electroporation of three-dimensional tissues. Moreover, a multi-channel phased-array excitation system allows for dynamic changes in the position, size, and shape of the electric field focal point, achieving "non-invasive scanning" electroporation without physically moving the electrodes.

[0027] When an excitation is applied, each micro-via outlet becomes a highly localized and more directional secondary electric field source. Furthermore, the pulses between multiple electrodes arranged at specific intervals also have a phase difference, which manipulates the interference behavior of these secondary electric field waves in space. This allows the electric field waves propagating from different micro-vias to have the same phase at a desired focal point (such as a point deep within a three-dimensional cell) through a specific phase difference, resulting in superposition and maximizing the electric field strength. In non-target areas, the electric field waves cancel each other out due to their opposite phases, weakening the electric field strength.

[0028] In this invention, a high-density through-hole array is formed on a flexible substrate. The electric field must be "emitted" through the through-holes. The through-hole array thus becomes a spatial modulation structure of the electric field, enabling the electric field generated by the phase-controlled electrode to form a designable focal point and action volume within the tissue.

[0029] This invention utilizes multi-electrode phase gradients and micro-via arrays of different forms to enable programmable adjustment of the focal position, focal size, and depth of the electric field in three-dimensional space through electrode phase and waveform parameters. This allows for the formation of a sufficiently large local high field strength at the target depth under a relatively low driving voltage, achieving focal and depth-controllable electroporation (including conventional electroporation and irreversible electroporation) of ex vivo tissue sections, while significantly reducing the field strength and damage risk in non-target areas.

[0030] Existing technologies are more geared towards traditional rigid electrodes or multipolar electrode structures in vivo.

[0031] In summary, the invention achieves focal high field strength at the target depth and field strength suppression in non-target areas through the synergistic design of micro-perforation array and phased-controlled multi-electrode, which has significant advantages in terms of drug delivery efficiency, safety and applicable scenarios. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0033] Figure 1A This is a schematic diagram of the structure of the present invention; Figure 1B This is a schematic diagram of the flexible electrode array module. Figure 2 Schematic diagram of the electric field principle of phased array; Figure 3 This is a functional principle block diagram of a multi-channel phase-controlled excitation module, illustrating the pulse generation unit, phase control module, and channel driving logic. Figure 4 The simulation results of the electric field focusing effect in the tissue profile are shown, illustrating the electric field intensity distribution and focal position under different phase control parameters; Figure 5 This is a schematic diagram of the attachment state of the device during use, demonstrating its adaptability and positioning on the skin or tissue surface; Figure 6A These are comparative images of stained sections before and after electroporation, used to illustrate the depth perforation effect and focus accuracy under different excitation strategies; Figure 6B This is a schematic diagram showing the variation of relative (electroporation) depth with different pulse voltages. Figure 6C This is a schematic diagram showing the variation of relative electroporation depth over different time periods; Figure 6D This is a schematic diagram illustrating the variation of relative electroporation depth with different pulse numbers and durations. Figure 7 A graph showing the dynamic change in resistance and a comparison of color depth; Figure 8 A bar chart showing the prediction error of the perforation focal depth; Figure 9 A statistical graph showing the proportion of PI-stained perforated cells in sections at different depths; Figure 10 A graph showing the changes in CD45-positive inflammation scores at different time points; Figure 11 This is a graph showing the change in skin surface temperature during stimulation.

[0034] The diagram is labeled as follows: 1-Controller (MCU), 2-Flexible electrode array module, 3-Multi-channel phased excitation module, 21-Flexible substrate, 22-Electrode, 23-Micro-via array. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0036] In this document, suffixes such as "module," "part," or "unit" used to denote elements are used only for the purpose of illustrative purposes and have no specific meaning in themselves. Therefore, "module," "part," or "unit" may be used interchangeably.

[0037] In this document, the terms "upper," "lower," "inner," "outer," "front," "rear," "one end," and "the other end," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the present invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0038] In this document, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," "connected," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, a direct connection, or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0039] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0040] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0041] Example 1: As Figure 1A , Figure 1B , Figure 2 , Figure 3 As shown, this embodiment provides a cell electroporation device, comprising: Controller 1 is configured to acquire excitation parameters, including voltage amplitude, pulse width, frequency, and phase delay.

[0042] Controller 1 is connected to a parameter setting interface, serving as the user's interaction point with the system. Users input the required control parameters through external interfaces such as an operation panel or software interface, and these parameters are then sent to Controller 1. In this embodiment, Controller 1 is an FPGA / MCU controller responsible for generating control commands, scheduling various tasks, and transmitting timing instruction excitation strategies to the next-level module, namely the multi-channel phase-controlled excitation module 3.

[0043] The flexible electrode array module 2 is configured to apply an electric field to target cells. Specifically, the flexible electrode array module 2 includes a flexible substrate 21 and a plurality of electrodes 22 disposed on the flexible substrate 21. The region where the electrodes 22 are located has a micro-via array 23, which is used to converge the electric field applied by the electrodes 22 and guide the direction of the electric field.

[0044] The flexible electrode array module 2 is the direct interface between the electroporation device and the target cells or tissues. It needs to stabilize the output electric field, adapt to the morphology of different cells and tissues, and optimize the distribution of the electric field through structural design to solve the problems of poor adhesion and electric field diffusion of traditional rigid electrodes 22.

[0045] The flexible substrate 21 serves as the base for supporting the electrodes 22 and the microvia array 23. It is typically made of PI (polyimide) film, PDMS (polydimethylsiloxane), or TPU (thermoplastic polyurethane), with a thickness of 80μm-120μm. It is flexible and stretchable, allowing it to adhere tightly to the surface of cell culture dishes, the inner walls of microfluidic chip channels, or be arranged around a support substrate for three-dimensional cell clusters, preventing unintended gaps between the electrodes 22 and the target cells. Furthermore, the substrate itself is an insulating material, preventing current crosstalk between the electrodes 22 and ensuring independent electric field output for each electrode 22.

[0046] Electrode 22 is a conductive unit disposed on the surface of flexible substrate 21, typically made of metal thin film or conductive polymer. In this embodiment, electrode 22 is ring-shaped and symmetrically distributed in a ring array, with the spacing between adjacent electrodes 22 being 0.5mm-2mm, adapted to the range of action of cell tissue; and each electrode 22 can be independently driven to cooperate with multi-channel phased excitation module 3.

[0047] In addition, as the output port of the electric field, it receives signals from the multi-channel phased excitation module and releases an electric field to the target cell. Because it supports independent driving, each electrode 22 can output an electric field with a specific phase delay. The maximum phase delay between the electrodes 22 is 360°, and the phase delay between adjacent electrodes 22 is equal. For example, six electrodes 22 can sequentially output electric fields with phases of 0°, 60°…300°, providing a basis for subsequent interference focusing of the electric field.

[0048] In some embodiments, there are an even number of electrodes 22, and the electrodes 22 arranged at intervals are connected together to form a group.

[0049] For example, the annular electrode 22 comprises six electrodes, with three connected together as a group. From the perspective of electric field distribution, the six electrodes 22 arranged at equal intervals along the circumference are electrically connected in groups, for example, electrodes 1, 3, and 5 form the first group, and electrodes 2, 4, and 6 form the second group. This creates two groups of equivalent virtual annular electrodes 22 spaced 120° apart in the circumferential direction. When pulses of different phases or polarities are applied to the two groups, the central region achieves highly symmetrical electric field focusing, preventing a situation where the field strength is excessively high in one direction, thereby improving the uniformity of the perforated area.

[0050] From a circuit implementation perspective, if all six electrodes 22 are driven independently, a large number of high-voltage channels are required, resulting in higher system complexity and cost. By electrically connecting every three electrodes 22 into a group, the high-voltage drive channels can be simplified to two or a few paths while maintaining good circumferential symmetry, reducing system complexity. Simultaneously, sufficient focusing capability and redundant electrode area are ensured, reducing single-point current density and the risk of electrode corrosion. Of course, in other embodiments, different numbers of electrodes 22, such as 4 or 8, and different grouping methods can be used; this invention does not impose any limitations.

[0051] In this embodiment, the microvia array 23 is the core structure for applying a precise electric field. The propagation of electric field lines preferentially selects regions with low dielectric resistance and concentrated channels. The channel size of the microvia is much smaller than the planar region of the substrate, and the dielectric environment of the microvia is more uniform. Therefore, the electric field lines output by the electrode 22 are confined within the microvia channels.

[0052] The dispersed electric field lines are concentrated in the micro-via channel, which significantly enhances the electric field intensity at the micro-via outlet and avoids the electric field from spreading and weakening on the substrate surface. The electric field lines propagate along the channel direction of the micro-via, that is, perpendicular to the direction of the flexible substrate 21, and can act directionally on the target cells on the other side of the substrate or penetrate into the three-dimensional cell cluster, which solves the problem of scattered electric field direction and insufficient deep cell effect of traditional electrode 22.

[0053] Because of the aforementioned structure, this invention enables localized electroporation of cells or cell populations at specified spatial locations or depths to achieve molecular introduction and local perturbation, thereby supporting research on spatial heterogeneity, cell interactions, and mechanisms. Specific applications include: Site-specific transfection of single cells / small number of cells: Plasmids, mRNA, siRNA, sgRNA / CRISPR complexes are introduced into selected cells or small clusters of cells in monolayer culture to achieve sparse labeling, intercellular control and cell heterogeneity studies.

[0054] Spatial patterning transfection: forming strip / dot matrix transfection regions on culture dishes or micropatterned substrates to study cell migration, population behavior, contact inhibition, boundary effects and morphogenesis.

[0055] Targeted intervention at co-culture interfaces: In co-culture systems such as immune cells-target cells, neurons-glial cells, and endothelial cells-pericytes, molecules are introduced only on one side of the interface or locally at the interface to analyze intercellular signal transduction and paracrine effects.

[0056] Local introduction within three-dimensional cell spheres / organoids: In three-dimensional culture of cell spheres, organoids, or hydrogels, reporter genes, lineage tracer barcodes, or editing tools are introduced at specific spatial layers (surface / middle / deep) to construct mosaic interventions for studying spatial partitioning functions.

[0057] Focal electroporation of ex vivo tissue sections: introducing fluorescent reporter, calcium indicator, optogenetic tools or gene editing systems into local areas of ex vivo sections such as brain slices and intestinal slices for the study of neural circuits, local microenvironment and tissue hierarchical structure.

[0058] Regionalized electroporation in embryonic / developmental models: Electroporation is performed on specific regions in embryonic or developmental models to introduce (e.g., reporter genes, morphogenetic regulators) for the study of developmental axes, local induction, and tissue differentiation.

[0059] Organelle / subcellular localization intervention (research-based terminology): With the goal of "delivering molecules enriched in the cell nucleus" or "delivering them to local high-field regions", conduct research on intranuclear delivery efficiency, nuclear membrane permeability and transcriptional regulation mechanisms (avoid describing it as clinically relevant "tumor core", etc.).

[0060] Local delivery establishes concentration gradients: Diffusionable signaling molecule encoding carriers or tracer molecules are introduced into local regions to form spatial gradients, which are used to validate chemotaxis, morphogenesis, cell polarity and signal diffusion models.

[0061] Positional control in high-throughput parameter screening: Divide multiple fixed-point regions on the same substrate, apply different electrical parameters and different loaded molecules to achieve parallel control within the same batch of samples, and reduce the impact of batch-to-batch differences on the conclusions.

[0062] Multi-region stepwise introduction (spatiotemporally resolved intervention): Different labels or regulatory molecules are sequentially introduced into different sites on the same sample via electroporation in chronological order, for cell fate tracking, temporal response and network dynamics studies.

[0063] More specifically, in this embodiment, the micro-vias in the micro-via array 23 penetrate the flexible substrate 21 and are arranged in a ring, a rectangle, or a radial pattern.

[0064] The ring arrangement can achieve symmetrical and uniform electric field convergence in the central region, making it suitable for single-point focusing or local target perforation, such as targeted intervention in the core of tumors in in vitro cell model studies such as tumor research and stem cell biology.

[0065] The array is regularly arranged and easy to process, and can form a uniform electric field distribution over a large area, making it suitable for the simultaneous processing of large cell populations or drug delivery.

[0066] The radially arranged electric field lines are concentrated towards the central region, exhibiting outstanding focusing ability. This makes it suitable for scenarios requiring deep penetration and directional delivery, such as the treatment of three-dimensional tissue structures or spherical cell clusters.

[0067] The three arrangement methods correspond to different application requirements, which can improve the adaptability and scalability of the present invention in different cell and tissue processing scenarios.

[0068] In this embodiment, the aperture of the micro-via array 23 is 50nm-500nm, and the spacing between the micro-vias is 1um-5um.

[0069] To address the problem of micropores being blocked by foreign objects, in some embodiments, the inner wall or orifice region of the micropore is provided with a stimuli-responsive sealing layer; such that in the non-working state, the sealing layer expands to block the micropore, while in the working state, the sealing layer contracts in response to external stimuli to open the micropore; the external stimuli include one or more of pH, electric field, or temperature.

[0070] In this invention, the micropores penetrate the flexible substrate 21, and the pore openings are directly visible from the cell / tissue side. The metal electrodes 22 do not penetrate the substrate but are deposited / patterned on one side of the substrate. An electrode window is reserved at the location of each micropore to keep the pore opening open, forming only a ring-shaped or matrix-shaped electrode pattern around the pore.

[0071] In terms of process flow, through-holes can be formed on the flexible substrate 21 first, and then metal can be deposited and stripped after leaving an opening area at the hole through a photolithography mask, so as to ensure that the metal only covers the area around the hole and does not fill the channel.

[0072] The micropores have diameters of only 50nm–500nm and spacings of 1μm–5μm, occupying a relatively small area compared to the entire electrode 22 region. The electrode 22 material employs conventional adhesion and conductive layer systems such as Cr / Au and Ti / Au, combined with plasma treatment and annealing processes to improve the adhesion between the metal and the flexible substrate 21. The adhesion of the electrode 22 layer is not significantly reduced due to the openings, and the micropores are not blocked by the electrode 22 material. Furthermore, a hydrogel or polymer thin layer responsive to stimuli such as pH, temperature, and electric field can be introduced into the inner wall or pore area of ​​the micropores as a sealing layer. In the non-working static state, this layer is in an expanded state, partially or completely sealing the pores to prevent the entry of contaminants such as macromolecules and cell debris. In the working state, by applying a specific electric field, changing the temperature, or changing the local pH value, the hydrogel shrinks or changes its permeability, thereby opening the gate and allowing ions and electric field lines to act on the underlying tissue through the micropores.

[0073] The controllable opening and closing structure helps improve the device's resistance to contamination and reusability, while also giving each microvia independent controllability.

[0074] The flexible substrate 21 ensures that the electrode 22 fits tightly with the target cell. Multiple independent electrodes 22 output electricity with phase difference. The micro-perforation array 23 converges and guides these electric field lines, ultimately forming an electric field with sufficient intensity and precise direction in the target cell region. This avoids excessive damage to surface cells and can act on deep cells or the interior of three-dimensional cell clusters, achieving a controllable electroporation effect.

[0075] The multi-channel phased excitation module 3, connected to the controller 1 and the flexible electrode array module 2, is configured to generate corresponding high-voltage pulses according to the voltage amplitude, pulse width, and frequency in the excitation parameters, and introduce a phase delay for each channel; the electrodes 22 corresponding to the channels are used to apply electric fields according to the phase delay, so that the electric fields applied by each electrode 22 interfere and superimpose with each other under the action of the micro-perforation, forming a fused electric field of the target radius at the target perforation depth, so as to electroperforate the target cells.

[0076] It is worth noting that depth stratification is not involved when the object is a two-dimensional monolayer adherent cell; when the object is a three-dimensional cell construct or tissue sample, "depth" is defined as the distance from the device attachment surface along the normal direction into the interior of the sample, which is used to characterize the position of the focused electric field.

[0077] For example, in tumor cell spheres (HeLa / MCF-7 / 4T1 cell spheres) or intestinal epithelial organoid samples with a diameter of approximately 200 μm–800 μm, the cells exhibit stratification and gradient distribution in the normal direction. The “target depth” of the focused electric field is used to set the cell population where electroporation mainly occurs at a distance of approximately 80 μm–150 μm from the attachment surface.

[0078] The multi-channel phase-controlled excitation module 3 is connected upward to the controller 1 to receive the excitation parameters sent by the main controller as the basis for signal generation; it is connected downward to the flexible electrode array module 2 to distribute the processed electrical signal to each independent electrode 22 channel and drive the electrode 22 to output an electric field.

[0079] The multi-channel phase-controlled excitation module 3 specifically includes a pulse generation unit (for example, it can be implemented using an electric field generator TX, see [link]). Figure 2 ) and phase control module (e.g., a phase adjuster, see See Figure 2 The pulse generation unit generates standard pulse waveforms through a timer and waveform buffer, and can flexibly configure parameters such as waveform frequency and width, providing a basic waveform template for subsequent phase adjustment. Its output range is a voltage amplitude of 10V-100V, a pulse width of 10µs-1000µs, and a frequency of 1Hz-2Hz.

[0080] The phase control module introduces corresponding phase delays to create phase differences for different channels according to the requirements of electric field focusing, and finally realizes electric field focusing in the spatial domain.

[0081] The multi-channel phase-controlled excitation module 3 has multi-channel drive control function, which distributes the phase-modulated signal to the corresponding power channel, and manages the mapping relationship and working status of each channel.

[0082] In this invention, the micro-vias penetrating the substrate discretize the electric field on the surface of electrode 22 into a large number of highly directional secondary electric field sources; each via outlet can be approximated as a small electric field emission unit. The position of the via in space is determined by the arrangement of the aperture array, while the phase of the electric field at the via is determined by the excitation phase of the electrode 22 where it is located.

[0083] Therefore, by introducing a specific phase difference between adjacent electrodes 22, the relative phase relationship of electric field waves emitted from different through holes within the tissue can be controlled, so that they coherently superimpose in the target area to form a focused high field strength, while canceling each other out in the non-target area, resulting in a significant reduction in the electric field.

[0084] The micro-aperture is responsible for where the electric field is emitted in space, while the phase difference is responsible for how these emissions are superimposed in space. Only by working together can three-dimensional programmable electric field focusing and depth control be achieved.

[0085] In addition, this embodiment also provides a method for performing cell electroporation using the above-mentioned cell electroporation device, comprising: S101, attach the flexible electrode array module 2 of the cell electroporation device to the target object.

[0086] Specifically, when processing adherent cells, the flexible electrode array module 2 is attached to the bottom / top surface of the cell culture dish or chip, so that the electric field is uniformly applied to the cell monolayer. When processing a population of suspended cells, the flexible electrode array module 2 is attached to the inner wall of the microfluidic chip channel to achieve controlled perforation of cells during the flow process; When processing three-dimensional cell clumps or organoid models, the flexible electrode array module 2 is arranged around the supporting substrate and achieves focused electroporation at the internal target depth through phased excitation.

[0087] S102, using the cell electroporation device to apply an electric field to the target object to electroporate the target cells on the target object.

[0088] Specifically, step S102 includes the following steps: S1021, the controller 1 of the cell electroporation device acquires the cell parameters configured by the user; the cell parameters include cell type and target perforation depth.

[0089] Users configure cell types and target perforation depths through human-computer interaction modules, such as touchscreens or keyboards. Controller 1 obtains the user-configured cell types and target perforation depths from these modules. Different cells have vastly different membrane potential thresholds and damage resistance; for example, tumor cells have lower membrane potentials and require less electric field strength for perforation. The target perforation depth determines the location of the electric field focus.

[0090] S1022, the controller 1 matches the corresponding excitation parameters in a pre-constructed cell-excitation parameter correspondence table according to the cell parameters; the excitation parameters include voltage amplitude, pulse width, frequency and phase delay.

[0091] Controller 1 calls a pre-built cell-excitation parameter mapping table to match the corresponding voltage amplitude, pulse width, frequency, and phase delay. This mapping table is based on empirical data obtained through prior experimental validation. For example, when HeLa cells are perforated at a depth of 2 mm, the optimal parameters are a voltage of 80 V, a pulse width of 200 μs, a frequency of 1 Hz, and a phase delay of 60°. This eliminates the need for manual user adjustments, improving efficiency and ensuring reproducibility across different experiments.

[0092] S1023, the multi-channel phased excitation module 3 of the cell electroporation device generates a corresponding high-voltage pulse based on the matched excitation parameters voltage amplitude, pulse width, and frequency, and introduces a phase delay for each channel; the electrodes 22 corresponding to the channels are used to simultaneously apply electric fields according to the phase delay, so that the electric fields applied by each electrode 22 interfere and superimpose with each other under the action of the micro-perforation, forming a fused electric field of the target radius at the target perforation depth, so as to perform electroporation on the target cells.

[0093] The multi-channel phased excitation module 3 generates a high-voltage pulse that meets the cell's requirements based on the matched voltage amplitude, pulse width, and frequency; and assigns a corresponding phase delay to each electrode 22 channel. Each electrode 22 synchronously outputs an electric field according to the phase delay, and combined with the guidance of the micropore array of the flexible electrode 22, the electric fields of different channels interfere in space. The electric fields are superimposed due to constructive interference to form a fused electric field at the target radius, while in non-target areas, the electric fields cancel each other out due to destructive interference, resulting in an intensity below the safety threshold and avoiding irrelevant cell damage.

[0094] Of course, in other scenarios, the fusion electric field obtained by the above method acts on the cell membrane of the target cell to form micropores. This is not only suitable for conventional cell electroporation, but also, according to actual needs, by adjusting the key parameters of the fusion electric field, to form irreversible micropores, thus applying to other scenarios such as tumor cell ablation.

[0095] Example 2: This embodiment provides a flexible adhesive electroporation device based on a six-channel independent excitation configuration, and verifies its electric field focusing effect in a skin tissue model using finite element simulation.

[0096] In terms of device structure, a 100 μm thick polyimide film is used as the flexible electrode substrate. Six independent driving electrodes are fabricated on the substrate using laser ablation and magnetron sputtering processes. Each electrode is symmetrically distributed in a ring array with a center-to-center spacing of approximately 1 mm. A micro-via array is uniformly distributed within each electrode region. The via diameter is 200 nm, the via spacing is 2 μm, and the total number of vias is approximately 5000 per electrode.

[0097] The multi-channel phased-array excitation module employs a 4 / 6-channel synchronous high-voltage pulse module with an output voltage range of 0–80V, a pulse duration of 200μs, and a repetition frequency of 2Hz. The controller uses an STM32H7 series MCU to achieve synchronous waveform output and phase control across multiple channels. In this embodiment, the multi-channel phased-array excitation module corresponds to four electrodes / six independent electrodes, and their phase differences are demonstratively set as follows: 0°, 90°, 180°, 270° (corresponding to four independent electrodes) or 0°, 60°, 120°, 180°, 240°, and 300° (corresponding to six independent electrodes). This symmetrical configuration enables clockwise electric field synthesis interference in tissue or cell models, resulting in a stable focusing effect. It should be noted that this phase difference is not the only limiting parameter; in practical applications, it can be flexibly adjusted according to the target depth and processing scenario. Furthermore, while the voltage amplitude of the four / six channels is set to be consistent in this embodiment, the system supports independent configuration of the voltage for each channel to meet the needs of different cell processing or tissue applications. Figure 3 The diagram shown is a block diagram of the module functions of the excitation system.

[0098] The 90° value in this embodiment is merely a demonstrative value for a four-channel ring-shaped symmetrical electrode; similarly, 60° is merely a demonstrative value for a six-channel ring-shaped symmetrical electrode, ensuring the directionality and symmetry of the electric field synthesis. In other cell processing scenarios, the phase difference is not a fixed value and can be adjusted according to the required focal depth and distribution. The example in this paper uses a 60° / 90° phase difference configuration, mainly based on the geometric structure of a ring-shaped six-channel / four-channel symmetrical distribution, which can form clockwise electric field synthesis interference, thereby forming a stable focusing area at the depth of the model tissue. This parameter is only a demonstrative setting and not the only limiting condition. In other application scenarios such as cell processing or organoid manipulation, the phase difference can be flexibly adjusted according to the target depth, processing area, and electric field distribution requirements. In the simulation verification of the embodiment, the voltage amplitude of each channel is kept consistent to verify the effect of phase modulation on the focusing effect; in practical applications, the system supports independent adjustment of the voltage of each channel, which can further achieve adaptation to different cell types and culture environments.

[0099] Electric field simulations were performed using COMSOL Multiphysics software to construct a two-dimensional cross-sectional model. In tissue application scenarios, the model was set as a homogeneous isotropic conductor (conductivity 0.2 S / m, thickness 6 mm) to verify the focusing principle. In cell processing scenarios, the model could be adjusted to reflect the conductivity parameters of culture medium or cell suspension (typical range 0.01–1.5 S / m), and heterogeneous distributions of cell populations or organoids could also be introduced to reflect a more realistic experimental environment.

[0100] In this embodiment, the voltage amplitudes of the four / six driving channels are set to the same value to highlight the role of phase difference in focusing; however, the system supports independent setting of the voltages of the four / six channels, thereby enabling flexible optimization of the electric field distribution based on cell type, culture conditions, and target depth. For example, a symmetrical voltage distribution can be used in adherent cell culture dishes, while a differentiated voltage distribution can be employed in three-dimensional organoids to enhance depth focusing.

[0101] For the six channels, the simulation results are as follows: Figure 4 As shown, a peak electric field intensity region (>1.0kV / cm) is formed at a tissue depth of approximately 2.8mm, exhibiting local convergence characteristics, with a focal radius of approximately 0.4mm, verifying the focusing effectiveness of micropore guidance + phased excitation.

[0102] The device measures 10mm x 1mm and possesses good flexibility and adhesion. Figure 5 The structural adaptation of the device when it is attached to the surface of skin tissue is demonstrated.

[0103] This embodiment illustrates that spatial electric field focusing can be achieved through the coordinated control of micropore array structure constraints and phase difference excitation electrical signals, enabling the formation of controllable perforation focal points, and providing a basis for subsequent verification on tissue physical models.

[0104] Example 3: This example is an experimental verification and depth controllability assessment of electroporation at the tissue scale.

[0105] This embodiment aims to verify the practical application effect of the phased-controlled electroporation system constructed in Embodiment 2 in ex vivo tissue samples, with a particular focus on the adjustability of the perforation depth, the accuracy of spatial positioning of the perforation area, and the selectivity of the effect of different parameter combinations on tissue. This embodiment combines fluorescence staining, electrode attachment experiments, and tissue section observation to form a complete verification process.

[0106] The tissue samples used were freshly obtained subcutaneous tissue from Wistar rats, with a thickness controlled within 4 mm. These tissues were cut into 10 mm × 10 mm pieces and pretreated with PBS (Phosphate Buffered Saline). A flexible electroporation device was attached to the tissue surface and secured with a medical-grade silicone ring frame to ensure uniform contact.

[0107] Using the four-channel system constructed in Example 1 or 2, the following parameters were set in different test groups: Group A (Focus Mode): See Figure 6A Different single-channel pulse voltages Fluorescence images of tissue sections at (10V, 15V, 20V, 30V) are shown, with red areas representing red fluorescent dye entering the cell nucleus after perforation and blue areas representing the cell nucleus. The pulse period T = 20ms, and all other pulse parameters remain consistent.

[0108] To avoid ambiguity, Figure 6A The voltage values ​​marked in the text are all single-channel output amplitudes. The synthetic excitation intensity in the target region can be represented by an equivalent superimposed voltage. Representation, approximately satisfying (For example correspond ). Scale bar: 100μm.

[0109] Group B (non-focused control): same voltage, all phases 0°; Group C (low voltage control): voltage 40V, other parameters are the same as Group A.

[0110] Immediately after 30 seconds of stimulation, tissue samples were incubated for 10 minutes in a staining solution containing PI (Propidium Iodide) dye. PI, a red fluorescent dye that can enter the cell nucleus after perforation, was used to assess the spatial distribution and depth of the perforation area. After staining, the samples were fixed with 4% paraformaldehyde and sectioned (10 μm thick) perpendicular to the electrode direction using a cryostat, and imaged using a laser confocal microscope. Here, 10 μm is the thickness of a single section.

[0111] The observation results show that ( Figures 6A-6D ): See Figure 6A In the rightmost image, group A, a distinct red high-fluorescence signal appears approximately 2.8 mm from the tissue surface. The perforation layer is concentrated, with a diameter of approximately 0.5 mm–0.6 mm, highly consistent with the simulated predicted focal position. Here, 2.8 mm is the depth accumulated from consecutive sections starting from the surface, also known as the absolute depth. This "depth" refers to the spatial position from the tissue surface, calculated using the consecutive section number n: Depth = n × 10 μm.

[0112] See Figure 6A In the second and third figures, in group A, when the pulse voltages are 15V and 20V respectively, the perforated area is close to the electrode surface, with a depth of less than 1mm and a diffuse distribution. See Figure 6A In the first image, in group A, no obvious PI signal was observed when the pulse voltage was 10V, indicating that effective perforation could not be induced under low voltage conditions.

[0113] Combination Figure 6A and Figure 6B It can be seen that, under the premise that other parameters are the same, with the pulse voltage... With the increase of , the relative depth of electroporation increases.

[0114] See Figure 6C It can be seen that, under the premise that other parameters are the same, for example, both are 30V and the number of pulses is 50, the relative electroporation depth (referred to as relative depth) increases with the increase of time.

[0115] See Figure 6D It can be seen that, under the premise of the same pulse voltage, the relative electroporation depth changes dynamically with both the duration and the number of pulses. For example, when the pulse voltage is 30V and the duration is 10ms, the relative electroporation depth increases with the increase of the number of pulses, but the electroporation depth also increases with the increase of the duration; similarly, when the pulse voltage is 30V and the number of pulses is the same, the relative electroporation depth also increases with the increase of the duration.

[0116] In this article, absolute depth refers to the actual distance from the electroporation site to the tissue surface, usually measured in μm. Relative depth refers to the ratio of absolute depth to the total tissue thickness at that location, typically expressed as a dimensionless value or percentage. For example, relative depth = absolute depth / total tissue thickness.

[0117] Further quantitative statistics were performed on the sliced ​​images: the absolute depth of the perforation focus was defined by the location of the PI signal peak. ), and calculate the proportion of PI-positive cells within that depth layer as the perforation rate ( The results showed that group A... The perforation rate in Group B was 73.4% ± 5.1%; the perforations in Group B were mainly distributed on the surface and diffused, with a perforation rate of 24.6% ± 3.8%; no stable PI positive layer was observed in Group C, suggesting that low voltage was insufficient to induce effective perforation.

[0118] This verifies that the phased excitation strategy not only increases the perforation depth but also significantly enhances the perforation efficiency in the focal region.

[0119] In summary, this embodiment demonstrates that the proposed microporous structure + phased excitation device can achieve a stable, deep, and spatially confined electroporation zone under real tissue conditions, possessing the potential for fine tissue intervention. Furthermore, the perforation focal position is in good agreement with the simulation results, confirming the depth controllability and spatial accuracy of this scheme in actual biological tissues.

[0120] Example 4: This embodiment is a system control verification based on phased waveform programming and punch response prediction.

[0121] To further verify the practicality of the device of this invention in dynamic control, parameter adaptation, and focus prediction, this embodiment constructs a complete multi-channel phased-array perforation system, covering excitation control, device driving, and tissue response feedback modules. The overall system structure includes: a controller (FPGA / MCU), a multi-channel phased-array excitation module (including a pulse generation unit, a phase control module, and a multi-channel driving module), a flexible electrode array module, and a tissue response detection module (for detecting current / resistance changes), etc.

[0122] In the experimental design, the target perforation depth was set to be between 2.0 mm and 3.8 mm, with a step of 0.2 mm, and a total of 10 sets of parameters were set.

[0123] Each excitation group uses a different combination of phase biases. The specific range and step size of the phase difference parameters are based on calculations from previous simulation models and are stored in the system control unit in the form of a lookup table. During the experiment, the controller calls the corresponding parameters according to the lookup table and automatically sets the excitation waveform to achieve gradual adjustment of the target perforation depth.

[0124] The phase difference ranges from 0° to 360°, and in practical applications, it needs to be selected in conjunction with the electrode geometry and the target focal depth. Typically, in a symmetrical electrode array, an evenly distributed six-channel 60° distribution, as in Example 2, is preferred to achieve stable focusing. The principle for setting the phase difference is to maximize the electric field intensity in the target area, and its value can be obtained through prior simulation or experimental calibration. In cell treatment scenarios, the phase difference is generally controlled between 30° and 90° to balance focusing efficiency and cell viability; in other scenarios, such as tissue ablation, a range of 60° to 120° can be used to achieve a deeper focusing effect.

[0125] The system simultaneously records the dynamic changes in the impedance of the tissue electrode interface before and after the pulse is applied, and uses the impedance change as a perforation signal for location verification.

[0126] Figure 7 Images of stained perforated tissue at different target depths (top) and corresponding electrode-tissue impedance dynamic curves (bottom) are shown. The results show that deeper targets (e.g., 3.6 mm) correspond to a slower impedance drop rate, the perforation layer is farther from the surface, and the tissue staining is more concentrated, indicating that the phase control combination set by the system can induce directional focusing perforation response at all target depths.

[0127] Further statistical analysis results are as follows Figure 8 As shown, the mean absolute error between the target depth and the measured perforation focal position is controlled between 0.18 mm and 0.38 mm, with an average of 0.27 mm and a standard deviation of 0.07 mm, verifying the repeatability and predictive stability of the system in depth control.

[0128] The phase-controlled driven flexible device with through-hole array constructed in this invention not only possesses precise spatial focusing capabilities but also enables predictive matching of perforation depth through a control system, providing a foundation for subsequent adaptive control and closed-loop feedback. Impedance drop signals within 1–3 seconds before perforation can serve as feedback, verifying the system's predictive ability regarding tissue response. The focusing position deviation after phase adjustment between different tissue samples is less than 10%, indicating that the device possesses a certain degree of individual adaptability.

[0129] This embodiment demonstrates that the present invention has stability in the formation of perforation focus under multiple parameter combinations, and that there is a modelable correlation between its output parameters and tissue response.

[0130] Example 5: This embodiment is an in vivo perforation verification experiment of a flexible adhesive electroporation device.

[0131] To verify the adaptability and functional feasibility of the flexible adhesive phased-array perforation system proposed in this invention under real physiological conditions, this embodiment designed and carried out an electroporation experiment on the surface of small animals to evaluate its performance in terms of target depth focusing, tissue safety and parameter response consistency.

[0132] Adult Wistar rats were used in the experiment. Their backs were shaved and cleaned. A 10mm × 10mm area was selected for attaching the flexible electrode array of this device, ensuring uniform contact. Excitation parameters were set as follows: six-channel phased-array excitation (0–300° clockwise increments), voltage 80V, pulse width 200μs, frequency 2Hz, and duration 60 seconds. Skin surface temperature was recorded in real time during the process.

[0133] Immediately after stimulation, tissue samples were collected, frozen sections were prepared along the vertical direction, and PI staining was performed to determine the proportion of perforated cells in different depth regions. Subsequently, tissue samples were collected at three time points: 0h, 24h, and 72h for CD45 immunostaining and quantification of inflammation scores.

[0134] like Figure 9 As shown, the highest perforation rate, reaching 71.2%, was observed in the 2.6–2.9 mm region; the perforation rate in the surface region (<1.0 mm) was less than 25%, while the signal attenuated significantly in the deep region (>3.2 mm). This result is consistent with the simulated predicted focus in Examples 1–3, indicating that the system can achieve focus consistency and depth focusing controllability under in-situ conditions.

[0135] like Figure 10As shown, tissue inflammation scores at different time points after stimulation revealed that the inflammatory response was mild immediately after stimulation (0h); it peaked at 24 hours, with the score increasing but not exceeding the moderate response threshold; and the inflammatory markers decreased significantly after 72 hours, approaching baseline levels. No obvious tissue necrosis or ulceration was observed, indicating that this method has good short-term biocompatibility.

[0136] like Figure 11 As shown, the skin surface temperature change curve during stimulation shows a peak temperature rise of 1.5°C, which is far below the thermal damage threshold (6°C), and the temperature rise is stable and controllable, further confirming that this piercing strategy is a typical non-thermal mechanism with good energy control capabilities.

[0137] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0138] Through the above description of the embodiments, those skilled in the art can clearly understand that the methods of the above embodiments can be implemented by means of software plus necessary general-purpose hardware platforms. Of course, they can also be implemented by hardware, but in many cases the former is a better implementation method. Based on this understanding, the technical solution of the present invention, or the part that contributes to the prior art, can be embodied in the form of a software product. This computer software product is stored in a storage medium (such as ROM / RAM, magnetic disk, optical disk) and includes several instructions to cause a computer terminal (which may be a mobile phone, computer, server, or network device, etc.) to execute the methods described in the various embodiments of the present invention.

[0139] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A cell electroporation device, characterized in that, include: The controller is configured to acquire preset excitation parameters; The excitation parameters include voltage amplitude, pulse width, frequency, and phase delay; The flexible electrode array module is configured to apply an electric field to the target cells; The flexible electrode array module includes a flexible substrate and a plurality of electrodes disposed on the flexible substrate; the region where the electrodes are located has a micro-via array, which is used to converge the electric field applied by the electrodes and guide the direction of the electric field; A multi-channel phase-controlled excitation module, connected to the controller and the flexible electrode array module, is configured to generate corresponding high-voltage pulses according to the voltage amplitude, pulse width and frequency in the excitation parameters, and to introduce a phase delay for each channel. Electric fields are applied to the electrodes corresponding to the channels with phase delay, so that the electric fields applied by each electrode interfere and superimpose with each other under the action of the micro-perforation, forming a fused electric field of the target radius at the target perforation depth, so as to electroporate the target cells. The method for performing cell electroporation using the aforementioned cell electroporation device includes: S101, attach the flexible electrode array module of the cell electroporation device to the target object; S102, using the cell electroporation device to apply an electric field to the target object to electroporate the target cells on the target object; Specifically, step S102 includes the following steps: S1021, the controller of the cell electroporation device acquires user-configured cell parameters; the cell parameters include cell type and target perforation depth; S1022, the controller matches the corresponding excitation parameters in a pre-built cell-excitation parameter correspondence table according to the cell parameters; the excitation parameters include voltage amplitude, pulse width, frequency, and phase delay; S1023, the multi-channel phased excitation module of the cell electroporation device generates a corresponding high-voltage pulse based on the matched excitation parameters voltage amplitude, pulse width, and frequency, and introduces a phase delay for each channel; the electrodes corresponding to the channels apply electric fields simultaneously according to the phase delay, so that the electric fields applied by each electrode interfere and superimpose with each other under the action of the micro-perforation, forming a fused electric field of the target radius at the target perforation depth, so as to perform electroporation on the target cells.

2. The cell electroporation device according to claim 1, characterized in that: The micro-vias in the micro-via array penetrate the flexible substrate and are arranged in a ring, a rectangle, or a radial pattern.

3. The cell electroporation device according to claim 1, characterized in that: The aperture of the micro-via array is 50nm-500nm, and the spacing between the micro-vias is 1um-5um.

4. The cell electroporation device according to claim 1, characterized in that: The inner wall or orifice region of the micro-vias in the micro-via array is provided with a stimuli-responsive sealing layer; such that in the non-working state, the sealing layer expands to block the micro-vias, while in the working state, the sealing layer contracts in response to external stimuli to open the micro-vias; the external stimuli include one or more of pH, electric field or temperature.

5. The cell electroporation device according to claim 1, characterized in that: The electrodes are arranged symmetrically in a ring array, with a spacing of 0.5 mm to 2 mm between adjacent electrodes; the maximum phase delay between the electrodes is... 360°, the phase delay between adjacent electrodes is equal.

6. The cell electroporation device according to claim 4, characterized in that: The number of electrodes is even, and the electrodes, which are arranged at intervals, are connected together to form a group.

7. The cell electroporation device according to claim 1, characterized in that: The flexible substrate is made of polyimide film, PDMS or TPU, with a thickness of 80um-120um.

8. The cell electroporation device according to claim 1, characterized in that: The multi-channel phase-controlled excitation module outputs a voltage amplitude of 10V-100V, a pulse width of 10us-1000us, and a frequency of 1Hz-2Hz.

9. A cell electroporation device according to claim 1, characterized in that: In step S01, when processing adherent cells, the flexible electrode array module is attached to the bottom / top surface of the cell culture dish or chip so that the electric field is uniformly applied to the cell monolayer. When processing a population of suspended cells, the flexible electrode array module is attached to the inner wall of the microfluidic chip channel to achieve controlled perforation of cells during the flow process; When processing three-dimensional cell clumps or organoid models, the flexible electrode array module is arranged around the supporting substrate and achieves focused electroporation at the internal target depth through phased excitation.

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