A microfluidic nanosecond electric pulse transfection device

Abstract

This invention discloses a microfluidic nanosecond electropulse transfection device, belonging to the field of cell engineering and biomedical equipment technology. It includes a nanosecond electropulse generation module, a PMMA microfluidic chip, a continuous fluid delivery module, and a collection beaker. The nanosecond electropulse generation module consists of a function generator, a converter, an oscilloscope, a high-voltage power supply, an energy storage capacitor, and a MOSFET switch. The dual-channel oscilloscope can simultaneously monitor control signals and high-voltage pulses. The microfluidic chip has embedded electrode pairs and is directly connected to the MOSFET switch over a short distance. The fluid module includes a magnetic stirrer, a feeding beaker, and a syringe pump, achieving uniform and continuous delivery of cell suspension. This microfluidic nanosecond electropulse transfection device, employing the above structure, can calibrate pulse parameters in real time, ensures uniform cell flow electric field duration, achieves high transfection repeatability and throughput, reduces sample contamination risk, and is suitable for large-scale transfection of exogenous molecules and exosome preparation.

Description

Technical Field

[0001] This invention relates to the field of cell engineering and biomedical equipment technology, and in particular to a microfluidic nanosecond electrical pulse transfection device. Background Technology

[0002] The introduction of exogenous genes into cells is a core experimental technique in cell biology, gene therapy, and biomedicine. Electroporation has become the mainstream transfection method due to its wide applicability to various cell types and ease of operation. Traditional benchtop electroporators mostly use a static electroporation tank structure, where cell suspensions are placed between electrodes for single electroporation. This results in weak batch processing capacity, uneven cell exposure time, and problems such as excessive cell breakdown and death in some areas and transfection failure in others, making it difficult to meet the experimental needs of continuous cell sample preparation.

[0003] Existing microfluidic electroporation devices mostly employ microsecond-level wide pulses. The long pulse duration and high-voltage energy can easily cause irreversible damage to the cell membrane, significantly reducing cell viability. Simultaneously, the long wiring of the high-voltage drive circuit leads to parasitic inductance and capacitance, severely distorting the pulse waveform and preventing the output of high-precision nanosecond pulses, thus hindering the achievement of gentle and efficient reversible electroporation. Conventional circuit designs lack a dual-channel synchronous monitoring structure, making it impossible to simultaneously calibrate the low-voltage control signal and the actual high-voltage pulse acting on the cells. Pulse parameters rely on theoretical settings, resulting in deviations in actual electric field strength and pulse width, leading to poor reproducibility in transfection experiments.

[0004] Furthermore, existing flow cytometry transfection equipment has a simple fluid delivery structure, leading to easy sedimentation and aggregation of cell mixtures and large fluctuations in cell concentration entering the microchannels, further exacerbating differences in transfection efficiency. The connection distance between the microchannel electrodes and high-voltage switching devices lacks optimization, resulting in significant pulse transmission loss and waveform distortion. Currently, there is a lack of a microfluidic nanosecond electroporation device that integrates precise nanosecond pulse generation, real-time dual-channel waveform calibration, and uniform continuous fluid delivery. This makes it impossible to simultaneously achieve high cell viability, stable transfection efficiency, and continuous sample processing, thus limiting the scale of cell transfection experiments. Summary of the Invention

[0005] The purpose of this invention is to provide a microfluidic nanosecond electroporation transfection device that solves the problems of existing flow electroporation equipment, such as difficulty in outputting high-precision nanosecond high-voltage pulses, easy distortion of pulse waveforms, and inability to synchronously verify control signals and actual discharge pulses. At the same time, it improves the shortcomings of traditional equipment, such as easy sedimentation of cell suspensions, inconsistent duration of cell interaction in the electric field, poor transfection uniformity, and high cell damage rate.

[0006] To achieve the above objectives, the present invention provides a microfluidic nanosecond electrical pulse transfection device, comprising a nanosecond electrical pulse generation module, a PMMA microfluidic chip, a continuous fluid delivery module, and a collection beaker; The nanosecond electrical pulse generation module is used to generate high-voltage electrical pulses with pulse widths of 1~1000ns. It consists of a function generator, a converter, an oscilloscope, a high-voltage power supply, an energy storage capacitor, and a MOSFET switch. The signal output port of the function generator is connected to the input of the converter via a coaxial cable. The output of the converter is connected to the gate of the MOSFET switch via a coaxial cable. The first channel of the oscilloscope is connected in parallel between the output of the converter and the gate of the MOSFET switch. The drain of the MOSFET switch serves as the high-voltage input and is connected to the lower end of the energy storage capacitor, while the upper end of the energy storage capacitor is connected to the positive output of the high-voltage power supply. The source of the MOSFET switch is connected to the anode of the PMMA microfluidic chip on one side and to a common ground on the other. The cathode of the microfluidic chip, the negative terminal of the high-voltage power supply, and the ground terminal of the oscilloscope are all connected to the common ground. The second channel of the oscilloscope is connected in parallel between the source of the MOSFET switch and the common ground. The PMMA microfluidic chip has a continuous microchannel inside, with electrode pairs embedded on the inner walls of both sides of the microchannel. The electrode pairs are directly electrically connected to the MOSFET switching source at a very short distance. The microchannel size is 200~500μm wide, 50~200μm deep, and 3000~10000μm long. The spacing between the electrode pairs is equal to the width of the microchannel. The continuous fluid delivery module consists of a magnetic stirrer, a feed beaker, and a syringe pump. The feed beaker containing a suspension of mixed cells and exogenous molecules is placed on the magnetic stirrer, and the outlet of the feed beaker is connected to the syringe inlet of the syringe pump via a medical silicone tube. The syringe outlet of the syringe pump is connected to the fluid inlet of the PMMA microfluidic chip via a silicone tube. The fluid outlet of the PMMA microfluidic chip is connected to a collection beaker via a silicone tube.

[0007] Preferably, the microchannel dimensions are 300μm wide, 100μm deep, and 5000μm long.

[0008] Preferably, the function generator can generate a low-voltage trigger signal, set the frequency, pulse width, duty cycle and trigger mode of the electrical pulse, and indirectly determine the final output nanosecond pulse parameters by controlling the on and off timing of the MOSFET switch.

[0009] Preferably, the converter is a signal matching and isolation device used to convert the low-voltage trigger signal output by the function generator into a drive voltage adapted to the MOSFET switch gate.

[0010] Preferably, the first channel of the oscilloscope is used to monitor the waveform, frequency, and pulse width of the low-voltage trigger signal and to verify whether the control signal is normal; the second channel of the oscilloscope is used to monitor the voltage amplitude, pulse width, rise time, and fall time of the high-voltage pulse actually applied to the cell at both ends of the PMMA microfluidic chip electrode, and to calibrate the pulse parameters.

[0011] Preferably, the high-voltage power supply outputs high-voltage DC to charge the energy storage capacitor at a constant voltage, and the output voltage of the high-voltage power supply determines the final pulse electric field strength; the energy storage capacitor is the core component of energy storage, and when the MOSFET switch is turned on, the stored energy can discharge rapidly in seconds, forming an instantaneous high-voltage pulse electric field at both ends of the microfluidic channel.

[0012] Preferably, the MOSFET switch is a metal-oxide-semiconductor field-effect transistor with a nanosecond-level response speed. It is controlled by a function generator trigger signal to control the start and end time of the energy storage capacitor discharge and output a nanosecond high-voltage pulse with precise width.

[0013] Preferably, the injection pump controls the delivery rate of the cell-exogenous molecule mixture suspension. The residence time of the cell-exogenous molecule mixture suspension in the microchannel electric field region is negatively correlated with the injection pump flow rate. By adjusting the injection pump flow rate, the electroporation effect of all cells is made uniform.

[0014] Preferably, when the cell-exogenous molecule mixture flows through the microchannel of the PMMA microfluidic chip, the nanosecond high-voltage pulses applied to the electrodes on both sides cause reversible electroporation of the cell membrane, allowing the exogenous molecules to enter the cell.

[0015] Therefore, the microfluidic nanosecond electrical pulse transfection device described above has the following beneficial effects: (1) This device adopts a 1~1000ns nanosecond high voltage pulse working mode. The pulse duration is much shorter than the traditional microsecond and millisecond level electroporation pulse. It only forms 1~10nm reversible micropores in the cell membrane. After the pulse ends, the cell membrane can quickly self-repair, avoiding leakage of cell contents. The thermal and electrolytic effects are weak, which greatly reduces cell apoptosis and stem cell damage and significantly improves cell survival rate. At the same time, the nanosecond pulse can act on the nuclear membrane to improve the efficiency of introducing exogenous nucleic acids such as plasmids and mRNA. (2) The continuous flow microfluidic processing structure is adopted. When the cell suspension flows through the microfluidic electrode area at a constant speed, the electroshock is completed instantaneously. The cell stays in the electric field for a very short time. In addition, the effective area of ​​the microfluidic electrode is small, and the amount of electrolysis products generated is extremely low, which effectively reduces the damage of electrochemical toxicity to cells. (3) Continuous flow cytometry enables high-throughput and reproducible cell sample processing. Cells are electroporated while flowing, eliminating the need for batch static electroporation operations in traditional equipment and improving experimental processing efficiency. At the same time, it reduces open operation steps such as centrifugation and liquid exchange, reducing the risk of microbial contamination of samples. (4) The nanosecond pulse generation module and the PMMA microfluidic chip are integrated into one, abandoning the traditional open electroporation tank structure. The functions of fluid delivery, cell positioning and pulse electric field application are integrated on the same platform, and the device structure is compact. The electric field distribution in the microchannel is uniform. The short-distance direct connection structure between the MOSFET and the electrode reduces waveform distortion, so that the electroporation conditions of all cells are uniform and the transfection effect is uniform and stable. (5) The integrated system can simultaneously realize functions such as in situ electroporation of cells and large-scale induction of exosomes, overcoming the defects of traditional electroporation equipment such as uneven electric field, high cell damage and inability to adapt to continuous cell preparation, and expanding the integrated application scenarios of cell gene introduction and exosome production. (6) The whole machine is equipped with a unified common ground and dual-channel waveform monitoring structure, which can suppress electromagnetic interference of high and low voltage circuits and protect precision testing instruments, and can also calibrate parameters such as pulse amplitude and pulse width in real time, adapting to laboratory-scale and standardized cell transfection operations.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the overall structure of a microfluidic nanosecond electrical pulse transfection device according to the present invention.

[0018] Figure label: 1. Function generator; 2. Converter; 3. Oscilloscope; 4. High voltage power supply; 5. Energy storage capacitor; 6. MOSFET switch; 7. Magnetic stirrer; 8. Feed beaker; 9. Injection pump; 10. PMMA microfluidic chip; 11. Collection beaker. Detailed Implementation

[0019] The following detailed description of embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0020] like Figure 1 As shown, a microfluidic nanosecond electrical pulse transfection device includes a nanosecond electrical pulse generation module, a PMMA microfluidic chip 10, a continuous fluid delivery module, and a collection beaker 11. The nanosecond electrical pulse generation module consists of a function generator 1, a converter 2, an oscilloscope 3, a high-voltage power supply 4, an energy storage capacitor 5, and a MOSFET switch 6; the continuous fluid delivery module includes a magnetic stirrer 7, a feed beaker 8, and a syringe pump 9.

[0021] The signal output port of function generator 1 is connected to the input of converter 2 via a coaxial cable. The output of converter 2 is connected to the gate of MOSFET switch 6 via a coaxial cable. Oscilloscope 3 has a first channel and a second channel. The first channel of oscilloscope 3 is connected in parallel between the output of converter 2 and the gate of MOSFET switch 6 to monitor the waveform, frequency and pulse width of low-voltage trigger signal in real time and verify whether the control signal is normal. The second channel of oscilloscope 3 is connected across the source of MOSFET switch 6 and the common ground of the whole machine to acquire the high-voltage pulses across the electrodes of PMMA microfluidic chip 10, measure the pulse amplitude, rising edge, falling edge and actual pulse width, and complete parameter calibration.

[0022] The positive terminal of the high-voltage power supply 4 is connected to the upper end of the energy storage capacitor 5, and the lower end of the energy storage capacitor 5 is connected to the drain of the MOSFET switch 6. The source of the MOSFET switch 6 is arranged in two paths: one path is electrically connected to the anode of the PMMA microfluidic chip 10, and the other path is connected to the common ground of the whole machine. The cathode of the PMMA microfluidic chip 10, the negative terminal of the high-voltage power supply 4, and the ground terminal of the oscilloscope 3 are all connected to the common ground of the whole machine, forming a complete and stable high and low voltage circuit. The PMMA microfluidic chip 10 has a continuous through-channel microfluidic channel. Electrode pairs are embedded on the inner walls of both sides of the microfluidic channel. The electrode pairs are directly electrically connected to the source of the MOSFET switch 6 with a short distance, reducing the parasitic inductance of the line. In this embodiment, the microfluidic channel is preferably 300μm wide, 100μm deep, and 5000μm long. The spacing between the electrode pairs is consistent with the width of the microfluidic channel, the electric field distribution in the channel is uniform, and cell blockage of the channel is avoided.

[0023] In the continuous fluid transport structure, the feed beaker 8, containing a suspension of cells and exogenous molecules, is placed on the platform of the magnetic stirrer 7. The magnetic stirrer 7 continuously stirs the mixture at a low speed to prevent cell sedimentation and aggregation. The outlet of the feed beaker 8 is connected to the syringe inlet mounted on the syringe pump 9 through a medical silicone tube. The syringe outlet is connected to the fluid inlet of the PMMA microfluidic chip 10 through a silicone tube. The syringe pump 9 precisely controls the volumetric flow rate of the mixed suspension, so that the cells pass through the electric field region of the microchannel at a uniform speed, ensuring that the electroporation time of each cell is consistent. The fluid outlet of the PMMA microfluidic chip 10 is connected to the collection beaker 11 through a silicone tube. The cell suspension after electroporation is uniformly collected into the collection beaker 11 for subsequent culture and detection.

[0024] The specific working process of this device is as follows: Preparation: Mix the digested stem cells with plasmid DNA to prepare a cell suspension of a certain concentration, pour it into the feed beaker 8 and turn on the magnetic stirrer 7 to stir at low speed; De-air bubbles: Use syringe pump 9 to pump PBS buffer into the entire flow path to remove air bubbles from all tubing and the PMMA microfluidic chip 10; Sample introduction: Switch to cell suspension, set the flow rate of the syringe pump 9 (usually 1~5 mL / h), and the cell suspension will begin to flow through the PMMA microfluidic chip 10 at a uniform speed; Electroporation: When the cell suspension flows stably through the electrode area of ​​the PMMA microfluidic chip 10, the nanosecond electrical pulse generation module is activated; the high-voltage power supply 4 charges the energy storage capacitor 5 at a constant voltage, and the function generator 1 outputs a low-voltage trigger signal with a pulse width of 1~1000ns. After voltage matching by the converter 2, the MOSFET switch 6 is periodically turned on, and the energy storage capacitor 5 releases a high-voltage nanosecond pulse instantaneously. The microfluidic electrode forms a short-time pulse electric field, forming reversible nanopores in the cell membrane to achieve the introduction of exogenous molecules; the control signal and high-voltage pulse are monitored synchronously through dual channels of the oscilloscope 3 throughout the process, and the output parameters of the function generator 1 are corrected as needed to achieve continuous, low-damage, and highly uniform cell flow cytometry electroporation. Collection: Transfected cells flow out of the PMMA microfluidic chip 10 and are dropped into a collection beaker 11 pre-filled with fresh culture medium; Post-processing: After collection, the cells are seeded into culture plates and placed in an incubator for further culture.

[0025] Therefore, this invention employs the aforementioned microfluidic nanosecond electroporation transfection device. A nanosecond electroporation generation module outputs narrow-pulse high-voltage pulses, which are then calibrated in real-time using a dual-channel oscilloscope. A short-distance direct connection between the MOSFET switch and the microfluidic chip electrodes reduces parasitic inductance and ensures a complete pulse waveform. Simultaneously, a continuous fluid delivery module consisting of a magnetic stirrer and a syringe pump achieves uniform and stable delivery of the cell suspension, preventing cell sedimentation and ensuring consistent cell interaction conditions within the microfluidic electric field. This device enables continuous flow, low-damage cell electroporation with high exogenous molecule introduction efficiency, high cell viability, good experimental reproducibility, high sample throughput, reduced contamination risk, and a compact overall structure. It can be widely applied in biomedical experimental scenarios such as cell gene transfection and large-scale exosome preparation.

[0026] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A microfluidic nanosecond electric pulse transfection device, characterized in that, Includes a nanosecond electrical pulse generation module, a PMMA microfluidic chip, a continuous fluid delivery module, and a collection beaker; The nanosecond electrical pulse generation module is used to generate high-voltage electrical pulses with pulse widths of 1~1000ns. It consists of a function generator, a converter, an oscilloscope, a high-voltage power supply, an energy storage capacitor, and a MOSFET switch. The signal output port of the function generator is connected to the input of the converter via a coaxial cable. The output of the converter is connected to the gate of the MOSFET switch via a coaxial cable. The first channel of the oscilloscope is connected in parallel between the output of the converter and the gate of the MOSFET switch. The drain of the MOSFET switch serves as the high-voltage input and is connected to the lower end of the energy storage capacitor, while the upper end of the energy storage capacitor is connected to the positive output of the high-voltage power supply. The source of the MOSFET switch is connected to the anode of the PMMA microfluidic chip on one side and to a common ground on the other. The cathode of the microfluidic chip, the negative terminal of the high-voltage power supply, and the ground terminal of the oscilloscope are all connected to the common ground. The second channel of the oscilloscope is connected in parallel between the source of the MOSFET switch and the common ground. The PMMA microfluidic chip has a continuous microchannel inside, with electrode pairs embedded on the inner walls of both sides of the microchannel. The electrode pairs are directly electrically connected to the MOSFET switching source at a very short distance. The microchannel size is 200~500μm wide, 50~200μm deep, and 3000~10000μm long. The spacing between the electrode pairs is equal to the width of the microchannel. The continuous fluid delivery module consists of a magnetic stirrer, a feed beaker, and a syringe pump. The feed beaker containing a suspension of mixed cells and exogenous molecules is placed on the magnetic stirrer, and the outlet of the feed beaker is connected to the syringe inlet of the syringe pump via a medical silicone tube. The syringe outlet of the syringe pump is connected to the fluid inlet of the PMMA microfluidic chip via a silicone tube. The fluid outlet of the PMMA microfluidic chip is connected to a collection beaker via a silicone tube.

2. The microfluidic nanosecond electropulse transfection device according to claim 1, characterized in that, The microchannel dimensions are 300μm wide, 100μm deep, and 5000μm long.

3. The microfluidic nanosecond electric pulse transfection device of claim 1, wherein, The function generator can generate a low-voltage trigger signal, set the frequency, pulse width, duty cycle and trigger mode of the electrical pulse, and indirectly determine the final output nanosecond pulse parameters by controlling the on and off timing of the MOSFET switch.

4. The microfluidic nanosecond electric pulse transfection device according to claim 3, wherein, The converter is a signal matching and isolation device used to convert the low-voltage trigger signal output by the function generator into a drive voltage that is compatible with the gate of the MOSFET switch.

5. The microfluidic nanosecond electric pulse transfection device of claim 1, wherein, The first channel of the oscilloscope is used to monitor the waveform, frequency, and pulse width of the low-voltage trigger signal and verify whether the control signal is normal; the second channel of the oscilloscope is used to monitor the voltage amplitude, pulse width, rise time, and fall time of the high-voltage pulse actually applied to the cell at both ends of the PMMA microfluidic chip electrode and to calibrate the pulse parameters.

6. The microfluidic nanosecond pulsed electric field transfection device of claim 1, wherein, The high-voltage power supply outputs high-voltage DC current to charge the energy storage capacitor at a constant voltage. The output voltage of the high-voltage power supply determines the final pulse electric field strength. The energy storage capacitor is the core component of energy storage. When the MOSFET switch is turned on, the energy storage capacitor discharges rapidly in seconds, forming an instantaneous high-voltage pulse electric field at both ends of the microfluidic channel.

7. The microfluidic nanosecond electropulse transfection device according to claim 1, characterized in that, MOSFET switches are metal-oxide-semiconductor field-effect transistors with nanosecond-level response speeds. They are controlled by a function generator trigger signal to control the start and end time of the energy storage capacitor discharge, and output nanosecond high-voltage pulses with precise widths.

8. The microfluidic nanosecond electric pulse transfection device of claim 1, wherein, The injection pump controls the delivery rate of the cell-exogenous molecule mixture suspension. The residence time of the cell-exogenous molecule mixture suspension in the microchannel electric field region is negatively correlated with the injection pump flow rate. By adjusting the injection pump flow rate, the electroporation effect of all cells can be made uniform.

9. The microfluidic nanosecond electric pulse transfection device of claim 1, wherein, When a suspension of cells and exogenous molecules flows through the microchannel of a PMMA microfluidic chip, nanosecond high-voltage pulses applied to the electrodes on both sides cause reversible electroporation of the cell membrane, allowing exogenous molecules to enter the cell.

Images