Gene transfection method and gene transfection device

Abstract

An embodiment of the present invention discloses a gene transfection method and a gene transfection device, belonging to the field of cell biology. The present invention discloses a gene transfection method, including the following processes: providing an ultrasonic generating device and a heat generating device, where the ultrasonic generating device can emit ultrasonic waves, and the heat generating device can directly heat the biological system to be transfected; the ultrasonic waves and the heat generating device simultaneously stimulate the biological system to be transfected. By setting the ultrasonic generating device and the heat generating device, the present invention enables the combined action of ultrasonic waves and heat on the biological system to be transfected, generating a combined action including various effects such as mechanical effects, cavitation effects, and microfluidic effects on the biological system to be transfected, further shortening the time required for cell transfection, causing less damage to cells, and thus improving the survival rate after transfection.

Description

Technical Field

[0001] The present invention relates to the field of cell biology, and particularly to a gene transfection method and a gene transfection device. Background Art

[0002] Transfection refers to the process of introducing exogenous genetic material (DNA or RNA) into eukaryotic cells and expressing its specific functions. Gene transfection methods can be classified into two major categories: viral vector methods and non-viral vector methods. Viral vectors utilize their ability to inject DNA fragments into host cells to achieve gene transfection. The gene to be delivered needs to be pre-packaged into replication-defective virus particles in advance. The viruses currently used include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses, etc. However, viral vectors can only transfect DNA fragments with relatively small molecular weights, and the preparation process is complex, costly, and there are risks such as random insertion sites, cytotoxicity, and induction of gene mutations. Non-viral vector methods include both chemical methods, such as liposome transfection, polymer transfection, etc., and physical methods, such as electroporation, sonoporation, optoporation, microinjection, mechanical compression, etc. Among chemical methods, the Lipofectamine TM 2000 reagent has the widest application range and is representative of liposome transfection technology. However, it has the drawback that it is difficult to excrete from the body once it enters the body, and may produce biological toxicity or immune reactions. Among physical methods, electroporation technology has the most extensive application and is considered to have the strongest transfection ability among all transfection technologies, and can transfect a variety of primary cells that are extremely difficult to transfect (such as immune cells, stem cells, nerve cells, etc.). However, it uses a high-energy electric field to act on living cells, has high cell damage, and is extremely prone to problems such as low cell survival rate or cell disability, which greatly limits its further expansion of the application range. Therefore, it is still crucial to explore new gene transfection technologies that are more economical, easy to operate, low in toxicity, and high in transfection efficiency.

[0003] Currently, the most commonly used non-viral vector gene transfection methods are electroporation technology and cationic liposome Lipofectamine TM 2000 technology. Sonoporation technology based on ultrasound has also been reported, but most of them need to rely on micron-sized ultrasound microbubbles to achieve. At the same time, some photothermal poration technologies based on thermal effects have also been proposed, but metal nanoparticles are required to achieve.

[0004] Previously, the present team applied for a gene transfection system and method (application number: CN 201810831296.X). This method utilizes the thermal effect generated by an ultrasonic generating device, establishes a temperature gradient through the intensity of ultrasonic waves, and different temperature gradients generate different thermal effects, thereby causing thermal perforation of recipient cells. However, gene transfection only using the thermal effect has the disadvantages of low transfection rate and low cell survival rate. Summary of the Invention

[0005] The object of the present invention is to overcome the above-mentioned defects existing in the prior art, and to provide a gene transfection method and a gene transfection device, which can improve the gene transfection rate and cell survival rate.

[0006] The first aspect of the present invention protects a gene transfection method, including the following process:

[0007] Providing an ultrasonic generating device and a heat generating device, the ultrasonic generating device can emit ultrasonic waves, and the heat generating device can directly heat the biological system to be transfected; the ultrasonic waves and the heat generating device simultaneously stimulate the biological system to be transfected to undergo transfection.

[0008] The second aspect of the present invention protects a gene transfection device, including an ultrasonic generating device, a heat generating device, a transfection container and a substrate. The transfection container is used to store the biological system to be transfected. The ultrasonic generating device and the heat generating device are both arranged on the substrate, and the ultrasonic generating device and the heat generating device are both connected to the transfection container;

[0009] The ultrasonic generating device can generate ultrasonic waves, and the ultrasonic waves can act on the transfection container;

[0010] The heat generating device can heat the transfection container, and the ultrasonic waves and the heat generator act on the transfection container simultaneously, so that the biological system to be transfected in the transfection container undergoes transfection.

[0011] Implementing the embodiments of the present invention will have the following beneficial effects:

[0012] By setting an ultrasonic generating device and a heat generating device, the present invention enables the combined action of ultrasonic waves and heat on the biological system to be transfected, generating a combined action including mechanical effects, cavitation effects, microfluidic effects, etc. on the biological system to be transfected, further shortening the time required for cell transfection and causing less damage to cells, thereby increasing their survival rate after transfection. The present invention can achieve gene transfection without the participation of microbubbles, with both high transfection efficiency and high cell survival rate. Description of the Drawings

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0014] Among them:

[0015] Figure 1 Schematic structural diagram of a specific embodiment of the present invention.

[0016] Figure 2 Schematic structural diagram of an ultrasonic generating device in a specific embodiment of the present invention.

[0017] Figure 3 Schematic structural diagram of a heat generating device in a specific embodiment of the present invention.

[0018] Figure 4 Schematic flow diagram of the operation of the device in the present invention.

[0019] In the figure, 10 is a gene transfection device; 11 is a main control module; 12 is a signal generating module; 13 is a power amplification module; 14 is a substrate; 15 is an ultrasonic generating device; 16 is a heat generating device; 17 is a receiving cavity; 151 is a piezoelectric substrate; 152 is an interdigital transducer. Specific embodiments

[0020] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0021] See Figure 1 , the first aspect of the present invention discloses a gene transfection method, including the following processes:

[0022] Step 1: Provide an ultrasonic generating device 15 and a heat generating device 16. The ultrasonic generating device 15 can emit ultrasonic waves, and the heat generating device 16 can directly heat the biological system to be transfected.

[0023] Step 2: The ultrasonic waves and the heat generating device 16 simultaneously stimulate the biological system to be transfected to cause transfection.

[0024] The simultaneous action of ultrasonic waves and heat on the biological system to be transfected can effectively stimulate the biological system to be transfected.

[0025] By providing the ultrasonic generating device 15 and the heat generating device 16, the present invention enables the combined action of ultrasonic waves and heat on the biological system to be transfected, generating a combined action including various effects such as mechanical effects, cavitation effects, and microfluidic effects on the biological system to be transfected, further shortening the time required for cell transfection, causing less damage to cells, and thus increasing the survival rate after transfection. The present invention can achieve gene transfection without the participation of microbubbles, with both high transfection efficiency and high cell survival rate.

[0026] The biological system to be transfected includes cells to be transfected, a culture medium for culturing the cells to be transfected, and genetic material (such as DNA plasmids or mRNA, etc.) to be introduced into the cells to be transfected. Under the action of ultrasonic waves, the biological system to be transfected can undergo physical processes such as expansion, contraction, oscillation, and collapse. The resulting shock waves, microjets, and other effects can cause pore-like structures to form on the surface of the cell membrane of the cells to be transfected. The drugs and genetic material to be introduced enter through the pore-like structures. When the parameters of the ultrasonic waves are appropriate, the structure of the cell membrane can fully recover after the ultrasonic wave action stops, and the cells to be transfected can continue to survive, indicating successful transfection, which is reversible perforation; otherwise, it is irreversible perforation, and irreversible perforation can lead to cell death. Similarly, under the action of heat, the receptor cells in the biological system to be transfected can produce a heat perforation effect, thereby changing the permeability of the receptor cell membrane, increasing the success rate of plasmid entry into the receptor cells, and making the gene transfection efficiency higher. When the temperature is appropriate, the structure of the cell membrane can fully recover after the heat action stops, and the cells to be transfected can continue to survive, indicating successful transfection, which is reversible perforation; otherwise, it is irreversible perforation, and irreversible perforation can lead to cell death.

[0027] The temporal and spatial distribution of the sound field can be achieved by adjusting parameters such as the amplitude, waveform, duty cycle, excitation time, etc. of each ultrasonic wave and by adjusting the arrangement of each ultrasonic wave.

[0028] The heat generating device 16 can be selected as a micro-resistance wire heater. The distribution of heat can be obtained by precisely designing the shape and arrangement of the micro-resistance wire heater, thereby obtaining the heating capacity and fine temperature control ability suitable for gene transfection requirements. For example, a metal wire with a width of several micrometers is coiled into a ring.

[0029] In a specific embodiment, the specific parameters of the ultrasonic waves acting on the biological system to be transfected include amplitude, duty cycle, and single excitation signal length, where the amplitude is 15V - 55V, the duty cycle is 25% - 75%, and the single excitation signal length is 1S - 15S. The time / space average temperature acting on the biological system to be transfected is 25°C - 75°C. The ultrasonic waves and temperature within this numerical range can improve the transfection efficiency and cell survival rate of the biological system to be transfected.

[0030] See Figure 1, in the second aspect of the present invention, a gene transfection device 10 is disclosed, which includes an ultrasonic generating device 15, a thermal generating device 16, a transfection container (not shown in the figure), and a substrate 14. The ultrasonic generating device 15 is an ultrasonic surface wave generator or an ultrasonic body wave generator. The thermal generating device 16 is selected from substances that can generate heat when energized, such as resistance wires, thin films, or semiconductors. The shape and material of the transfection container are not limited as long as it can accommodate the biological system to be transfected. Both the ultrasonic generating device 15 and the thermal generating device 16 are arranged on the substrate 14, and both are connected to the transfection container. The ultrasonic generating device 15 and the thermal generating device 16 can be simultaneously arranged at the bottom of the transfection container, or simultaneously arranged on the side wall of the transfection container, or separately arranged at the bottom and side wall of the transfection container, and the specific setting method is selected according to the cells to be transfected.

[0031] The ultrasonic generating device 15 can generate ultrasonic waves, and the ultrasonic waves can act on the transfection container;

[0032] The thermal generating device 16 can heat the transfection container. The ultrasonic waves and the thermal generating device 16 act on the transfection container simultaneously, causing the biological system to be transfected in the transfection container to undergo transfection. Separately setting ultrasonic waves and a certain amount of heat alone can both improve the transfection efficiency and survival rate of cells. The present invention further enhances the cell transfection efficiency and survival rate by combining ultrasonic waves and heat.

[0033] See Figure 2 , in a specific embodiment, the ultrasonic generating device 15 includes a piezoelectric substrate 151 and an interdigital transducer 152. The piezoelectric substrate 151 is selected from one of single crystal niobate, lead zirconate titanate piezoelectric ceramics (PZT), zinc oxide piezoelectric thin films, and aluminum nitride piezoelectric thin films. In order to obtain a large electromechanical coupling coefficient, lithium niobate with 128° YX double-sided polishing is selected as the piezoelectric substrate 151 in this embodiment. The interdigital transducer 152 receives an excitation signal to drive the piezoelectric substrate 151 to vibrate, and the vibration of the piezoelectric substrate 151 drives the transfection container to vibrate, generating ultrasonic waves. Usually, an interdigital electrode layer is plated on the piezoelectric substrate 151, and the interdigital electrode layer is etched using micro-nano processing technology to form the interdigital transducer 152. By setting appropriate processing parameters such as the coating thickness, exposure time, coating thickness, and angle, as well as selecting appropriate device structure parameters such as the metal film material, number of finger pairs, and acoustic aperture size, an interdigital transducer 152 capable of generating the required ultrasonic surface waves is prepared, so that its emission frequency, insertion loss, device bandwidth, and other indicators can meet the needs of gene transfection. The piezoelectric substrate 151 is arranged on the substrate 14;

[0034] The interdigital transducer 152 is arranged on the piezoelectric substrate 151, the transfection container is connected to the piezoelectric substrate 151, the interdigital transducer 152 drives the piezoelectric substrate 151 to vibrate, and the piezoelectric substrate 151 generates ultrasonic waves, and the generated ultrasonic waves act on the transfection container.

[0035] See Figure 2 Figure 2 , in a specific embodiment, the number of ultrasonic generating devices 15 is more than two, and each ultrasonic generating device 15 is arranged around the transfection system to be transfected. The heat generating device 16 is arranged below the transfection system to be transfected. The ultrasonic waves generated by each ultrasonic generating device 15 act on the transfection system in a superposed manner, and the heat generating device 16 generates a temperature gradient field and acts on the transfection system.

[0036] The transfection container is arranged above the ultrasonic generating device 15, and the ultrasonic waves emitted by adjacent ultrasonic generating devices 15 are superposed in the area where the heat generating device 16 is located. In this way, there are both the superposed ultrasonic wave action and the heating action of the heat generating device 16 in the transfection container, and the two actions are combined.

[0037] The area where the ultrasonic waves are superposed is the position marked by the circles in the first to third columns in the figure. The ultrasonic waves emitted by the ultrasonic generating device 15 are superposed and then act on the transfection container together with the heat generating device 16.

[0038] See Figure 2 and Figure 4 Figure 4 , in a specific embodiment, the ultrasonic generating devices 15 are arranged on the substrate 14 in a matrix arrangement. In each row of ultrasonic generating devices 15, a heat generating device 16 is arranged between adjacent ultrasonic generating devices 15;

[0039] The transfection container includes a container body, and several accommodating cavities 17 are provided on the container body. The accommodating cavities 17 are arranged in one-to-one correspondence with the heat generating devices 16.

[0040] See Figure 1 Figure 1 , in a specific embodiment, it further includes a main control module 11 and a signal generating module 12. The main control module 11 is electrically connected to the signal generating module 12, and the signal generating module 12 is electrically connected to the ultrasonic generating device 15 and the heat generating device 16 respectively; the main control module 11 is used to set the first parameters of the ultrasonic generating device 15 and the second parameters of the heat generating device 16 respectively. The first parameters may specifically be the amplitude, waveform, duty cycle, excitation time, etc. of the ultrasonic waves generated by the ultrasonic generating device 15;; the signal generating module 12 is used to generate a first excitation signal according to the first parameters, generate a second excitation signal according to the second parameters, and output the first excitation signal to the ultrasonic generating device 15 and the second excitation signal to the heat generating device 16.

[0041] The ultrasonic generating device 15 converts the first excitation signal to generate ultrasonic waves, and the heat generating device 16 converts the second excitation signal to heat the transfection container.

[0042] The first parameter includes the amplitude, waveform, duty cycle, single excitation signal length, and center frequency of the ultrasonic wave. The amplitude of the ultrasonic wave is 0.001V - 1000V; the waveform of the ultrasonic wave is selected from sine wave, square wave, triangular wave, trapezoidal wave, or irregular waveform; the duty cycle of the ultrasonic wave is 0.001% - 99.999%; the single excitation signal length of the ultrasonic wave is 0.001s - 100min; the center frequency of the ultrasonic wave is 0.001Hz - 1000MHz.

[0043] In a specific embodiment, it further includes a power amplification module 13. The power amplification module 13 is electrically connected to the signal generation module 12, and the power amplification module 13 is also electrically connected to the ultrasonic generating device 15 and the heat generating device 16 respectively; the power amplification module 13 is used to amplify the first excitation signal and the second excitation signal emitted by the signal generation module 12.

[0044] See Figure 3 , in a specific embodiment, the heat generating device 16 is a micro-resistance wire, and the micro-resistance wires are arranged to form a heating body in the shape of a quasi-return character.

[0045] In a specific embodiment, it further includes a frequency detection module and a temperature detection module. The frequency detection module and the temperature detection module are both arranged in the transfection container, and the frequency detection module and the temperature detection module are both electrically connected to the main control module. The frequency detection module detects the ultrasonic frequency in the transfection container and transmits the numerical value of the ultrasonic frequency to the main control module 11, and the temperature detection module detects the temperature in the transfection container and transmits the numerical value of the temperature to the main control module 11.

[0046] The following are specific embodiments.

[0047] Embodiment 1

[0048] A manufacturing method of the ultrasonic generating device 15 includes the following steps:

[0049] (1) Glue coating: On the surface of the piezoelectric substrate 151 material that has been completely cleaned, spin-coat the positive photoresist AZ4620 at 5000 rpm for 30 s, and after glue coating, place it on a heating plate at 120 °C and bake for 3 min. Use a step profiler to test the thickness of the photoresist, and the thickness of the photoresist is approximately 5 μm.

[0050] (2) Exposure and development: According to the shape and structure of the interdigital transducer 152 in the ultrasonic generating device 15, design a film with corresponding patterns. Cover the film above the photoresist for exposure, and the light-transmitting part is cured. After using AZ400 developer to dissolve the uncured part, place it on a heating plate at 150 °C and bake for 10 min.

[0051] (3) Sputtering: Perform magnetron sputtering on the substrate that has completed the pattern transfer, so that a metal film with a thickness of about 200 nm is formed on its surface.

[0052] (4) De-gumming: Place the substrate wafer with the metal film grown thereon in an acetone solution, and use the ultrasonic vibration generated by an ultrasonic cleaner to strip the photoresist, thus completing the fabrication of the micro-nano scale ultrasonic generating device 15.

[0053] The manufacturing method of the thermal generating device 16 includes the following steps:

[0054] (1) Coating: On the surface of the piezoelectric substrate 151 material that has been thoroughly cleaned, spin-coat the positive photoresist AZ4620 at 5000 rpm for 30 s. After coating, place it on a heating plate at 120 °C and bake for 3 min. Use a profiler to measure the thickness of the photoresist, and the thickness of the photoresist is approximately 5 μm.

[0055] (2) Exposure and development: Design a film with corresponding patterns according to the shape and structure of the thermal generating device 16. Cover the film above the photoresist for exposure, and the light-transmitting part is cured. After using AZ400 developer to dissolve the uncured part, place it on a heating plate at 150 °C and bake for 10 min.

[0056] (3) Sputtering: Perform magnetron sputtering on the substrate wafer that has completed the pattern transfer, so that a metal film with a thickness of about 200 nm is formed on its surface.

[0057] (4) De-gumming: Place the substrate wafer with the metal film grown thereon in an acetone solution, and use the ultrasonic vibration generated by an ultrasonic cleaner to strip the photoresist, thus completing the fabrication of the micro-nano scale thermal generating device 16.

[0058] According to the above manufacturing method, in this embodiment, multiple ultrasonic generating devices 15 and thermal generating devices 16 are fabricated on the substrate 14 at one time. The spatial distribution of these ultrasonic generating devices 15 and thermal generating devices 16 is in the same horizontal plane. The ultrasonic waves generated by the ultrasonic generating devices 15 form a gradient sound field region, and this region coincides with the gradient temperature field region generated by the thermal generating devices 16. The transfection container is placed within the coincidence range and is simultaneously affected by the sound field and the temperature field, and thus finally realizes the gene transfection effect of cells. Among them, two ultrasonic generating devices 15 and one thermal generating device 16 form a group, and the distance between each group of ultrasonic generating devices 15 is 1.5 cm.

[0059] In this embodiment, the main control module 11 is a computer. In this embodiment, the computer sets the first parameter and transmits the signal to the signal generation module 12. The signal generation module 12 generates a first excitation signal with an amplitude of 10 mV, a duty cycle of 50%, a length of 5 seconds, and a center frequency of 24 MHz. The signal generation module 12 sends the first excitation signal to the power amplification module 13. The power amplification module 13 is a dual-channel digital power amplifier with a rated amplification power of 2 W. The power amplification module 13 amplifies the above low-voltage sine wave signal into two high-voltage sine wave signals with an amplitude of 20 V and other parameters consistent with the original signal, and outputs them. These two high-voltage sine wave signals are used as excitation signals and are output to the ultrasonic generation device 15 to respectively stimulate its operation. The ultrasonic generation device 15 converts its corresponding excitation signal to generate ultrasonic waves. The emitted ultrasonic waves undergo coherent superposition in the region between the two ultrasonic generation devices 15 to form a gradient sound field.

[0060] In this embodiment, the computer simultaneously controls a constant current power supply with a rated current of 100 mA, and the output of this power supply stimulates the thermal generation device 16 to operate. When the thermal generation device 16 is continuously stimulated, a gradient temperature field of 34 °C to 39 °C is formed.

[0061] This embodiment also includes a frequency detection module. A time-gated electrical pulse signal frequency measurement device is used to measure the actual frequency of the above sine wave signal, and the frequency reading is fed back to the computer, and the computer determines whether it is necessary to adjust the parameters used to generate the sine wave signal. The parameter adjustment includes slightly increasing or decreasing the set value of the excitation signal frequency in the parameters so that the actual frequency of the sine wave excitation signal is closer to the inherent center frequency of the ultrasonic generation device 15.

[0062] This embodiment also includes a temperature detection module. A semiconductor temperature sensor is used to measure the actual temperature within the temperature field range, and the temperature reading is fed back to the computer.

[0063] The biological system to be transfected in this embodiment includes human breast cancer cells MDA-MB-231 as the transfection target and green fluorescent DNA plasmid as the transfection substance.

[0064] Comparative Example 1

[0065] This comparative example only uses the thermal generation device 16 and does not use the ultrasonic generation device 15 compared with Embodiment 1, and other conditions are the same as those in Embodiment 1.

[0066] Comparative Example 2

[0067] This comparative example only uses the ultrasonic generation device 15 and does not use the thermal generation device 16 compared with Embodiment 1, and other conditions are the same as those in Embodiment 1.

[0068] The detection results of cell transfection efficiency and cell viability in Example 1, Comparative Example 1, and Comparative Example 2 are as follows:

[0069] The cell transfection efficiency in Example 1 is 95%, and the cell viability is 90%.

[0070] The cell transfection efficiency in Comparative Example 1 is 10%, and the cell viability is 97%.

[0071] The cell transfection efficiency in Comparative Example 2 is 80%, and the cell viability is 93%.

[0072] From the above results, it can be seen that when the ultrasonic generating device 15 and the thermal generating device 16 act simultaneously, the cell transfection efficiency is significantly increased, and at the same time, it has a high cell viability, and its performance is better than that of setting the ultrasonic generating device 15 alone or the thermal generating device 16 alone.

[0073] The above-disclosed are only the preferred embodiments of the present invention. Of course, the scope of the rights of the present invention cannot be limited thereby. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope covered by the present invention.

Claims

1. A gene transfection device, characterized in that, it includes an ultrasonic generating device, a heat generating device, a transfection container and a substrate; wherein, the ultrasonic generating device includes a piezoelectric substrate and an interdigital transducer disposed on the piezoelectric substrate, and the piezoelectric substrate is mounted on the substrate; the transfection container is connected to the piezoelectric substrate; the ultrasonic generating device and the heat generating device are both disposed on the substrate and are both connected to the transfection container; the interdigital transducer is used to drive the piezoelectric substrate to vibrate, generate ultrasonic waves and act on the transfection container; the heat generating device is used to heat the transfection container; the transfection container is used to store the biological system to be transfected, and the ultrasonic waves generated by the ultrasonic generating device and the heat generated by the heat generating device act on the biological system to be transfected in the transfection container at the same time, so that gene transfection occurs in the biological system; wherein, the specific parameters of the ultrasonic waves acting on the biological system to be transfected include amplitude, duty cycle and single excitation signal length, wherein the amplitude is 15V - 55V, the duty cycle is 25% - 75%, and the single excitation signal length is 1s - 15s; the time / space average temperature acting on the biological system to be transfected is 25°C - 75°C.

2. The gene transfection device according to claim 1, characterized in that, the number of the ultrasonic generating devices is more than two, each of the ultrasonic generating devices is arranged around the biological system to be transfected, the heat generating device is arranged below the biological system to be transfected, and the ultrasonic waves generated by each of the ultrasonic generating devices act on the transfection system in a superimposed manner, and the heat generating device generates a temperature gradient field acting on the transfection system.

3. The gene transfection device according to claim 1, characterized in that, the ultrasonic generating devices are arranged on the substrate in a matrix arrangement, and in each row of the ultrasonic generating devices, a heat generating device is arranged between adjacent ultrasonic generating devices.

4. The gene transfection device according to claim 1, characterized in that, the transfection container includes a container body, and a plurality of accommodating cavities are provided on the container body, and the accommodating cavities are arranged in one-to-one correspondence with the heat generating devices.

5. The gene transfection device according to any one of claims 1 - 4, characterized in that, it further includes a main control module and a signal generating module, the main control module is electrically connected to the signal generating module, and the signal generating module is electrically connected to the ultrasonic generating device and the heat generating device respectively; the main control module is used to respectively set a first parameter of the ultrasonic generating device and a second parameter of the heat generating device; the signal generating module is used to generate a first excitation signal according to the first parameter, generate a second excitation signal according to the second parameter, output the first excitation signal to the ultrasonic generating device, and output the second excitation signal to the heat generating device; the ultrasonic generating device converts the first excitation signal to generate the ultrasonic waves, and the heat generating device converts the second excitation signal to heat the transfection container.

6. The gene transfection device according to claim 5, characterized in that, It further includes a power amplification module, which is electrically connected to the signal generation module, and is also respectively electrically connected to the ultrasonic generating device and the heat generating device; the power amplification module is used to amplify the first excitation signal and the second excitation signal emitted by the signal generation module.

7. The gene transfection device according to claim 6, wherein, it further includes a frequency detection module and a temperature detection module. The frequency detection module and the temperature detection module are both arranged in the transfection container, and are both electrically connected to the main control module. The frequency detection module detects the ultrasonic frequency in the transfection container and transmits the numerical value of the ultrasonic frequency to the main control module. The temperature detection module detects the temperature in the transfection container and transmits the numerical value of the temperature to the main control module.

8. A gene transfection method, wherein, it includes the following processes: providing the gene transfection device according to any one of claims 1-7; placing the biological system to be transfected in a transfection container in contact with the piezoelectric substrate; driving the interdigital transducer to generate ultrasonic waves in the piezoelectric substrate and act on the biological system in the transfection container; at the same time, driving the heat generating device to heat the biological system so that gene transfection occurs in the biological system.

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