Microscale electroporation method based on single-cell force-electric coupling characteristics

Abstract

The application relates to a microscale electroporation method based on single-cell force-electric coupling characteristics, which realizes real-time sensing of electrical characteristics without optical labeling by acquiring the electrical impedance signal of cell deformation in the first detection area of a microchannel and extracting electrical characteristic parameters. By matching the electrical characteristic parameters with a pre-constructed database and determining the corresponding optimal electroporation parameter group, the defect that a fixed parameter mode cannot adapt to single-cell heterogeneity is overcome. By applying the optimal electroporation parameter group to apply an electric pulse when the cell reaches the electroporation area, precise electroporation for single-cell individuals is realized. By acquiring the electrical impedance signal of the second detection area after electroporation and evaluating the electroporation effect based on the signal change amount before and after electroporation, real-time quantitative judgment of the electroporation quality is realized. Without complex optical equipment, the application overcomes the defect that the single-cell electroporation efficiency and survival rate are difficult to balance due to the fixed parameter mode.

Description

Technical Field

[0001] This application relates to the fields of microfluidics and cell manipulation technology in the biomedical engineering industry, and in particular to a microscale electrotransfection method based on the force-electric coupling characteristics of single cells. Background Technology

[0002] Electroporation is a physical transfection technique that uses electrical pulses to create reversible or irreversible micropores on the cell membrane, thereby enabling exogenous molecules to enter the cell. Due to its wide applicability, high efficiency and independence from viral vectors, it is widely used in gene editing, cell therapy and vaccine development.

[0003] Currently, scholars both domestically and internationally have conducted extensive research on electrotransfection pulse parameters, systematically examining the influence of parameters such as electric field strength, pulse waveform, pulse duration, and number of pulses on the electrotransfection efficiency of different cell types, and thereby screening for optimal electrotransfection conditions suitable for specific cell lines. However, these methods generally employ a "pre-experiment mode," that is, after screening a fixed set of optimal parameters for a certain cell type, all cells of the same type are batch-processed using this set of parameters. This technical approach implicitly assumes a fundamental assumption: that cells of the same type have consistent biological characteristics. But in reality, even cells from the same source exhibit significant differences in size, morphology, cell cycle, and mechanical properties; this single-cell heterogeneity is an inherent property of cell populations. Therefore, the batch processing mode with fixed parameters cannot ensure that every cell is under optimal electrotransfection conditions, making it difficult to simultaneously achieve transfection rate and survival rate.

[0004] To address these issues, some researchers have attempted to combine optical equipment such as microscopes to adjust electroporation parameters in real time based on optical information such as cell morphology and size. While these methods have improved the specificity of regulation to some extent, they still suffer from drawbacks such as complex equipment, difficulty in system integration, potential impact of optical labeling on cell viability, and limited throughput.

[0005] In recent years, related studies have confirmed a clear correlation between cellular mechanical and electrical properties, providing a theoretical basis for acquiring cellular mechanical states in real time without complex optical equipment. However, current technology has not yet translated this theory into an engineered solution capable of measuring single-cell mechanical properties in real time and adaptively adjusting electrotransfer parameters accordingly.

[0006] In summary, how to achieve real-time adaptive control of electrotransfection parameters to address the mechanical heterogeneity of single cells without the need for complex optical equipment is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] Based on this, in order to address the technical problem that traditional methods cannot achieve real-time adaptive control of electrotransfection parameters for single-cell mechanical heterogeneity without the need for complex optical equipment, this application provides a microscale electrotransfection method based on the mechanical-electrical coupling characteristics of single cells.

[0008] This application provides a microscale electrotransfection method based on the single-cell force-electric coupling characteristics, including: The electrical impedance signal generated by the deformation of the channel cross-section when the target single cell passes through the first detection area of ​​the microchannel is obtained, and this electrical impedance signal is defined as the first target electrical impedance signal; The electrical characteristic parameters of the target single cell are extracted based on the first target impedance signal; The electrical characteristic parameters are matched with a pre-constructed mapping database. The mapping database includes multiple reference data, each of which includes the mechanical characteristic parameters of a reference single cell, the electrical characteristic parameters of a reference single cell, the optimal electrotransfection parameter set of a reference single cell, and the mapping relationship between the mechanical characteristic parameters of the reference single cell and the electrical characteristic parameters of the reference single cell and the optimal electrotransfection parameter set of the reference single cell. The optimal electroporation parameter set corresponding to the target single cell is determined based on the matching results; When the target single cell reaches the electrotransfection zone within the microchannel, an electrical pulse is applied to the target single cell using the optimal electrotransfection parameter set to perform electrotransfection; The electrical impedance signal of the target single cell as it passes through the second detection area of ​​the microchannel after electrotransfection is obtained due to the deformation caused by the change in the cross-section of the channel, and this electrical impedance signal is defined as the second target electrical impedance signal; The effectiveness of electrotransfection and cell viability were assessed based on the change between the first and second target impedance signals.

[0009] This application relates to a microscale electrotransfection method based on the mechano-electric coupling characteristics of single cells. By acquiring the electrical impedance signal of the target single cell when it deforms due to changes in the channel cross-section in the first detection zone of the microchannel and extracting its electrical characteristic parameters, real-time sensing of the electrical characteristics of single cells without optical labeling is achieved. By matching the electrical characteristic parameters with a pre-constructed mapping database containing mapping relationships between mechanical characteristic parameters, electrical characteristic parameters, and optimal electrotransfection parameter sets, the corresponding optimal electrotransfection parameter set is determined, overcoming the limitation of fixed parameter modes in adapting to single-cell heterogeneity. By applying an electrical pulse using the optimal electrotransfection parameter set when the target single cell reaches the electrotransfection zone, precise electrotransfection of individual single cells is achieved. By acquiring the electrical impedance signal of the second detection zone after electrotransfection and evaluating the electrotransfection effect and cell viability based on the signal changes before and after electrotransfection, real-time quantitative evaluation of electrotransfection quality is achieved. By combining the above steps, this application achieves adaptive electrotransfection parameter control and effect verification for single-cell mechanical heterogeneity without the need for complex optical equipment, overcoming the shortcomings of fixed parameter mode which makes it difficult to balance single-cell electrotransfection efficiency and survival rate. Attached Figure Description

[0010] Figure 1 This is a flowchart illustrating a microscale electrotransfection method based on the force-electric coupling characteristics of single cells, provided as an embodiment of this application.

[0011] Figure 2 This is a schematic diagram of the structure of a microscale electrotransfection system based on the single-cell force-electric coupling characteristics, provided as an embodiment of this application.

[0012] Figure 3 This is a schematic diagram of the structure of a microfluidic chip in a microscale electrotransfection system based on the single-cell force-electric coupling characteristics, provided as an embodiment of this application.

[0013] Figure 4 This is a schematic diagram of the structure of a microchannel and recovery device in a microscale electrotransfection system based on the single-cell force-electric coupling characteristics, provided as an embodiment of this application.

[0014] Figure 5 This is a schematic diagram of the structure of a microchannel and recovery device in a microscale electrotransfection system based on the single-cell force-electric coupling characteristics, provided as another embodiment of this application.

[0015] Figure 6 This is a schematic diagram illustrating the correspondence between the curves of each region of the microchannel and the time-resistance component, and the curves of each region of the microchannel and the time-capacitance component, in a microscale electrotransfection method based on the single-cell force-electric coupling characteristics provided in an embodiment of this application.

[0016] Figure 7This is a graph of the time-resistance component curve after processing in a microscale electrotransfection method based on the single-cell force-electric coupling characteristics provided in an embodiment of this application.

[0017] Figure 8 This is a graph of the time-capacitance component curve after processing in a microscale electrotransfection method based on the single-cell force-electric coupling characteristics provided in an embodiment of this application.

[0018] Figure label: 10-Pressure drive device; 20-Flow rate sensor; 30-Microfluidic chip; 310-Inlet region; 320-Filtration region; 330-Microchannel; 331-Inlet straight pipe section; 332-Funnel-shaped tapering section; 333-Equal width straight pipe contraction section; 334-Funnel-shaped expanding section; 335-Outlet straight pipe section; 340-Outlet region; 351-First electrode plate; 352-Second electrode plate; 353-Third electrode plate; 354-Fourth electrode plate; 355 - Fifth electrode; 356 - Sixth electrode; 357 - Seventh electrode; 358 - Eighth electrode; 360 - First detection area; 370 - Second detection area; 380 - Electroporation area; 381 - Parallel plate electroporation electrode; 381a - Upper electrode; 381b - Lower electrode; 40 - Cell impedance meter; 50 - Data processing device; 60 - Recovery device; 610 - Micropillar array sorting structure; 621 - First collection container; 622 - Second collection container. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0020] This application provides a microscale electrotransfection method based on the single-cell force-electric coupling characteristics. It should be noted that the microscale electrotransfection method based on the single-cell force-electric coupling characteristics provided in this application is applied to a microscale electrotransfection system based on the single-cell force-electric coupling characteristics.

[0021] Furthermore, the microscale electrotransfection method based on the single-cell force-electric coupling characteristics provided in this application does not limit the executing entity. Optionally, the executing entity of the microscale electrotransfection method based on the single-cell force-electric coupling characteristics provided in this application can be a data processing device in a microscale electrotransfection system based on the single-cell force-electric coupling characteristics. For the sake of brevity, the microfluidic system will be referred to as "system" in all subsequent descriptions.

[0022] like Figure 1As shown, in one embodiment of this application, the microscale electrotransfection method based on the single-cell force-electric coupling characteristics includes the following steps S100 to S700: S100: Obtain the impedance signal caused by the deformation of the channel cross-section when the target single cell passes through the first detection area of ​​the microchannel, and define the impedance signal as the first target impedance signal.

[0023] S200, extract the electrical characteristic parameters of the target single cell based on the first target impedance signal.

[0024] S300, the electrical characteristic parameters are matched with a pre-constructed mapping database. The mapping database includes multiple reference data, each of which includes the mechanical characteristic parameters of a reference single cell, the electrical characteristic parameters of a reference single cell, the optimal electrotransfection parameter set of the reference single cell, and the mapping relationship between the mechanical characteristic parameters of the reference single cell and the optimal electrotransfection parameter set of the reference single cell.

[0025] S400, determine the optimal electroporation parameter set corresponding to the target single cell based on the matching results.

[0026] S500, when the target single cell reaches the electrotransfection zone within the microchannel, an electrical pulse is applied to the target single cell using the optimal electrotransfection parameter set to perform electrotransfection.

[0027] S600, acquire the electrical impedance signal of the target single cell as it passes through the second detection area of ​​the microchannel after electrotransfection, which is deformed due to the change in the cross-section of the channel, and define the electrical impedance signal as the second target electrical impedance signal.

[0028] S700 assesses electrotransfection efficacy and cell viability based on the change between the first and second target impedance signals.

[0029] Specifically, in S100, the microchannel is a fluid channel with a variable cross-section structure, whose cross-sectional dimensions change along the cell flow direction. When a target single cell is suspended in the buffer solution and flows through the first detection zone of the microchannel with the buffer solution, the cell deforms due to the compression of the channel wall as the channel cross-section gradually transitions from a large cross-section in the straight inlet section to a small cross-section in the funnel-shaped tapering section. During the deformation process, the cell membrane area increases and the cytoplasmic conductive pathway narrows; these changes are directly reflected in the impedance signal when the cell passes through the first detection zone.

[0030] The microchannel has two detection zones: a first detection zone, which is the zone through which cells pass before electroporation, and a second detection zone, which is the zone through which cells pass after electroporation, and also has multiple electrodes.

[0031] As cells pass through the first detection zone, the cell impedance meter applies AC excitation signals to multiple electrode pads within the zone and acquires the impedance change signals caused by the cell's passage. The acquired impedance change signals are synchronously demodulated by a lock-in amplifier within the cell impedance meter, extracting the resistance component in phase with the AC excitation signal and the capacitance component quadrated with it, resulting in time-resistance and time-capacitance component curves, respectively. To facilitate signal comparison and feature extraction, after subtracting background impedance, the ordinates of the time-resistance and time-capacitance component curves are normalized for each time interval, ultimately yielding the processed time-resistance and time-capacitance component curves.

[0032] The horizontal axis of the processed time-resistance component curve represents time, and the vertical axis represents the normalized resistance value, as shown below. Figure 7 As shown, Figure 7 The horizontal axis is in seconds. Figure 7 The ordinate is dimensionless because it represents the normalized resistance value. The time-resistance component curve after treatment is used to characterize changes in the electrical properties of the cytoplasm.

[0033] The processed time-capacitance component curve has time on the x-axis and the normalized capacitance value on the y-axis, as shown below. Figure 8 As shown, Figure 8 The horizontal axis is in seconds. Figure 8 The ordinate is dimensionless because it represents the normalized capacitance value. The processed time-capacitance component curve is used to characterize changes in the dielectric properties of the cell membrane.

[0034] Subsequently, the processor inside the cell impedance meter performs feature extraction on the processed time-resistance component curve and the processed time-capacitance component curve, respectively, to obtain characteristic parameters such as the normalized resistance component peak value, the normalized capacitance component peak value, and cell migration time. The lock-in amplifier and processor are internal structures of the cell impedance meter and are not the focus of this application, therefore they are not shown in the figure.

[0035] As the cell passes through the second detection zone, the cell impedance meter applies an AC excitation signal to multiple electrode pads within the zone and acquires the impedance change signal caused by the cell's passage. The acquired impedance change signal is synchronously demodulated by a lock-in amplifier within the cell impedance meter, extracting the resistance component in phase with the AC excitation signal and the capacitance component quadrated with it, thus obtaining the time-resistance component curve and the time-capacitance component curve, respectively. This principle is similar to that of the cell passing through the first detection zone and will not be elaborated further here.

[0036] like Figure 7 As shown, the three normalized resistance component peaks on the left are from the first detection area, corresponding to the normalized resistance component peaks between the first and second electrode plates, between the second and third electrode plates, and between the third and fourth electrode plates, respectively. Figure 7 As shown, the three normalized resistance component peaks on the right are from the second detection area, corresponding to the normalized resistance component peaks between the fifth and sixth electrode plates, the normalized resistance component peaks between the sixth and seventh electrode plates, and the normalized resistance component peaks between the seventh and eighth electrode plates, respectively.

[0037] Figure 8 Similarly, the explanation will not be repeated.

[0038] Extracting the electrical characteristic parameters of the target single cell in S200 involves selecting a representative resistance peak from multiple normalized resistance component peaks that can represent the electrical characteristic parameters of the target single cell, and selecting a representative capacitance peak from multiple normalized capacitance component peaks that can represent the electrical characteristic parameters of the target single cell.

[0039] Optionally, the signal between the third and fourth electrodes is used to represent the cell's electrical properties. At this point, the cell is within the contraction section of a straight tube of equal width, where the channel cross-section is narrowest, cell deformation reaches its maximum and tends to stabilize; the cytoplasmic channel is compressed to its narrowest point, and the peak value of the resistance component reaches its maximum; the cell membrane area is stretched to its maximum, and the peak value of the capacitance component reaches its maximum. The signal amplitude between the third and fourth electrodes is the largest, and the signal-to-noise ratio is the highest, best reflecting the electrical property parameters of the cell under extreme deformation. Therefore, the normalized peak value of the resistance component corresponding to the signal between the third and fourth electrodes, and the normalized peak value of the capacitance component corresponding to the signal between the third and fourth electrodes, are used as the electrical property parameters of the target single cell extracted in S200. The electrical property parameters of the target single cell extracted in S200 are the electrical property parameters of the target single cell before electrotransfection.

[0040] Similarly, in S700, when evaluating the electrotransfection effect and cell viability based on the change between the first and second target impedance signals, it is also necessary to first extract the electrical characteristic parameters of the target single cell after electrotransfection from the second target impedance signal. At this time, the peak values ​​of the normalized resistance and capacitance components corresponding to the signals between the fifth and sixth electrodes are selected as the electrical characteristic parameters of the target single cell after electrotransfection. Since the target single cell has not yet recovered its deformation, the changes in resistance and capacitance are caused by electrotransfection, excluding interference from deformation recovery. Therefore, the peak values ​​of the normalized resistance and capacitance components corresponding to the signals between the fifth and sixth electrodes are used as the electrical characteristic parameters of the target single cell after electrotransfection.

[0041] Finally, by subtracting the electrical characteristic parameters of the target single cell before electrotransfection and the electrical characteristic parameters of the target single cell after electrotransfection, the difference obtained is the change between the first target impedance signal and the second target impedance signal. This change is used to evaluate the electrotransfection effect and cell viability.

[0042] In this embodiment, by acquiring the impedance signal of the target single cell when it deforms due to changes in the channel cross-section in the first detection area of ​​the microchannel and extracting the electrical characteristic parameters, real-time sensing of the electrical characteristics of single cells without optical labeling is achieved. By matching the electrical characteristic parameters with a pre-constructed mapping database containing the mapping relationship between mechanical characteristic parameters, electrical characteristic parameters, and the optimal electrotransfection parameter set, the corresponding optimal electrotransfection parameter set is determined, overcoming the defect that the fixed parameter mode cannot adapt to the heterogeneity of single cells. By applying an electrical pulse to the optimal electrotransfection parameter set when the target single cell arrives at the electrotransfection area, precise electrotransfection and cell survival status for individual single cells are achieved. By acquiring the impedance signal of the second detection area after electrotransfection and evaluating the electrotransfection effect based on the signal change before and after electrotransfection, real-time quantitative evaluation of electrotransfection quality is achieved. In summary, this application achieves adaptive electrotransfection parameter control and effect verification for the mechanical heterogeneity of single cells without the need for complex optical equipment, overcoming the defect that the fixed parameter mode makes it difficult to balance the efficiency and survival rate of single-cell electrotransfection.

[0043] In one embodiment of this application, the microchannel is provided with an inlet straight tube section, a funnel-shaped tapering section, a straight tube contraction section of equal width, a funnel-shaped expanding section and an outlet straight tube section in sequence along the cell flow direction.

[0044] The electro-dyeing zone is provided within the equal-width straight tube contraction section.

[0045] A first electrode plate, a second electrode plate, and a third electrode plate are sequentially arranged from the end of the inlet straight pipe section to the end of the funnel-shaped tapering section. A fourth electrode plate and a fifth electrode plate are sequentially arranged in the equal-width straight pipe contraction section. A sixth electrode plate, a seventh electrode plate, and an eighth electrode plate are sequentially arranged from the end of the funnel-shaped expanding section to the end of the outlet straight pipe section.

[0046] The first detection area is formed by the channel area covered by the first electrode sheet to the fourth electrode sheet, the second detection area is formed by the channel area covered by the fifth electrode sheet to the eighth electrode sheet, and the electrotransfer area is located in the channel area between the fourth electrode sheet and the fifth electrode sheet.

[0047] Specifically, the structural design of the microchannel is the physical foundation for the entire microscale electrotransfection method based on the single-cell force-electric coupling characteristics. The inlet straight tube section is the initial region after cells enter the microchannel; its cross-sectional size is relatively large, allowing cells to remain in their natural state without deformation. The distance L1 between the inlet of the inlet straight tube section and the first electrode is set to 100 micrometers. This distance ensures that the cells have stabilized their flow state before entering the detection zone, achieving single-row orderly flow. The cross-section of the funnel-shaped tapering section gradually decreases from the large size of the inlet straight tube section to the small size of the constant-width straight tube contraction section. As cells flow through this region, they experience gradually increasing compression, resulting in controllable progressive deformation. The cross-sectional size of the constant-width straight tube contraction section remains constant, allowing cells to maintain a stable deformed state. This region also contains the electrotransfection zone for applying electrical pulses to cells in a deformed state. The cross-section of the funnel-shaped expanding section gradually increases from the small size of the constant-width straight tube contraction section to the large size of the outlet straight tube section, allowing cells to gradually recover their deformed state in this region. The outlet straight section is the last region before the cells leave the microchannel, and its cross-sectional dimensions are restored to the same as those of the inlet straight section.

[0048] The arrangement of the electrode pads is closely related to the division between the detection zone and the electrotransfection zone. The first electrode pad is placed at the end of the inlet straight tube section, the second electrode pad is placed at the junction of the inlet straight tube section and the funnel-shaped tapering section, the third electrode pad is placed at the junction of the funnel-shaped tapering section and the constant-width straight tube contraction section, and the fourth electrode pad is placed within the constant-width straight tube contraction section near the upstream of the electrotransfection zone. The channel area covered by the first to fourth electrode pads constitutes the first detection zone, which covers the entire process of cell deformation from initial compression to complete deformation, enabling the acquisition of electrical impedance signals throughout the deformation process. The fifth electrode pad is placed within the constant-width straight tube contraction section near the downstream of the electrotransfection zone, the sixth electrode pad is placed at the junction of the constant-width straight tube contraction section and the funnel-shaped expanding section, the seventh electrode pad is placed at the junction of the funnel-shaped expanding section and the outlet straight tube section, and the eighth electrode pad is placed at the end of the outlet straight tube section. The channel area covered by the fifth to eighth electrode plates constitutes the second detection zone. This zone encompasses the entire process of cell recovery from the onset of deformation after electrotransfection to complete recovery, enabling the acquisition of electrical impedance signals during the recovery process. The electrotransfection zone is located within the equal-width straight tube contraction section between the fourth and fifth electrode plates, i.e., between the first and second detection zones. Cells complete the electrotransfection operation in this region.

[0049] The spacing between each electrode is set according to the equivalent cell diameter A to ensure detection accuracy and electrotransfection effect. Optionally, the distance L2 between the first and second electrodes is equal to 1.2 times A, ensuring that the cell remains in a stable single-cell state when passing between these two electrodes. The distance L3 between the second and third electrodes is equal to 1.5 times A, ensuring that the cell has sufficient space to complete deformation during the contraction phase. The distance L4 between the third and fourth electrodes is equal to 2 times A, ensuring that the cell remains in a stable deformation state in the initial region of the equal-width straight tube contraction phase. The distance L5 between the fourth electrode and the beginning of the electrotransfection zone is equal to 50 micrometers, and the distance L7 between the end of the electrotransfection zone and the fifth electrode is equal to 50 micrometers. The distance L8 between the fifth and sixth electrodes is equal to 2 times A. The distance L9 between the sixth and seventh electrodes is equal to 1.5 times A. The distance L10 between the seventh and eighth electrodes is equal to 1.2 times A. The above setup ensures that cells can be completely detected and processed as they pass through the detection and electrotransfection zones.

[0050] Optionally, the equivalent channel diameter D1 of the inlet straight tube section and the equivalent channel diameter D3 of the outlet straight tube section are both equal to 2.5 times A, with wider channels ensuring that cells are in a free state when entering and leaving. The equivalent channel diameter D2 of the equal-width straight tube constriction section is equal to 0.85 times A, with narrower channels ensuring that cells are subjected to sufficient compression deformation in this region.

[0051] In this embodiment, by setting up a channel structure in the microchannel with an inlet straight tube section, a funnel-shaped tapering section, a straight tube contraction section of equal width, a funnel-shaped expanding section, and an outlet straight tube section connected in sequence, the cells undergo a deformation process during flow, with the cross-section changing from large to small, maintaining equal width, and then changing from small to large. An electrotransfection zone is set in the straight tube contraction section of equal width. At the same time, the first to eighth electrode plates are arranged at specific positions, and the spatial relationship between the first detection zone, the second detection zone, and the electrotransfection zone is defined. This realizes the integration of the entire process of cell deformation-impedance detection-electrotransfection-re-detection within a single microchannel, providing a structural basis for continuous, high-throughput manipulation of single cells.

[0052] In one embodiment of this application, S300 includes the following S310 to S320, namely, matching the electrical characteristic parameters with a pre-built mapping database includes the following S310 to S320: S310, calculate the weighted Euclidean distance between the electrical characteristic parameters of the target single cell and the electrical characteristic parameters of the reference single cell in each reference data of the mapping database.

[0053] S320: Select the data with the smallest weighted Euclidean distance as the matching result.

[0054] Specifically, the weighted Euclidean distance calculation method described in S310 is as follows: The electrical characteristic parameters of the target single cell are extracted to form a multi-dimensional feature vector. This feature vector contains values ​​for multiple feature dimensions, such as the peak value of the normalized resistance component, the peak value of the normalized capacitance component, and migration time. Each reference data in the mapping database also contains the electrical characteristic parameters of the corresponding reference single cell, forming a feature vector of the same dimension. To quantify the similarity in electrical characteristics between the measured cell and each reference single cell in the database, we characterize this similarity by calculating the weighted Euclidean distance. The weighted Euclidean distance is an improved Euclidean distance metric. Its core idea is to assign different weights to different feature dimensions when calculating the distance, thereby reflecting the differences in the importance of each feature dimension in the similarity evaluation.

[0055] In S320, after calculating the weighted Euclidean distance between the measured feature vector and the feature vectors of all reference data in the database, the data are sorted in ascending order of weighted Euclidean distance value. The smaller the weighted Euclidean distance, the higher the similarity in electrical properties between the measured cell and the reference cell. The data with the smallest weighted Euclidean distance is selected as the matching result; the reference single cell corresponding to this data is the cell whose electrical properties are closest to the target single cell to be tested.

[0056] In this embodiment, by calculating the weighted Euclidean distance between the electrical characteristic parameters of the target single cell and the electrical characteristic parameters of each reference data in the mapping database, and taking the smallest distance as the matching result, a quantitative and ranking objective evaluation of the similarity between the measured cells and the existing reference data is realized, avoiding the uncertainty brought about by subjective matching, and improving the accuracy and repeatability of determining the optimal electrotransfection parameter set.

[0057] In one embodiment of this application, S310 includes the following S311, namely, calculating the weighted Euclidean distance between the electrical characteristic parameters of the target single cell and the electrical characteristic parameters of the reference single cell in each reference data of the mapping database, including the following S311: S311. For each characteristic dimension of the electrical characteristic parameter, calculate the difference between the measured characteristic value and the database characteristic value, divide the difference by the standard deviation of the characteristic dimension in multiple repeated experiments, multiply by the corresponding weight coefficient, and then take the square root of the sum of squares of the multiplication results of each characteristic dimension to obtain the weighted Euclidean distance.

[0058] Specifically, S311 is a further refinement and formalization of the weighted Euclidean distance calculation method in S310. The weighted Euclidean distance can be calculated using the following formula 1: Formula 1.

[0059] Where d is the weighted Euclidean distance between the measured feature vector and the database feature vector, and a smaller d value indicates a higher similarity. i is the number of the feature dimension included in the electrical characteristic parameter. N is the total number of feature dimensions included in the electrical characteristic parameter. is the normalized eigenvalue of the i-th feature dimension, which is extracted from the first target impedance signal. This is the feature value of the i-th feature dimension under the corresponding combination of mechanical parameters in the database. This value is pre-calibrated experimentally and stored in the database. This represents the population standard deviation of the i-th feature dimension for this type of cell under natural conditions. This value reflects the degree of fluctuation of this feature dimension under experimental conditions. The larger the standard deviation, the worse the measurement repeatability of this dimension, and the smaller its weight in distance calculation. is the weight coefficient for the i-th feature dimension, which is preset according to the degree of influence of the feature dimension on the electroporation effect.

[0060] For example, suppose the electrical characteristic parameters include three feature dimensions: normalized peak resistance, normalized peak capacitance, and migration time. For a certain reference single cell, its normalized peak resistance is 0.75, normalized peak capacitance is 0.60, and migration time is 2.1 milliseconds, while the corresponding values ​​for a reference data in the mapping database are 0.70, 0.55, and 2.0 milliseconds, respectively. The differences between the three dimensions are 0.05, 0.05, and 0.1 milliseconds, respectively. Assuming the population standard deviations of the three dimensions in multiple repeated experiments are 0.02, 0.03, and 0.05 milliseconds, respectively, the normalized differences are 2.5, 1.67, and 2, respectively. Multiplying by their respective weight coefficients, assuming the weights of the three dimensions are 0.5, 0.3, and 0.2, the products are 1.25, 0.5, and 0.4, respectively. The square root of the sum of the squares of the three products is the weighted Euclidean distance, which is the similarity measure between the measured cell and the reference cell. In this example, the weighted Euclidean distance is approximately 1.4. The process involves iterating through all reference data in the database, calculating the weighted Euclidean distance for each reference, and selecting the one with the smallest weighted Euclidean distance as the matching result.

[0061] In this embodiment, the difference between the measured feature value and the database feature value is calculated for each feature dimension of the electrical characteristic parameter. This difference is then divided by the standard deviation of the feature dimension in multiple repeated experiments and multiplied by the corresponding weight coefficient. Finally, the square root of the sum of squares of the calculation results for each dimension is taken. This achieves the dedimensionalization and normalization of each feature dimension in the similarity measurement, effectively eliminating the bias influence of features of different magnitudes on the matching results. At the same time, the importance of each feature dimension is flexibly adjusted by the weight coefficient, which significantly improves the robustness and accuracy of the matching algorithm.

[0062] In one embodiment of this application, before S100, that is, before the acquisition of the electrical impedance signal caused by the deformation of the channel cross-section when the target single cell passes through the first detection area of ​​the microchannel, and before the electrical impedance signal is defined as the first target electrical impedance signal, a mapping database is constructed.

[0063] The constructed mapping database includes the following W100 to W800: W100, create a blank mapping database.

[0064] W200 selects a reference single cell from a sample library containing a preset number of reference single cells.

[0065] W300 drives the reference single cell through the first detection area of ​​the microchannel, collects the first reference impedance signal generated by the deformation of the channel cross-section when the reference single cell passes through the first detection area of ​​the microchannel, and extracts the reference electrical characteristic parameters.

[0066] W400, within the electrotransfection zone of the microchannel, applies an electrical pulse to the reference single cell using an electrotransfection parameter group consisting of multiple electrotransfection parameters, and acquires a second reference impedance signal that is deformed due to changes in the channel cross-section when the reference single cell passes through the second detection zone after electrotransfection.

[0067] The W500 determines whether reference single-cell electrotransfection was successful and whether the reference single cells are viable based on the change between the first and second reference impedance signals.

[0068] W600: When the reference single cell simultaneously meets the conditions of successful electrotransfection and survival, the electrotransfection parameter set corresponding to the reference single cell is determined as the optimal electrotransfection parameter set. A mapping relationship is established between the mechanical property parameters of the reference single cell, the reference electrical property parameters, and the optimal electrotransfection parameter set. Based on the mechanical property parameters of the reference single cell, the reference electrical property parameters, the optimal electrotransfection parameter set of the reference single cell, and the mapping relationship between the mechanical property parameters of the reference single cell, the reference electrical property parameters, and the optimal electrotransfection parameter set of the reference single cell, a reference data is generated.

[0069] W700 stores the reference data into the mapping database.

[0070] W800, return to W200, that is, return to the step of selecting a reference single cell from the sample library containing a preset number of reference single cells, until the preset number of reference single cells have completed the storage of reference data.

[0071] Specifically, the construction of the mapping database is a fundamental preliminary step in the entire microscale electrotransfection method based on the single-cell force-electric coupling characteristics. Its purpose is to establish a mapping relationship from "electrical characteristic parameters" to "optimal electrotransfection parameter set", so that in subsequent practical applications, only the electrical characteristic parameters of the target single cell need to be measured to quickly obtain the corresponding optimal electrotransfection parameter set through matching in the mapping database.

[0072] In W100, a blank mapping database is created. This mapping database is used to store each piece of reference data generated subsequently. The mapping database can adopt a relational database structure, containing multiple data tables to store different types of parameter information.

[0073] In W200, a reference single cell is selected from a sample library containing a predetermined number of reference single cells. The reference single cells in the sample library are pre-screened cells with clearly known mechanical properties. These mechanical properties include elastic modulus, cell membrane surface viscosity, and migration time, which can be pre-determined through independent mechanical testing methods. The predetermined number refers to the total number of reference single cells in the sample library; this number should be large enough to ensure that the database covers a sufficiently diverse range of cell mechanical properties. Reference single cells are selected one by one from the sample library for subsequent processing until all reference single cells have been processed.

[0074] In W300, the reference single cell is driven through the first detection area of ​​the microchannel. This step is the same as the process of acquiring the first target impedance signal of the target single cell in S100, the difference being that the object of processing is a reference single cell instead of the target single cell. The reference single cell deforms due to the change in the channel cross-section as it passes through the first detection area, and the cell impedance meter collects the impedance signal during its passage; this signal is defined as the first reference impedance signal. Reference electrical characteristic parameters are extracted from the first reference impedance signal, using the same method as in S200 for extracting electrical characteristic parameters from the first target impedance signal, including extracting characteristic parameters such as the peak value of the normalized resistance component, the peak value of the normalized capacitance component, and the migration time.

[0075] In the W400, within the electrotransfection zone of the microchannel, an electrotransfection parameter set consisting of multiple electrotransfection parameters is applied to the reference single cell. The electrotransfection parameter set includes parameters in four dimensions: pulse voltage amplitude, pulse width, number of pulses, and pulse interval. After applying the electrotransfection parameter set, an impedance signal is acquired again when the reference single cell passes through the second detection zone; this signal is defined as the second reference impedance signal.

[0076] In W500, the success of electrotransfection is determined by the change between the first and second reference impedance signals. The criterion for successful electrotransfection is the change in impedance signals before and after electrotransfection, specifically the change in resistance and capacitance components. The change in resistance reflects the degree of change in cytoplasmic resistance, while the change in capacitance reflects the degree of change in cell membrane capacitance. Successful electrotransfection should result in the formation of reversible micropores in the cell membrane, allowing exogenous molecules to enter the cell while maintaining the integrity of the cell membrane structure and enabling it to repair itself after the electrotransfusion pulse. In this state, the impedance change is characterized by a moderate decrease in both the resistance and capacitance components.

[0077] The survival of a cell is determined by the change between the first and second reference impedance signals. If the resistance or capacitance components decrease significantly, it indicates cell death.

[0078] In W600, the electrotransfection parameter set corresponding to a reference cell is determined as the optimal electrotransfection parameter set only when the reference cell simultaneously meets both the conditions of successful electrotransfection and survival. This dual verification mechanism ensures the quality of each reference data in the database. Subsequently, a mapping relationship is established between the mechanical property parameters, reference electrical property parameters, and the optimal electrotransfection parameter set of the reference cell, and this information is integrated into a complete reference data set. In W700, this reference data is stored in the mapping database. In W800, it returns to W200 to continue processing the next reference cell until the preset number of reference cells in the sample library have been processed.

[0079] In this embodiment, a mapping database is pre-constructed before running the entire process on the test cell (i.e., the target single cell), and a complete process of polling each reference single cell in the sample library is adopted. This includes collecting the first reference impedance signal and reference electrical characteristic parameters, applying the electrotransfection parameter set and collecting the second reference impedance signal, determining whether the transfection was successful and whether the cell survived based on the signal changes before and after electrotransfection, and establishing a mapping relationship and storing it in the database only when both successful transfection and cell survival are met. After storage, the reference single cells are sorted by a microcolumn array for post-verification and classification collection, ensuring that each reference data in the mapping database comes from a reliable experiment that has been doubly verified, providing a high-quality data foundation for subsequent matching.

[0080] In one embodiment of this application, W400 includes W410 to W420, namely, applying an electrical pulse to the reference single cell using an electrotransfection parameter group consisting of multiple electrotransfection parameters within the electrotransfection zone of the microchannel, and acquiring a second reference impedance signal that is deformed due to changes in the channel cross-section when the reference single cell passes through the second detection zone after electrotransfection, including W410 to W420 as follows: W410, for the reference single cell, randomly selects one parameter value from each of the preset pulse voltage amplitude range, preset pulse width range, preset pulse number range, and preset pulse interval time range, and combines the four randomly selected parameter values ​​to form the electroporation parameter group.

[0081] W420, perform electrotransfection on the reference single cell using the electrotransfection parameter set.

[0082] Specifically, the four preset ranges described in W410 are baseline parameter ranges predetermined based on the biological characteristics of this cell type. The preset range for pulse voltage amplitude refers to the range of voltage amplitudes that the electroporation device can output, determined based on the voltage range applicable to this cell type in previous experiments. The preset range for pulse width refers to the range of duration for a single electrical pulse. The preset range for the number of pulses refers to the range of the number of electrical pulses applied during one electroporation process. The preset range for pulse interval time refers to the range of the interval time between two adjacent electrical pulses. A parameter value is randomly selected from each of these four preset ranges, and then the four randomly selected parameter values ​​are combined into a complete electroporation parameter set. The purpose of random selection is to conduct a broad exploration within the parameter space and avoid missing certain parameter combinations due to human preference.

[0083] The four preset ranges can be obtained from academic materials. Since the parameters are selected within these four preset ranges, they will not deviate from the actual situation. For example, if the reference single cell type is HeLa human cervical cancer cells, a random value is selected from the preset range of pulse voltage amplitude, which is 2000 volts / cm; a random value is selected from the preset range of pulse width, which is 30 microseconds; a random value is selected from the preset range of pulse count, which is 1; no value is needed for the preset range of pulse interval time, because the pulse count is 1, and the pulse interval time is 0. In summary, a pulse voltage amplitude of 2000 volts / cm, a pulse width of 30 microseconds, a pulse count of 1, and a pulse interval time of 0 together constitute an electroporation parameter set. It should be noted that in the field of electroporation, electric field strength is uniformly used to characterize pulse parameters, with the unit being volts / cm. Electric field strength is the ratio between voltage and electrode spacing. Different devices have different electrode spacings, and only by using volts / cm can experimental conditions be uniformly compared and reproduced.

[0084] The cell type determines different preset ranges, and the different electroporation parameters are due to different mechanical property parameters.

[0085] In W420, the electrotransfection parameter set developed in W410 is used to perform electrotransfection on reference single cells. Electrotransfection is performed by applying an electrical pulse waveform that conforms to the characteristics of the electrotransfection parameter set to the reference single cells through the parallel plate electrotransfection electrodes in the electrotransfection zone.

[0086] In this embodiment, by randomly selecting parameter values ​​within the preset ranges of pulse voltage amplitude, pulse width, pulse number, and pulse interval time and combining them to form an electrotransfection parameter set, an efficient traversal exploration of the multidimensional parameter space is achieved. This allows the reference data to cover diverse electrotransfection conditions, accumulating sufficient training samples for subsequent fitting of the mapping function from mechanical property parameters to the optimal electrotransfection parameters through multivariate nonlinear regression or neural networks.

[0087] In one embodiment of this application, after W700, the method further includes, that is, after storing the reference data into the mapping database, the method further includes, W710: W710 controls the reference single cell to be sorted through a micropillar array sorting structure located downstream of the second detection area. The micropillar array sorting structure is composed of multiple cylinders arranged in a matrix. In the micropillar array sorting structure, the spacing between two adjacent cylinders is set to 0.8 to 1.2 times the equivalent diameter of the cell.

[0088] Specifically, in the W710, the reference single cells are sorted by a micropillar array sorting structure located downstream of the second detection area for post-processing verification and classification of the reference single cells already in the database. The micropillar array sorting structure utilizes the correlation between cell mechanical properties and electrotransfection results. Cells that have successfully undergone electrotransfection have micropores in their cell membranes, leading to partial cytoplasmic leakage, decreased overall cell stiffness, and increased deformability. When these cells flow through the micropillar array, due to the decreased stiffness, they are more likely to deform between the micropillars and shift laterally, thus entering specific collection channels. Cells that have not successfully transfected or have died maintain their original stiffness or have changed stiffness in different ways, resulting in different shift behaviors when flowing through the micropillar array. By sorting cells using the micropillar array, cells with different electrotransfection results can be directed to different collection channels. The sorted cells can then be further applied to fluorescence detection, live / dead staining, or culture analysis.

[0089] The micropillar array sorting structure consists of a base plate and multiple cylinders fixedly connected to the base plate. The cylinders are arranged in a matrix on the base plate, meaning they are equidistant along and perpendicular to the cell flow direction with specific row and column spacing. The spacing between adjacent cylinders, whether in the row or column direction, is set to 0.8 to 1.2 times the equivalent cell diameter A. This spacing ensures that cells must pass through the gaps between the cylinders as they flow through the micropillar array.

[0090] In this embodiment, by controlling the reference single cell to pass through a micropillar array sorting structure composed of multiple cylinders arranged in a matrix, and setting the spacing between adjacent cylinders to 0.8 to 1.2 times the equivalent diameter of the cell, the mechanical selectivity principle of lateral displacement when the cell flows through the micropillar array after successful electrotransfection causes a decrease in cell stiffness is utilized. This achieves rapid sorting without the need for complex sorting instruments, simplifies the sorting process, and avoids the potential impact of exogenous labels on cell viability.

[0091] In one embodiment of this application, S500 includes S510 to S530, namely, when the target single cell reaches the electrotransfection zone within the microchannel, applying the optimal electrotransfection parameter set to the target single cell to perform electrotransfection, including the following S510 to S530: S510, determine the moment when the target single cell leaves the first detection area based on the first target impedance signal when the target single cell passes through the first detection area.

[0092] S520, calculate the predicted time when the target single cell arrives at the electrotransfection zone based on the time when the target single cell leaves the first detection zone, the cell flow rate when the target single cell leaves the first detection zone, and the distance between the outlet of the first detection zone and the electrotransfection zone.

[0093] S530, when the predicted time arrives, output an electrical pulse waveform according to the pulse voltage amplitude, pulse width, pulse number and pulse interval time in the optimal electrotransfection parameter group to perform electrotransfection.

[0094] Specifically, in S510, when the target single cell passes through the first detection zone, the cell impedance meter continuously acquires its impedance signal. Once the target single cell has completely left the first detection zone, the impedance signal drops from its peak value when the target single cell passed through back to the background baseline level. By monitoring this characteristic of the normalized impedance peak falling back to the baseline and remaining there for a period of time, it is possible to accurately determine that the cell has left the first detection zone. Because the cell has just left the fourth electrode at this time, this moment is recorded as T4, which is the moment the cell leaves the first detection zone.

[0095] In S520, the cell flow rate of the target single cell as it leaves the first detection area can be monitored in real time by a flow rate sensor. The distance between the outlet of the first detection area and the electroporation area is a fixed structural parameter of the microchannel, which is determined during the chip design stage. Figure 5 L5 in the first detection zone. The time required for a target single cell to move from the exit of the first detection zone to the electrotransfection zone is equal to the distance L5 divided by the cell flow rate when the target single cell leaves the first detection zone. Therefore, the predicted time for the cell to arrive at the electrotransfection zone is... It can be calculated using Formula 2.

[0096] Formula 2.

[0097] in, This is the predicted time when the target single cell reaches the electrotransfection zone. L5 represents the moment when the target single cell leaves the first detection zone, and L5 represents the distance between the exit of the first detection zone and the electroporation zone. The cell flow rate when the target single cell leaves the first detection zone.

[0098] In S530, at the predicted time Upon arrival, the controller triggers the high-voltage pulse generator, outputting an electrical pulse waveform according to the optimal electrotransfer parameter set determined in S400. The optimal electrotransfer parameter set includes the pulse voltage amplitude... Pulse width Number of pulses N and pulse interval time The pulse generator generates corresponding electrical pulse waveforms based on these parameters, which are applied to the target single cell through a pair of vertically opposed and parallel electrode plates in the electrotransfection zone. This pair of electrode plates includes an upper electrode plate and a lower electrode plate. The extension direction of the upper electrode plate is parallel to the extension direction of the lower electrode plate. The extension direction of the upper electrode plate is parallel to the cell flow direction. The distance between the upper and lower electrode plates is equal to the equivalent diameter of the channel of the constricted section of the equal-width straight tube. The distance between the outlet of the first detection zone and the electrotransfection zone is the distance between the fourth electrode plate and the left end of the upper electrode plate in the electrotransfection zone, i.e. Figure 5 L5 in the middle.

[0099] The length of the upper electrode plate is Figure 6 The upper electrode (L6) must be long enough to ensure that all cell types can complete the full electrical pulse application process as they pass through the electrotransfection zone. The lower electrode is also L6 in length. Different cell types may have different pulse widths and pulse counts in their optimal electrotransfection parameter sets, resulting in varying total electrotransfection times. Setting the electrotransfection zone to a sufficient length ensures that even cells requiring the longest duration can complete the full electrical pulse application during their passage through the zone.

[0100] In this embodiment, the moment when the target single cell leaves the first detection area is determined based on the first target impedance signal. The predicted moment when the cell arrives at the electrotransfection area is calculated by combining the background flow rate and the distance between the outlet of the first detection area and the electrotransfection area. At the predicted moment, the electrical pulse waveform is output according to the pulse voltage amplitude, pulse width, pulse number and pulse interval time in the optimal electrotransfection parameter set. This achieves precise time synchronization between cell arrival and electrical pulse application, avoiding electrotransfection failure or cell damage caused by time deviation.

[0101] In one embodiment of this application, S700 includes S710 to S740 as follows: the step of evaluating the electrotransfection effect and cell viability based on the change between the first target impedance signal and the second target impedance signal includes S710 to S740 as follows: S710, extract the peak value of the first resistance component and the peak value of the first capacitance component when passing through the first detection area before electroporation.

[0102] S720, extracts the peak value of the second resistance component and the peak value of the second capacitance component when passing through the second detection area after electrotransfection.

[0103] S730, calculate the resistance change between the peak value of the first resistance component and the peak value of the second resistance component, and the capacitance change between the peak value of the first capacitance component and the peak value of the second capacitance component.

[0104] S740, the electroporation effect and cell survival status are determined based on the change in resistance and the change in capacitance.

[0105] Specifically, in S710, the peak values ​​of the resistance and capacitance components before electrotransfection are extracted from the first target impedance signal. The peak value of the resistance component reflects the cytoplasmic resistance characteristics of the cell under deformed conditions, and the peak value of the capacitance component reflects the cell membrane capacitance characteristics of the cell under deformed conditions. These two peak parameters were already obtained when extracting the electrical characteristic parameters in S200, and can be directly used in this step.

[0106] In S720, the peak values ​​of the resistance and capacitance components after electrotransfection are extracted from the second target impedance signal. The second target impedance signal is collected when cells pass through the second detection zone after electrotransfection, at which time the cells are in the process of deformation recovery. The peak value of the resistance component after electrotransfection reflects the change in cytoplasmic resistance after electropulse treatment, and the peak value of the capacitance component reflects the change in cell membrane capacitance after electropulse treatment.

[0107] In S730, the change in resistance before and after electrotransfer is calculated. The change in resistance is equal to the absolute value of the difference between the peak value of the first resistance component and the peak value of the second resistance component, divided by the peak value of the first resistance component, and then multiplied by 100%, see Formula 3.

[0108] Formula 3. Wherein, This represents the change in resistance. This represents the peak value of the first resistance component. The first resistance component peak value is the normalized resistance component peak value corresponding to the signal between the third and fourth electrode plates. The second resistance component peak value is the normalized resistance component peak value corresponding to the signal between the fifth and sixth electrode plates.

[0109] The change in capacitance is equal to the absolute value of the difference between the peak value of the first capacitance component and the peak value of the second capacitance component, divided by the peak value of the first capacitance component, and then multiplied by 100%, see Formula 4.

[0110] Formula 4. Wherein, This represents the change in capacitance. This represents the peak value of the first capacitance component. The peak value of the second capacitance component is the normalized peak value of the capacitance component corresponding to the signal between the third and fourth electrode plates. The peak value of the second capacitance component is the normalized peak value of the capacitance component corresponding to the signal between the fifth and sixth electrode plates.

[0111] The change in resistance reflects the degree of change in cytoplasmic resistance before and after electroporation, while the change in capacitance reflects the degree of change in cell membrane capacitance before and after electroporation.

[0112] In S740, based on the specific values ​​of the changes in resistance and capacitance, the electrotransfection status is determined to be one of three possible outcomes: cell death, successful transfection with cell survival, or unsuccessful electrotransfection with cell survival. The threshold values ​​for these three states are explained in detail in subsequent sections S741 to S743.

[0113] In this embodiment, by extracting the peak values ​​of the first resistance component and the first capacitance component when passing through the first detection zone before electrotransfection, and the peak values ​​of the second resistance component and the second capacitance component when passing through the second detection zone after electrotransfection, the resistance change and capacitance change are calculated, and the electrotransfection status is determined based on the changes. The complex electrotransfection effect is quantified into calculable resistance and capacitance change indicators, realizing real-time, quantitative, and objective evaluation of the electrotransfection effect and cell viability status, and providing quantifiable data basis for subsequent possible adjustment and improvement of electrotransfection parameter sets.

[0114] In one embodiment of this application, S740 includes the following S741 to S743, namely, determining the electrotransfection effect and cell viability based on the change in resistance and the change in capacitance includes the following S741 to S743: S741, when the change in resistance is greater than or equal to a first threshold or the change in capacitance is greater than or equal to a second threshold, cell death is determined.

[0115] S742, when the change in resistance is between the third threshold and the first threshold and the change in capacitance is between the fourth threshold and the second threshold, it is determined that the electrotransfection is successful and the cells are alive.

[0116] S743, when the change in resistance is less than or equal to the third threshold and the change in capacitance is less than or equal to the fourth threshold, it is determined that electrotransfection was unsuccessful and the cells are viable. The third threshold is less than the first threshold, and the fourth threshold is less than the second threshold.

[0117] Specifically, S741 to S743 establish a three-level classification judgment logic based on the changes in resistance and capacitance. The first threshold, second threshold, third threshold, and fourth threshold are empirical thresholds pre-calibrated through a large number of experiments. In a specific embodiment of this application, the first threshold is set to 60%, the second threshold is set to 40%, the third threshold is set to 20%, and the fourth threshold is set to 10%.

[0118] In S741, when the resistance change is greater than or equal to the first threshold, i.e., 60%, that is... When the capacitance change is greater than or equal to the second threshold of 40%, it indicates a sharp drop in cytoplasmic resistance, which usually signifies irreversible rupture of the cell membrane, leading to massive leakage of cytoplasm and cell death. Similarly, when the capacitance change is greater than or equal to the second threshold of 40%, it indicates a significant drop in cytoplasmic resistance. When this occurs, it indicates a sharp increase in cell membrane capacitance, which also signifies severe damage to the cell membrane structure and cell death. Since changes in resistance and capacitance reflect two different states—the cytoplasm and the cell membrane, respectively—if either indicator reaches the death threshold, cell death can be determined. Therefore, the two conditions are logically related by an "OR" condition. Continuing with the example above, that is… or At that time, cell death is determined.

[0119] In S742, when cells do not meet the death criteria, and the change in resistance is between the third threshold and the first threshold, and the change in capacitance is between the fourth threshold and the second threshold, electrotransfection is considered successful and the cells are viable. Continuing with the above example, that is... and At this point, successful electrotransfection and cell survival are considered achieved. In this state, the changes in electrical resistance and capacitance are within a suitable range, indicating the formation of an appropriate number of reversible micropores on the cell membrane. Exogenous molecules can enter the cell through these micropores, while the cell membrane structure has not been irreversibly damaged, allowing it to repair itself after the electrical pulse ends. This is the ideal electrotransfection result.

[0120] In S743, when cells do not meet the death criteria, and the change in electrical resistance is less than or equal to the third threshold (20%) and the change in capacitance is less than or equal to the fourth threshold (10%), electrotransfection is considered unsuccessful and the cells are viable. Continuing with the above example, that is... and At this point, it is determined that electrotransfection was unsuccessful and the cells are still alive. The changes in resistance and capacitance in this state are both very small, indicating that the applied electric field strength is insufficient to form effective micropores on the cell membrane, preventing exogenous molecules from entering the cell.

[0121] Only cells that are determined to have been successfully electrotransfected and are viable, i.e., cells in the S742 state, will have their corresponding electrotransfection parameter sets stored in the database as the optimal electrotransfection parameter sets for that cell. Cells determined to be dead or unsuccessfully transfected will not have their corresponding electrotransfection parameter sets stored in the database.

[0122] In this embodiment, by setting a first threshold, a second threshold, a third threshold, and a fourth threshold, and determining cell death based on a change in resistance greater than or equal to the first threshold or a change in capacitance greater than or equal to the second threshold, determining successful transfection based on a change in resistance between the third threshold and the first threshold and a change in capacitance between the fourth threshold and the second threshold, and determining unsuccessful transfection based on a change in resistance less than or equal to the third threshold and a change in capacitance less than or equal to the fourth threshold, a clear and explicit three-level classification judgment logic is constructed. This makes the evaluation of electrotransfection results have an objective and unified quantitative standard, eliminating the subjectivity and uncertainty of manual interpretation.

[0123] In one embodiment of this application, S500 further includes the following steps during the application of the electrical pulse: S540 monitors the position of the target single cell in real time during the electrotransfection process. When the target single cell moves out of the electrotransfection area during the interval between two adjacent electrical pulses, the output of subsequent electrical pulses is terminated.

[0124] Specifically, because the target single cell continues to move forward, the cell remains fluid even during the application of the electrical pulses. For a multi-pulse strategy, where the number of pulses N is greater than 1, there is a pulse interval between adjacent electrical pulses. During the pulse interval, the cell may continue to move forward a distance.

[0125] The time required for a cell to pass through the electrotransfection zone can be calculated based on the flow velocity of the target single cell within the zone and the length of the zone along the flow direction. The length of the electrotransfection zone along the flow direction is the length of the upper electrode. If the target single cell moves out of the electrotransfection zone during the interval between two adjacent electrical pulses, the output of subsequent electrical pulses is terminated. Specifically, when the time required for a cell to pass through the electrotransfection zone is less than or equal to the sum of the total time occupied by the executed pulse sequence and the current interval time, it indicates that the cell has moved out of the electrotransfection zone before the arrival of the subsequent pulse. If an electrical pulse is continued to be applied at this time, since there are no cells in the electrotransfection zone, the electrical pulse will act on the blank buffer solution instead of the cells, which not only wastes energy but may also cause unnecessary wear and tear on the electrode. Therefore, the system automatically terminates subsequent pulses to avoid spatiotemporal discharge in the absence of cells.

[0126] The flow rate of the target single cell in the electrotransfection zone is numerically equal to the cell flow rate of the target single cell when it leaves the first detection zone.

[0127] This mechanism ensures that the electrical pulse is applied only when the cells are within the electrotransfection zone, improving the efficiency of electrotransfection and the lifespan of the equipment.

[0128] In this embodiment, by monitoring the position of the target single cell in real time during the application of the electrical pulse and terminating the subsequent pulse when the cell is removed from the electrotransfection area, ineffective discharge in the cell-free state is avoided, thereby improving the accuracy of electrotransfection and the safety of the equipment.

[0129] In one embodiment of this application, before S100, i.e. before acquiring the impedance signal when the target single cell passes through the first detection area of ​​the microchannel, the method further includes: S010, measure the background impedance.

[0130] Specifically, S010 includes: preparing a blank control solution, which is a cell-free liquid with the exact same composition as the cell suspension. Before injecting the cell suspension containing the target single cell, the blank control solution is first introduced into the microchannel. Impedance data is continuously collected for a sufficient period using a cell impedance meter to obtain a time-background impedance curve.

[0131] Background impedance mainly originates from the double-layer capacitance Cdl formed at the electrode-electrolyte interface and the impedance Z1 of the solution itself. Since the blank control solution does not contain cells, the impedance signal measured at this time is the background impedance signal. The principle of background impedance measurement in S010 is the same as that in S100. The collected background impedance signal is synchronously demodulated by the lock-in amplifier set inside the cell impedance meter, and the resistance component in phase with the AC excitation signal and the capacitance component orthogonal to it are extracted to obtain the time-background resistance component curve and the time-background capacitance component curve, respectively. The time axis of the time-background resistance component curve corresponds to the time axis of the time-resistance component curve in S100. Therefore, the measured resistance at each time point in S100 can be subtracted from the background resistance at the same time point to obtain the impedance change value caused by cells alone. That is, the background resistance is subtracted from the ordinate of the time-resistance component curve obtained in S100, and then normalized to obtain the processed time-resistance component curve.

[0132] Similarly, the processed time-capacitance component curve also needs to have the background capacitance subtracted from the ordinate and then normalized to obtain the processed time-capacitance component curve.

[0133] In this embodiment, by pre-measuring the background impedance before cell injection and subtracting it during the actual measurement, systematic errors such as electrode polarization, solution background conductivity, and temperature changes are eliminated, thereby improving the accuracy and reliability of impedance signal measurement.

[0134] In one embodiment of this application, the length L6 of the electrotransfection region along the flow direction is determined based on the cell motility and a preset maximum pulse width. L6 is also the length of the upper electrode plate.

[0135] Because different cells have different mechanical properties, the pulse width in the corresponding optimal electroporation parameter set is different. The number of pulses, N, also varies, leading to differences in the total time required for different cells to complete electrotransfection. Before the cells reach the electrotransfection zone, it is impossible to know in advance the specific values ​​of pulse width and pulse number in the optimal electrotransfection parameter set for that cell, and therefore it is impossible to know in advance the total time required for the cells to complete electrotransfection.

[0136] To ensure that each cell type completes the full electrical pulse application process as it passes through the electrotransfection zone, the zone must be sufficiently long. Specifically, the length L6 of the electrotransfection zone should meet the following requirements: Where V6 is the flow rate of the target single cell in the electroporation zone. The total time required to complete the preset maximum number of pulses. The flow rate of the target single cell in the electrotransfection zone is numerically equal to the cell flow rate of the target single cell when it leaves the first detection zone.

[0137] pass The limitation ensures that the length of the electrotransfection zone is sufficient to guarantee that even cells requiring the longest duration can complete the application of all electrical pulses during their passage through the electrotransfection zone.

[0138] In one embodiment of this application, the mechanism by which the impedance signal is formed when a cell passes through a microchannel is as follows.

[0139] When cells flow through microchannels, the total impedance of the cells as they pass through the channels Mainly composed of the double-layer capacitance at the electrode-solution interface Cell membrane capacitance Cytoplasmic resistance and solution impedance Together they constitute a whole, satisfying the following formula 5: Formula 5.

[0140] in, This represents the total impedance as the cell passes through the channel. Cytoplasmic resistance, For imaginary units, The angular frequency at which the AC excitation is applied, For cell membrane capacitance, The double-layer capacitance formed at the electrode-electrolyte interface. This represents the solution impedance.

[0141] The cell membrane is composed of a phospholipid bilayer and has insulating properties, thus it can be considered a capacitor. Cell membrane capacitance. satisfy ,in The dielectric constant of the cell membrane, For cell membrane area, This refers to the cell membrane thickness. When a cell is compressed and deformed as a whole within the funnel-shaped constriction of a microchannel, a spherical cell is stretched into an ellipsoid, and the cell membrane area... Increased, thus leading to increased cell membrane capacitance The corresponding increase is observed in cytoplasmic resistance. This reflects the internal resistance characteristics of the cell. When the cell is squeezed in the contraction channel, the cross-sectional area of ​​the cell decreases and the current path increases, resulting in an increase in cytoplasmic resistance.

[0142] Lock-in amplification technology allows for the simultaneous extraction of resistance and capacitance components from the total impedance signal. The resistance component primarily characterizes changes in cytoplasmic conductivity, while the capacitance component primarily characterizes changes in cell membrane capacitance. When cells deform within microchannels, both resistance and capacitance components undergo corresponding changes. These changes, after feature extraction, can be quantitatively used to deduce the cell's mechanical properties.

[0143] In this application, a constant amplitude AC voltage excitation method is used, that is, an AC voltage of fixed frequency or swept frequency is applied to the electrodes, and the impedance change is inferred by measuring the current change caused by the passage of a cell. Compared with the constant current excitation method, the constant voltage excitation can provide a more stable electric field distribution when the cell passes through the detection area, which is beneficial to improving the accuracy of impedance measurement.

[0144] In one embodiment of this application, the microscale electrotransfection method based on the single-cell force-electric coupling characteristics is implemented by the following microscale electrotransfection system based on the single-cell force-electric coupling characteristics.

[0145] For the sake of brevity, all reference numerals for hardware structures such as devices, apparatuses, and equipment appearing in this application are only indicated in this embodiment, that is, in the embodiment of the microscale electrotransfection system based on the single-cell force-electric coupling characteristics. They are not indicated in the aforementioned embodiments of the microscale electrotransfection method based on the single-cell force-electric coupling characteristics, nor in subsequent embodiments.

[0146] like Figure 2 As shown, the microscale electrotransfection system based on the single-cell force-electric coupling characteristics includes a pressure driving device 10, a flow rate sensor 20, a microfluidic chip 30, a cell impedance meter 40, a data processing device 50, and a recovery device 60.

[0147] The pressure-driven device 10 is sealed to the inlet region 310 of the microfluidic chip 30. The pressure-driven device 10 establishes a stable pressure difference between the inlet and outlet of the microchannel 330, driving the cell suspension to form a uniform background flow. The pressure-driven device 10 can be a syringe pump, which pushes a piston to apply positive pressure to the fluid at the inlet of the microchannel 330. This positive pressure is higher than atmospheric pressure, while the outlet of the microchannel 330 is open to the atmosphere, thus creating a pressure difference between the inlet and outlet of the microchannel. This pressure difference is the driving force for the flow of the cell suspension.

[0148] The flow rate sensor 20 is installed in the microchannel 330 to monitor the flow rate in the microchannel 330 in real time and feed the flow rate data back to the data processing device 50 for real-time calculation of the predicted time when cells arrive at the electrotransfection zone.

[0149] The microfluidic chip 30 is fabricated on polydimethylsiloxane using standard soft lithography. The microfluidic chip 30 integrates microchannels 330, with multiple electrode pads sequentially arranged at the bottom of the channels for applying a constant amplitude AC voltage excitation and acquiring the impedance signal as cells pass through. Figure 3 As shown, the microfluidic chip 30 includes, in sequence, an inlet region 310, a filtration region 320, a microchannel 330, and an outlet region 340 along the cell flow direction.

[0150] like Figure 5 As shown, the microchannel 330 includes an inlet straight pipe section 331, a funnel-shaped tapering section 332, a straight pipe contraction section of equal width 333, a funnel-shaped expanding section 334, and an outlet straight pipe section 335. Figure 4 As shown, an electrotransfer zone 380 is provided on the equal-width straight tube contraction section 333, and the electrotransfer zone 380 integrates a parallel plate electrotransfer electrode 381. Figure 5 As shown, the parallel plate electroporation electrode 381 includes an upper electrode 381a and a lower electrode 381b. (As...) Figure 5 and Figure 6 As shown, the length of the inlet straight pipe section 331 is L1 + L2, and the equivalent diameter of the channel of the inlet straight pipe section 331 is D1. The length of the funnel-shaped tapering section 332 is L3. The length of the equal-width straight pipe contraction section 333 is L4 + L5 + L6 + L7 + L8. The equivalent diameter of the channel of the equal-width straight pipe contraction section 333 is D2. The length of the funnel-shaped expanding section 334 is L9. The length of the outlet straight pipe section 335 is L10 + L11. The equivalent diameter of the channel of the outlet straight pipe section 335 is D3. D1 equals D3, and D1 is greater than D2.

[0151] like Figure 5As shown, a first electrode plate 351, a second electrode plate 352, and a third electrode plate 353 are sequentially arranged from the end of the inlet straight pipe section 331 to the end of the funnel-shaped tapering section 332. A fourth electrode plate 354 and a fifth electrode plate 355 are sequentially arranged within the equal-width straight pipe tapering section 333. A sixth electrode plate 356, a seventh electrode plate 357, and an eighth electrode plate 358 are sequentially arranged from the end of the funnel-shaped expanding section 334 to the end of the outlet straight pipe section 335. The channel area covered by the first electrode plate 351 to the fourth electrode plate 354 constitutes the first detection area 360. The channel area covered by the fifth electrode plate 355 to the eighth electrode plate 358 constitutes the second detection area 370. Figure 4 and Figure 6 As shown, the length of the first detection region 360 is L2 + L3 + L4. The length of the electroporation region 380 is L6. The length of the second detection region 370 is L8 + L9 + L10.

[0152] The cell impedance meter 40 is electrically connected to each electrode plate and is used to apply AC excitation of a fixed frequency or swept frequency to each electrode plate and to collect the current changes caused by the passage of cells. The cell impedance meter 40 extracts the resistance and capacitance components from the collected signal using lock-in amplification technology and converts them into normalized electrical parameter signals.

[0153] The data processing device 50 is connected to the cell impedance meter 40, the flow rate sensor 20, and the pressure drive device 10, and is responsible for real-time acquisition, filtering, feature extraction, database matching, and calculation of electroporation parameters of the impedance signal. The data processing device 50 is also used to generate control commands based on the matching results, and control the high-voltage pulse generator of the electroporation zone 380 to output electrical pulses according to the calculated optimal electroporation parameter set.

[0154] The recovery device 60 is located downstream of the outlet region 340 of the microfluidic chip 30. For example... Figure 4 As shown, the recovery device 60 includes a micropillar array sorting structure 610 disposed downstream of the outlet area 340, and a first collection container 621 and a second collection container 622 respectively connected to the micropillar array sorting structure 610. The micropillar array sorting structure 610 consists of a base plate and multiple cylinders fixedly connected to the base plate. The multiple cylinders are arranged in a matrix on the base plate, and the spacing between two adjacent cylinders is set to 0.8 to 1.2 times the equivalent diameter of the cells. The first collection container 621 is used to collect cells that have been successfully electrotransfected, and the second collection container 622 is used to collect cells that have not been successfully electrotransfected.

[0155] In one embodiment of this application, the above-mentioned S-series steps and W-series steps cooperate with each other in actual operation to form a complete adaptive electroporation process.

[0156] The W-series steps pertain to the database construction phase and are completed before the actual electroporation of the target single cells. In the W-series steps, a large number of reference single cells with known mechanical properties are selected from the sample library. These cells undergo detection in the first detection zone, electroporation processing in the second detection zone, and sorting by a micropillar array. The mapping relationships between the mechanical properties, electrical properties, and optimal electroporation parameter sets of the reference single cells that simultaneously meet the criteria of successful electroporation and survival are stored in a mapping database. The output of the W-series steps is a mapping database containing multiple reference data.

[0157] The S-series steps pertain to the practical application phase and are executed after the W-series steps. In the S-series steps, the target single cell is injected into the microchannel. The first target impedance signal is acquired through the first detection zone, and electrical characteristic parameters are extracted. These parameters are then matched against the mapping database constructed in the W-series steps to find the most similar reference data. The corresponding optimal electrotransfection parameter set is read, and this parameter set is applied to the electrotransfection zone. Finally, the second target impedance signal is acquired through the second detection zone, and the electrotransfection effect is evaluated. The output of the S-series steps is the personalized electrotransfection treatment of the target single cell and its effect evaluation.

[0158] The connection between the two stages lies in the mapping database. The mapping database built in the W-series steps serves as the basis for the matching operations in the S-series steps.

[0159] In practice, the W-series and S-series steps can be performed sequentially on the same microscale electrotransfection system based on the single-cell mechano-electric coupling characteristics. First, a mapping database is constructed using reference single cells to complete the W-series steps. Then, the microchannels are cleaned, and the target single cells are injected to perform the S-series steps. Alternatively, the W-series steps can be completed in the laboratory beforehand, and the constructed database can be stored in a data processing device. In subsequent practical applications, the mapping database can be directly invoked to execute the S-series steps.

[0160] In one embodiment of this application, the electrical characteristic parameters in S200 include migration time.

[0161] Migration time refers to the time interval from when a cell enters the first detection zone to when it completely leaves the first detection zone. The length of the migration time depends on two factors: first, the background flow rate V0. The background flow rate V0 is the flow rate of the blank control solution in the microchannel of S010, i.e., the flow rate of the suspension without individual cells. The higher the background flow rate V0, the faster the cells pass through the first detection zone, and the shorter the migration time. Second, the resistance to cell movement caused by deformation as they pass through the first detection zone. The greater the cell stiffness, the more difficult the deformation, and the longer the time it takes to pass through the first detection zone.

[0162] Under experimental conditions where the background flow velocity V0 remains constant, migration time is related to the resistance to cell movement during deformation and can serve as an auxiliary characteristic parameter reflecting cell mechanical properties in matching. For two measurements with the same background flow velocity V0, the difference in migration time can reflect differences in mechanical properties such as cell stiffness; therefore, migration time has significant distinguishing value in database matching. The background flow velocity V0 can be detected using another flow rate sensor.

[0163] Even if the peak values ​​of the resistivity and capacitance components of two cells are very close, a significant difference in migration time indicates that their mechanical properties are different, and they should be matched with different reference data. Therefore, migration time can be used as a special electrical characteristic parameter to distinguish two different cells with similar peak values ​​of resistivity and capacitance components. In the weighted Euclidean distance calculation of S311, migration time participates in the distance calculation as an independent feature dimension, and its weight coefficient is preset according to the degree of influence of migration time on electrotransfection effect.

[0164] In one embodiment of this application, W500 is followed by: W610, when the reference single-cell electrotransfection fails or the reference single cell does not survive, the electrotransfection parameter set corresponding to the reference single cell is determined as the failed electrotransfection parameter set. A mapping relationship is established between the mechanical property parameters of the reference single cell, the electrical property parameters of the reference single cell, and the failed electrotransfection parameter set. Based on the mechanical property parameters of the reference single cell, the electrical property parameters of the reference single cell, the failed electrotransfection parameter set of the reference single cell, and the mapping relationship between the mechanical property parameters of the reference single cell, the electrical property parameters of the reference single cell, and the failed electrotransfection parameter set of the reference single cell, a failure data is generated.

[0165] W620 stores the failure data into a failure database.

[0166] In one embodiment of this application, after W200 and before W300, the following is also included: W210, determine whether there are other reference single cells with similar mechanical properties to the reference single cell that have experienced electroporation failure or cell non-viability.

[0167] If, in case of electroporation failure or cell non-viability, there are no other reference single cells with similar mechanical properties to the reference single cell, then W300 is executed, and subsequent W400 adopts the embodiments of W410 to W420.

[0168] If, in case of electroporation failure or cell non-viability, other reference single cells with similar mechanical properties to the reference single cell exist (W230), then W300 is executed, and subsequent W400 adopts the embodiments of W430 to W470.

[0169] Optionally, the W210 includes: W211, calculate the weighted Euclidean distance between the electrical characteristic parameters of the reference single cell and the electrical characteristic parameters of the reference single cell in each reference data of the mapping database.

[0170] W212 selects the reference single cell within the reference data with the smallest weighted Euclidean distance as the approximate single cell.

[0171] W213 determines whether the weighted Euclidean distance is less than the preset weighted Euclidean distance threshold.

[0172] W214, if the weighted Euclidean distance is less than the preset weighted Euclidean distance threshold, it is determined that there are no other reference single cells with similar mechanical property parameters to the reference single cell that have failed electroporation or are not viable, and W220 is executed.

[0173] Specifically, the reference data in the mapping database consists of data samples from successfully electrotransfected cells that survive. If a reference data point with a weighted Euclidean distance less than a preset weighted Euclidean distance threshold is found, it indicates that the similarity between the target single cell and the approximate single cell is sufficiently high. This means that there is a reliable data point in the reference database, and no other reference single cells with similar mechanical properties to the aforementioned reference single cell have failed electrotransfection or failed to survive. Therefore, W220 is executed. Subsequently, W400 will use the electrotransfection parameters composed of four random values, as in the examples of W410 to W420. Even if electrotransfection ultimately fails or the cells die, there will still be at least one reliable data point in the reference database to support this.

[0174] Optionally, the W210 also includes: W215, if the weighted Euclidean distance is greater than or equal to the preset weighted Euclidean distance threshold, it is determined that there are other reference single cells with similar mechanical property parameters to the reference single cell that have failed electroporation or are not viable, and W230 is executed.

[0175] Specifically, similarly, if no reference data with a weighted Euclidean distance less than a preset weighted Euclidean distance threshold is found, it indicates that the similarity between all valid reference data and the reference single cell is insufficient. In this case, other reference single cells with similar mechanical properties to the reference single cell may have experienced electroporation failure or cell non-viability, and W230 is executed. Examples from W430 to W470 will be used subsequently in W400.

[0176] The following describes embodiments of W430 to W470.

[0177] In one embodiment of this application, W400 further includes: W430 calculates the weighted Euclidean distance between the electrical characteristic parameters of the reference single cell and the electrical characteristic parameters of the reference single cell in each failure data of the failure database.

[0178] W440 selects the reference single cell within the failed data with the smallest weighted Euclidean distance as the approximate single cell.

[0179] W450 adjusts the electrical characteristic parameter set of an approximate single cell.

[0180] In one embodiment of this application, W450 includes: W451, based on the difference between the mechanical property parameters of the reference single cell and the mechanical property parameters of the approximate single cell, determines at least one electrotransfection parameter to be adjusted.

[0181] W452 determines the adjustment direction for each electroporation parameter to be adjusted.

[0182] W453 determines the specific adjustment amount for each electroporation parameter to be adjusted.

[0183] Specifically, in W451, three mechanical property parameters are mainly used to determine which electrotransfection parameters need to be adjusted and which do not. These three mechanical property parameters are cell elastic modulus, cell membrane surface viscosity, and cell migration time.

[0184] Optionally, W451 includes: W451a1, calculate the difference between the elastic modulus of the reference single cell and the elastic modulus of the approximate single cell to obtain the elastic modulus difference.

[0185] W451a2 determines whether the difference in elastic modulus is less than the threshold value for adjusting elastic modulus.

[0186] W451a3 If the difference in elastic modulus is less than or equal to the threshold value for adjusting elastic modulus, then it is determined that the pulse voltage amplitude is not the electrotransfer parameter to be adjusted, and W451b1 is executed.

[0187] W451a4 If the difference in elastic modulus is greater than the threshold value for adjusting elastic modulus, then the pulse voltage amplitude is determined to be the electrotransfer parameter to be adjusted, and W451b1 is executed.

[0188] W451b1 calculates the difference between the cell membrane surface viscosity of the reference single cell and the cell membrane surface viscosity of the approximate single cell to obtain the cell membrane surface viscosity difference value.

[0189] W451b2 determines whether the difference in cell membrane surface viscosity is less than the threshold value for adjusting cell membrane surface viscosity.

[0190] If the difference in cell membrane surface viscosity is less than or equal to the threshold for adjusting cell membrane surface viscosity (W451b3), then the pulse width is determined to be an electroporation parameter to be adjusted, and W451c1 is executed.

[0191] If the difference in cell membrane surface viscosity is greater than the threshold for adjusting cell membrane surface viscosity, then the pulse width is determined to be the electrotransfection parameter to be adjusted, and W451c1 is executed.

[0192] W451c1 calculates the difference between the migration time of the reference single cell and the migration time of the approximate single cell to obtain the migration time difference.

[0193] W451c2 determines whether the migration time difference is less than the starting migration time threshold.

[0194] W451c3 If the migration time difference is less than or equal to the starting migration time threshold, then it is determined that the number of pulses is not the electrotransfer parameter to be adjusted, and W452 is executed.

[0195] W451c4 If the migration time difference is greater than the starting migration time threshold, then the number of pulses is determined to be the electrotransfection parameter to be adjusted, and W452 is executed.

[0196] Optionally, in W452, the adjustment direction of the cell elastic modulus is such that the larger the difference in elastic modulus, the larger the pulse voltage amplitude. In W452, the adjustment direction of the pulse width is such that the larger the difference in cell membrane surface viscosity, the larger the pulse width. In W452, the adjustment direction of the migration time is such that the larger the difference in migration time, the more pulses are applied.

[0197] Optionally, W453 includes: W453a determines the adjustment amount for each electrotransfer parameter to be adjusted based on the correspondence table between the numerical range of the mechanical property parameter difference and the adjustment amount of the electrotransfer parameter.

[0198] Table 1 - Correspondence between the numerical range of the mechanical property parameter difference and the adjustment amount of the electroporation parameter. δ0<δ≤δ1 M=1 δ1<δ≤δ2 M=2 δ2<δ M=3 In Table 1, δ represents the difference in mechanical property parameters. δ0 is the initial threshold for adjusting the mechanical property parameters. δ1 is the first threshold for the mechanical property parameters. δ2 is the second threshold for the mechanical property parameters. δ3 is the third threshold for the mechanical property parameters. M is the adjustment step size for the electrotransfer parameters.

[0199] When the difference in mechanical property parameters δ is substituted with the difference in elastic modulus, δ0 is substituted with the initial elastic modulus threshold, which is 5% of the reference elastic modulus value. δ1 is substituted with the first elastic modulus difference threshold, which is 15% of the reference elastic modulus value. δ2 is substituted with the second elastic modulus difference threshold, which is 30% of the reference elastic modulus value. M is substituted with the number of adjustment steps for the voltage pulse amplitude. One adjustment step for the voltage pulse amplitude is 5% of the reference voltage pulse amplitude, that is, when M equals 1, the adjustment amount of the voltage pulse amplitude is 5% of the reference voltage pulse amplitude. The reference elastic modulus value can be set to 2 kPa, which can be obtained from comprehensive academic information databases or public scientific data centers for the corresponding cell type. The reference voltage pulse amplitude can be set to 2000 V / cm, which can be obtained from comprehensive academic information databases or public scientific data centers for the corresponding cell type.

[0200] When the mechanical property parameter difference δ is substituted with the cell membrane surface viscosity difference, δ0 is substituted with the threshold value for adjusting the cell membrane surface viscosity difference, which is 10% of the baseline cell membrane surface viscosity value. δ1 is substituted with the first cell membrane surface viscosity difference threshold value, which is 20% of the baseline cell membrane surface viscosity value. δ2 is substituted with the second cell membrane surface viscosity difference threshold value, which is 40% of the baseline cell membrane surface viscosity value. M is substituted with the number of pulse width adjustment steps, where one pulse width adjustment step is 10% of the baseline pulse width. That is, when M equals 1, the pulse width adjustment is 10% of the baseline pulse width value. The baseline cell membrane surface viscosity value can be set to 30 Pa·s, i.e., 30 Pascals per second, and can be obtained from comprehensive academic information databases or public scientific data centers for the corresponding cell type. The baseline pulse width value can be set to 30 µs, i.e., 30 microseconds, and can be obtained from comprehensive academic information databases or public scientific data centers for the corresponding cell type.

[0201] When the mechanical property parameter difference δ is substituted with the migration time difference, δ0 is substituted with the initial migration time difference threshold, which is 10% of the baseline migration time. δ1 is substituted with the first migration time difference threshold, which is 25% of the baseline migration time. δ2 is substituted with the second migration time difference threshold, which is 50% of the baseline migration time. M is substituted with the number of pulse count adjustment steps, where one adjustment step is the pulse count plus 1, rounded down. That is, when M equals 1, the adjustment amount of the pulse count is the baseline pulse count plus 1. The baseline migration time can be set to 0.02s, i.e., 0.02 seconds, and can be obtained from a comprehensive academic information database or a public scientific data center for the corresponding cell type. The baseline pulse count can be set to 1, i.e., 1 time, and can be obtained from a comprehensive academic information database or a public scientific data center for the corresponding cell type. In this embodiment, by updating the mapping database according to the experimental results, continuous learning and closed-loop optimization based on measured data are realized, thereby continuously improving the parameter matching accuracy of subsequent cells.

[0202] The technical features of the above embodiments can be combined arbitrarily, and the execution order of the method steps is not restricted. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0203] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.

Claims

1. A microscale electrotransfection method based on the mechano-electric coupling characteristics of single cells, characterized in that, include: The electrical impedance signal generated by the deformation of the channel cross-section when the target single cell passes through the first detection area of ​​the microchannel is obtained, and this electrical impedance signal is defined as the first target electrical impedance signal; The electrical characteristic parameters of the target single cell are extracted based on the first target impedance signal; The electrical characteristic parameters are matched with a pre-constructed mapping database. The mapping database includes multiple reference data, each of which includes the mechanical characteristic parameters of a reference single cell, the electrical characteristic parameters of a reference single cell, the optimal electrotransfection parameter set of a reference single cell, and the mapping relationship between the mechanical characteristic parameters of the reference single cell and the electrical characteristic parameters of the reference single cell and the optimal electrotransfection parameter set of the reference single cell. The optimal electroporation parameter set corresponding to the target single cell is determined based on the matching results; When the target single cell reaches the electrotransfection zone within the microchannel, an electrical pulse is applied to the target single cell using the optimal electrotransfection parameter set to perform electrotransfection; The electrical impedance signal of the target single cell as it passes through the second detection area of ​​the microchannel after electrotransfection is obtained due to the deformation caused by the change in the cross-section of the channel, and this electrical impedance signal is defined as the second target electrical impedance signal; The effectiveness of electrotransfection and cell viability were assessed based on the change between the first and second target impedance signals.

2. The microscale electrotransfection method based on the single-cell force-electric coupling characteristics according to claim 1, characterized in that, The microchannel is arranged sequentially along the cell flow direction as an inlet straight tube section, a funnel-shaped tapering section, a straight tube contraction section of equal width, a funnel-shaped expanding section, and an outlet straight tube section. The electro-dyeing zone is provided within the equal-width straight tube contraction section; A first electrode plate, a second electrode plate, and a third electrode plate are sequentially arranged from the end of the inlet straight pipe section to the end of the funnel-shaped tapering section. A fourth electrode plate and a fifth electrode plate are sequentially arranged in the equal-width straight pipe contraction section. A sixth electrode plate, a seventh electrode plate, and an eighth electrode plate are sequentially arranged from the end of the funnel-shaped expanding section to the end of the outlet straight pipe section. The first detection area is formed by the channel area covered by the first electrode sheet to the fourth electrode sheet, the second detection area is formed by the channel area covered by the fifth electrode sheet to the eighth electrode sheet, and the electrotransfer area is located in the channel area between the fourth electrode sheet and the fifth electrode sheet.

3. The microscale electrotransfection method based on the single-cell force-electric coupling characteristics according to claim 1, characterized in that, The step of matching the electrical characteristic parameters with a pre-built mapping database includes: Calculate the weighted Euclidean distance between the electrical characteristic parameters of the target single cell and the electrical characteristic parameters of the reference single cell in each reference data in the mapping database; The data with the smallest weighted Euclidean distance is selected as the matching result.

4. The microscale electrotransfection method based on the single-cell force-electric coupling characteristics according to claim 3, characterized in that, The calculation of the weighted Euclidean distance between the electrical characteristic parameters of the target single cell and the electrical characteristic parameters of the reference single cell in each reference data in the mapping database includes: For each feature dimension of the electrical characteristic parameter, calculate the difference between the measured feature value and the database feature value. Divide the difference by the standard deviation of the feature dimension in multiple repeated experiments, multiply by the corresponding weight coefficient, and then take the square root of the sum of squares of the multiplication results of each feature dimension to obtain the weighted Euclidean distance.

5. The microscale electrotransfection method based on the single-cell force-electric coupling characteristics according to claim 1, characterized in that, Before acquiring the electrical impedance signal generated by the deformation of the channel cross-section when the target single cell passes through the first detection area of ​​the microchannel, and defining this electrical impedance signal as the first target electrical impedance signal, the method further includes: Build a mapping database; The construction of the mapping database includes: Create an empty mapping database; Select a reference single cell from a sample library containing a preset number of reference single cells; The reference single cell is driven through the first detection area of ​​the microchannel, and the first reference impedance signal generated by the deformation due to the change of the channel cross section when the reference single cell passes through the first detection area of ​​the microchannel is collected, and the reference electrical characteristic parameters are extracted. Within the electrotransfection zone of the microchannel, an electrotransfection parameter group consisting of multiple electrotransfection parameters is applied to the reference single cell to obtain a second reference impedance signal that is deformed due to the change in the channel cross-section when the reference single cell passes through the second detection zone after electrotransfection. The success of reference single-cell electrotransfection and the viability of reference single cells are determined by the change between the first and second reference impedance signals. When the reference single cell simultaneously meets the conditions of successful electrotransfection and survival, the electrotransfection parameter set corresponding to the reference single cell is determined as the optimal electrotransfection parameter set. A mapping relationship is established between the mechanical property parameters of the reference single cell, the reference electrical property parameters, and the optimal electrotransfection parameter set. Based on the mechanical property parameters of the reference single cell, the reference electrical property parameters of the reference single cell, the optimal electrotransfection parameter set of the reference single cell, and the mapping relationship between the mechanical property parameters of the reference single cell, the reference electrical property parameters, and the optimal electrotransfection parameter set of the reference single cell, a reference data is generated. The reference data is stored in the mapping database; Return to the step of selecting a reference single cell from a sample library containing a preset number of reference single cells, until the preset number of reference single cells have all completed the storage of reference data.

6. The microscale electrotransfection method based on the single-cell force-electric coupling characteristics according to claim 5, characterized in that, Within the electrotransfection region of the microchannel, an electrotransfection parameter group consisting of multiple electrotransfection parameters is applied to the reference single cell to apply an electrical pulse, and a second reference impedance signal is obtained as the reference single cell deforms due to changes in the channel cross-section when passing through the second detection region after electrotransfection. This includes: For the reference single cell, one parameter value is randomly selected from each of the preset pulse voltage amplitude range, preset pulse width range, preset pulse number range, and preset pulse interval time range. The four randomly selected parameter values ​​are combined to form the electroporation parameter group. The reference single cell was electrotransfected using the electrotransfection parameter set.

7. The microscale electrotransfection method based on the single-cell force-electric coupling characteristics according to claim 5, characterized in that, After storing the reference data in the mapping database, the method further includes: The reference single cell is controlled to be sorted by a micropillar array sorting structure located downstream of the second detection area; the micropillar array sorting structure is composed of multiple cylinders arranged in a matrix; in the micropillar array sorting structure, the spacing between two adjacent cylinders is set to 0.8 to 1.2 times the equivalent diameter of the cell.

8. The microscale electrotransfection method based on the single-cell force-electric coupling characteristics according to claim 1, characterized in that, When the target single cell reaches the electrotransfection zone within the microchannel, applying an electrical pulse to the target single cell using the optimal electrotransfection parameter set to perform electrotransfection includes: The moment when the target single cell leaves the first detection area is determined based on the first target impedance signal when the target single cell passes through the first detection area. Based on the time when the target single cell leaves the first detection area, the cell flow rate when the target single cell leaves the first detection area, and the distance between the outlet of the first detection area and the electroporation area, the predicted time when the target single cell arrives at the electroporation area is calculated. When the predicted time arrives, an electrical pulse waveform is output according to the pulse voltage amplitude, pulse width, pulse number and pulse interval time in the optimal electrotransfection parameter set to perform electrotransfection.

9. The microscale electrotransfection method based on the single-cell force-electric coupling characteristics according to claim 1, characterized in that, The assessment of electrotransfection efficacy and cell viability based on the change between the first and second target electrical impedance signals includes: Extract the peak value of the first resistance component and the peak value of the first capacitance component when passing through the first detection area before electroporation; Extract the peak value of the second resistance component and the peak value of the second capacitance component when passing through the second detection area after electroporation; Calculate the change in resistance between the peak value of the first resistance component and the peak value of the second resistance component, and the change in capacitance between the peak value of the first capacitance component and the peak value of the second capacitance component; The electrotransfection effect and cell survival status are determined based on the changes in resistance and capacitance.

10. The microscale electrotransfection method based on the single-cell force-electric coupling characteristics according to claim 9, characterized in that, The determination of electroporation effectiveness and cell viability based on the changes in resistance and capacitance includes: When the change in resistance is greater than or equal to a first threshold or the change in capacitance is greater than or equal to a second threshold, it is determined as cell death; When the change in resistance is between the third threshold and the first threshold and the change in capacitance is between the fourth threshold and the second threshold, the cell is considered to have been successfully electrotransfected and is alive. When the change in resistance is less than or equal to the third threshold and the change in capacitance is less than or equal to the fourth threshold, it is determined that the electrotransfection was unsuccessful and the cells survived; the third threshold is less than the first threshold and the fourth threshold is less than the second threshold.

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