An ultrasonic perforation eukaryotic and prokaryotic cell in vitro transfection and transformation device

Abstract

This invention provides an ultrasonic perforation device for in vitro transfection and transformation of eukaryotic and prokaryotic cells, comprising: a well plate with a plurality of receiving wells; a driving mechanism; a water tank for holding deionized water; the bottom of the well plate being immersed in the deionized water; a heating element; at least two ultrasonic transducers of different frequencies, disposed below the well plate with their emitting ends immersed in the deionized water; the driving mechanism driving the well plate to move horizontally, so that the receiving well to be tested is located in the far-field region directly above the corresponding ultrasonic transducer. Because the receiving wells are located in the far-field region of the ultrasonic transducer's sound field, the sound intensity of the ultrasonic transducer's sound field changes little with spatial position during transfection, resulting in stable spatial characteristics of the sound field. This facilitates relatively stable ultrasonic cavitation intensity and ultrasonic perforation effect, and the cell membrane's repairable ultrasonic perforation leads to high cell transfection efficiency and high cell viability. Placing the emitting ends of the ultrasonic transducers in deionized water avoids the risk of contamination from direct contact with the cell suspension.

Description

Technical Field

[0001] This invention relates to the field of transfection equipment technology, and in particular to an ultrasonic perforation device for in vitro transfection and transformation of eukaryotic and prokaryotic cells. Background Technology

[0002] In bioengineering applications, target genes are often introduced into recipient cells to facilitate the detection of their expression or to study other functions of exogenous genes. Their biological functions are studied by observing the transient or stable expression of the target gene in in vitro cultured cells. This is also one of the fundamental links in disciplines or industries such as cell engineering, genetic engineering, and gene engineering. As a result, technologies such as gene therapy and cell factories have also flourished.

[0003] There are various ways to introduce exogenous genes (or drugs and proteins, etc.) into cells. The method of introducing them into prokaryotic cells is generally called transformation; the method of introducing them into eukaryotic cells using viral vectors is called transduction; and the technique of introducing them into eukaryotic cells using non-viral vectors is called transfection. Non-viral gene introduction can be broadly divided into chemical and physical methods. The principle of chemical introduction is to use polymers, liposomes, cationic substances, etc., to form complexes with negatively charged nucleic acids, which bind to the cell membrane surface and enter the cell through endocytosis or membrane fusion. Physical introduction methods mainly use mechanical force or thermal effects to temporarily disrupt the cell membrane, introducing the target gene into eukaryotic cells. These methods mainly include microinjection, gene gun method, laser perforation method, electroporation, and ultrasound transfection. Viral transduction is highly efficient, but because the exogenous gene must integrate into the chromosome of the transfected cell to be expressed, it carries the risk of carcinogenicity and teratogenicity, has poor safety, and the system is small, only able to introduce nucleic acid molecules such as DNA and RNA, and the design and experimental cycle is also long. Among chemical transfection methods, liposome transfection is relatively mature in commercialization, but it also has drawbacks such as being able to introduce only nucleic acid molecules, high cost, high cytotoxicity, and a tendency to induce inflammatory reactions. Among physical transfection methods, electrotransfection is widely used in in vitro experiments, but its further development is limited by problems such as low cell viability, the need for cumbersome pretreatment, and the difficulty in scaling up the transfection system.

[0004] Ultrasonic transfection boasts numerous advantages, including ease of operation, high safety, minimal cell damage, low cost, and the ability to introduce various molecules such as nucleic acids, drugs, and proteins into recipient cells. It also facilitates the scaling up of reaction systems, making its application prospects very broad. The main principle of ultrasonic transfection is acoustic cavitation, which refers to the process of creating reversible pores in the cell membrane using the cavitation effect of ultrasound. The cavitation effect of ultrasound refers to the vibration, growth, and collapse of micro-gas nuclei in a liquid caused by ultrasound reaching its intensity threshold. This process is typically very rapid and intense, accompanied by instantaneous local high temperature, high pressure, and microjets generated by the collapse of bubbles. These effects temporarily disrupt the cell membrane and create pores ranging from tens to hundreds of nanometers, allowing exogenous genes to enter the target cells, achieving the goal of gene delivery. Furthermore, the combined use of ultrasonic transfection with ultrasound contrast agents can achieve even higher transfection efficiency. The addition of ultrasound contrast agents provides a large number of cavitation nuclei, significantly enhancing the cavitation effect, and the bubble size of a few micrometers is well-suited for ultrasound transfection experiments using megahertz-level ultrasound.

[0005] However, existing ultrasound transfection instruments mainly use handheld ultrasound probes. When the ultrasound probe is directly immersed in the cell culture medium, it is prone to cell contamination, metal ion contamination, and probe corrosion. Furthermore, the spatial position of the non-contact ultrasound probe and the cells to be transfected cannot be relatively fixed, making it impossible to maintain consistent ultrasound parameters at the cell location. In the near-field region of the ultrasound transducer, the sound field parameters generated by the ultrasound probe (transducer) vary drastically with spatial position. In the sound field generated by a megahertz-level ultrasound transducer, the peak negative pressure of the sound field within a space of a few micrometers along the axis will vary by more than 20 times between its minimum and maximum values, thus hindering the provision of stable experimental conditions. In addition, the ambient temperature of the entire transfection system is not stabilized during ultrasound treatment, which is also detrimental to the cells being in an optimal physiological activity and transfection-friendly state.

[0006] In view of the technical bottlenecks of the above-mentioned transfection methods and equipment, the main objective of this invention is to address the current technical deficiencies in the key step of transfection in cell engineering by developing a new ultrasonic perforation in vitro cell transfection device and establishing a mature transfection operation process with high transfection efficiency and low cost applicable to different cell systems. Summary of the Invention

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions.

[0008] This invention provides an ultrasonic-perforated eukaryotic and prokaryotic cell in vitro transfection and transformation device, comprising:

[0009] The well plate has several wells for holding cell suspensions, ultrasound contrast agents, and plasmid mixtures.

[0010] A drive mechanism for driving the perforated plate to move in at least one direction in the horizontal plane;

[0011] A water tank for holding deionized water; the bottom of the perforated plate is immersed in the deionized water;

[0012] Heating element, used to heat deionized water;

[0013] At least two ultrasonic transducers of different frequencies are disposed below the orifice plate with their emitting ends immersed in deionized water.

[0014] The driving mechanism drives the orifice plate to move in the horizontal plane so that the receiving orifice to be tested is located in the far field region directly above the corresponding ultrasonic transducer for transfection or conversion.

[0015] Preferably, the ultrasonic transducer is selected from at least two of the following: a first ultrasonic transducer with a frequency of (800±80) kHz, a second ultrasonic transducer with a frequency of (1±0.1) MHz, a third ultrasonic transducer with a frequency of (2±0.2) MHz, and a fourth ultrasonic transducer with a frequency of (40±4) kHz.

[0016] Preferably, the perforated plate is a six-hole plate or a twelve-hole plate; the vertical distance between the center of the bottom surface of the receiving hole and the upper surface of the ultrasonic transducer is 160mm-200mm.

[0017] Preferably, the plurality of the receiving holes are distributed in multiple rows and columns.

[0018] Preferably, the drive mechanism includes an X-axis module and a Y-axis module.

[0019] Preferably, the outer surface of the water tank is covered with an insulation layer.

[0020] Preferably, the heating element is a resistance heating tube, which is located in the lower receiving hole inside the water tank.

[0021] Preferably, it also includes a temperature sensor to obtain temperature information at the bottom of the receiving hole.

[0022] Preferably, it also includes an outer shell and a drain pipe; the outer shell includes a hinged box body and a box cover; one end of the drain pipe is connected to the inside of the water tank, and the other end extends out of the outer shell.

[0023] Preferably, it further includes:

[0024] A control panel is located on one side of the housing;

[0025] Several adjustable feet are provided at the bottom of the housing.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] This invention provides an ultrasonic perforation device for in vitro transfection and transformation of eukaryotic and prokaryotic cells. The receiving orifice is located in the far-field region of the ultrasonic transducer's acoustic field. During transfection, the acoustic intensity of the ultrasonic transducer's acoustic field changes little with spatial position, and the spatial characteristics of the acoustic field are stable. This helps to ensure relatively stable ultrasonic cavitation intensity and ultrasonic perforation effect. Furthermore, the cell membrane's repairable ultrasonic perforation results in high cell transfection efficiency and high cell viability. In addition, the transmitting end of the ultrasonic transducer is placed in deionized water. The ultrasonic transducer resonates and propagates ultrasound waves through the deionized water into the mixture in the receiving orifice. Under the action of an ultrasonic contrast agent, the cells undergo ultrasonic perforation, thereby achieving transfection. This scheme avoids the risk of contamination from direct contact between the ultrasonic transducer and the culture medium in the cell suspension, and the ultrasonic transducer is easy to clean and disinfect.

[0028] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0029] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0030] Figure 1 This is a three-dimensional structural diagram of the device body when the lid is open according to the present invention. Figure 1 ;

[0031] Figure 2 This is an exploded view of a partial structure of the device body of the present invention;

[0032] Figure 3 This is a partial three-dimensional structural diagram of the device body of the present invention;

[0033] Figure 4 This is a three-dimensional structural diagram of the device body when the lid is open according to the present invention. Figure 2 ;

[0034] Figure 5 This is a sound field intensity distribution diagram of a 1MHz ultrasonic transducer in one embodiment of the present invention.

[0035] In the diagram: 100, the main body of the device;

[0036] 10. Orifice plate; 11. Receiving hole;

[0037] 20. Drive mechanism; 21. X-axis module; 211. Second slider; 212. X-axis guide rail; 22. Y-axis module; 221. First slider; 222. Y-axis guide rail; 23. Bracket; 231. Support platform; 232. Connecting plate; 24. Guide assembly; 241. Linear guide rail; 242. Third slider;

[0038] 31. Water tank; 311. First clearance hole; 312. First through hole; 32. Heating element; 33. Insulation layer; 331. Second clearance hole; 34. Drain pipe;

[0039] 41. First ultrasonic transducer; 42. Second ultrasonic transducer; 43. Third ultrasonic transducer;

[0040] 50. Outer shell; 51. Box body; 52. Box lid;

[0041] 60. Control Panel;

[0042] 70. Adjust the feet;

[0043] 80. Base plate; 81. First base; 82. Second base; 83. Mounting base;

[0044] 91. Displacement drive circuit; 92. Circuit board. Detailed Implementation

[0045] The invention will now be described in further detail with reference to the accompanying drawings, which will make the foregoing and other objects, features, aspects, and advantages of the invention more apparent, enabling those skilled in the art to practice it upon referring to the text of the specification. In the drawings, shapes and dimensions are enlarged for clarity, and the same reference numerals are used throughout the figures to indicate the same or similar parts. In the following description, terms such as center, thickness, height, length, front, back, rear, left, right, top, bottom, upper, lower, etc., are used based on the orientation or positional relationship shown in the drawings. In particular, “height” corresponds to the dimension from top to bottom, “width” corresponds to the dimension from left to right, and “depth” corresponds to the dimension from front to back. These relative terms are for ease of explanation and are not generally intended to require a specific orientation. Terms relating to attachment, connection, etc. (e.g., “connection” and “attachment”) refer to the relationship in which these structures are directly or indirectly fixed or attached to each other by an intermediate structure, and to movable or rigid attachments or relationships, unless otherwise explicitly stated.

[0046] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0047] Example 1

[0048] This invention provides an ultrasonic-perforated eukaryotic and prokaryotic cell in vitro transfection and transformation device, such as... Figure 1 , Figure 2 As shown, the device includes a device body 100, which includes:

[0049] The well plate 10 is provided with a plurality of receiving holes 11; the receiving holes 11 are used to hold a mixture of cell suspension, ultrasound contrast agent and plasmid;

[0050] Drive mechanism 20 is used to drive the perforated plate 10 to move in at least one direction in the horizontal plane;

[0051] Water tank 31 is used to hold deionized water; the bottom of the perforated plate 10 is immersed in the deionized water;

[0052] Heating element 32 is used to heat deionized water, thereby regulating the temperature at the bottom of the well plate 10 to provide a suitable temperature for the cells, so that the cells are in good condition and the cell transfection efficiency is improved.

[0053] At least two ultrasonic transducers of different frequencies are disposed below the orifice plate 10 and their emitting ends are immersed in deionized water.

[0054] The driving mechanism 20 drives the orifice plate 10 to move in the horizontal plane so that the receiving hole 11 to be tested is located in the far field region directly above the corresponding ultrasonic transducer for transfection or conversion.

[0055] In this embodiment, the device body 100 is equipped with well plates 10 to hold different transfection mixtures. Before transfection, the cells only need to undergo simple digestion and resuspension. After adding the cell suspension, ultrasound contrast agent, and plasmid into the corresponding wells 11, the ultrasound-perforated in vitro transfection process can be automatically completed within the device body 100. During transfection, the wells are located in the far-field region of the ultrasound transducer's sound field. The sound intensity of the sound field changes little with spatial position, and the spatial characteristics of the sound field are stable, which helps to ensure relatively stable ultrasound cavitation intensity and ultrasound perforation effect. Moreover, the cell membrane's repairable ultrasound perforation results in high cell transfection efficiency and high cell survival rate. In addition, the transmitting end of the ultrasound transducer is placed in deionized water. The ultrasound transducer resonates and transmits ultrasound waves through the deionized water to the mixture in the wells 11. Under the action of the ultrasound contrast agent, the cells undergo ultrasound perforation, thereby achieving transfection. This scheme can avoid the risk of contamination caused by direct contact between the ultrasound transducer and the culture medium in the cell suspension, and the ultrasound transducer is easy to clean and disinfect. The device body 100 is equipped with a first driving circuit to obtain an average stable ultrasonic intensity in the far field region using different ultrasonic transducers.

[0056] Specifically, the preparation of the mixture to be transfected includes the following steps:

[0057] (1) HeLa cells were cultured in a carbon dioxide incubator at 37°C and 5% carbon dioxide atmosphere in a 10-well plate until 60%-70% adhered. The culture medium was DMEM medium (Gibco) containing 1% antibiotics (Gibco) and 10% fetal bovine serum (Gibco). The 10-well plate was a six-well plate.

[0058] (2) After washing the cells twice with PBS buffer, digest the cells with trypsin-EDTA for 1 min, add culture medium and gently pipette the adherent cells off the wall, then centrifuge the obtained cell suspension at 1000 rpm at room temperature for 5 min. Aspirate the centrifugation waste liquid, add 2 mL of culture medium and gently pipette to resuspend the cells, and then add them to well 10;

[0059] (3) Prepare ultrasound contrast agent (SonoVue, Bracco) with 5 mL of PBS buffer. Add pBOBi-EGFP enhanced green fluorescent plasmid to the ultrasound contrast agent well plate 10 at a concentration of 100 μL / well to make the final concentration 20 μg / mL. Gently shake the well plate 10 to mix it well to obtain the mixture to be transfected for transfection.

[0060] In one embodiment, such as Figure 1 , Figure 2 As shown, the ultrasonic transducer is selected from at least two of the following: a first ultrasonic transducer 41 with a frequency of (800±80) kHz, a second ultrasonic transducer 42 with a frequency of (1±0.1) MHz, a third ultrasonic transducer 43 with a frequency of (2±0.2) MHz, and a fourth ultrasonic transducer with a frequency of (40±4) kHz, to accommodate transfection operations requiring different frequencies. Specifically, the first ultrasonic transducer 41 with a frequency of (800±80) kHz, the second ultrasonic transducer 42 with a frequency of (1±0.1) MHz, and the third ultrasonic transducer 43 with a frequency of (2±0.2) MHz are suitable for eukaryotic cell transfection, while the fourth ultrasonic transducer with a frequency of (40±4) kHz is suitable for prokaryotic cell transformation.

[0061] Preferably, the frequency of the first ultrasonic transducer 41 is 800 kHz, the frequency of the second ultrasonic transducer 42 is 1 MHz, and the frequency of the third ultrasonic transducer 43 is 2 MHz. Specifically, the first driving circuit drives the three ultrasonic transducers to obtain a stable average ultrasonic intensity in their far-field region, with an irradiation time of 10 s to 3 min and an ultrasonic intensity of 0 to 4 W / cm². 2 (Spatial mean and temporal peak value) High-frequency signals can achieve a duty cycle of 5%-100% after pulse width modulation, which can meet the experimental conditions of different cell lines, different cell states and densities.

[0062] Further, the well plate 10 is a six-well plate or a twelve-well plate; the vertical distance between the center of the bottom surface of the receiving well 11 and the upper surface of the ultrasonic transducer is 160mm-200mm. Specifically, by designing the size of the well plate 10 and the vertical distance between the well plate 10 and the ultrasonic transducer, the far-field regions of the above three types of ultrasonic transducers are matched, so that the mixture to be transfected in the receiving well 11 can be moved into the far-field region of the ultrasonic transducer under the drive of the driving mechanism 20, thereby stabilizing the ultrasonic cavitation intensity and ultrasonic perforation effect and improving the cell transfection efficiency. Preferably, the well plate 10 is a six-well plate, and the vertical distance between the center of the bottom surface of the receiving well 11 and the upper surface of the ultrasonic transducer is 200mm, so that the six-well plate is always located in the far-field region of the ultrasonic transducer; by driving the driving mechanism 20, each receiving well 11 on the well plate 10 can be adjusted to the far-field region directly above the corresponding ultrasonic transducer.

[0063] In one embodiment, the plurality of receiving holes 11 are arranged in multiple rows and columns, and are compactly arranged.

[0064] Furthermore, such as Figure 2 As shown, the driving mechanism 20 includes an X-axis module 21 and a Y-axis module 22; the X-axis module 21 is used to drive the perforated plate 10 to move along the X-axis, and the Y-axis module 22 is used to drive the perforated plate 10 to move along the Y-axis, thereby realizing two-dimensional motion to adjust the perforated plate 10 to be located in different rows or columns of receiving holes 11 so as to be in the transfection region, wherein the transfection region refers to the far-field region directly above the corresponding ultrasonic transducer.

[0065] In one specific embodiment, such as Figures 1 to 3 As shown, the perforated plate 10 is connected to the drive end of the Y-axis module 22 via the bracket 23, and the Y-axis module 22 is fixed to the drive end of the X-axis module 21.

[0066] Furthermore, the bracket 23 is made of 6061 aluminum, which is lightweight and has a certain strength.

[0067] Furthermore, the two ends of the bracket 23 are bent to form a support platform 231 and a connecting plate 232 respectively. The perforated plate 10 is installed on the upper surface of the support platform 231, and the connecting plate 232 is fixed to the drive end of the Y-axis module 22.

[0068] Furthermore, such as Figure 2 , Figure 3 As shown, the Y-axis module 22 is equipped with a matching first slider 221 and Y-axis guide rail 222. The first slider 221 is connected to the drive end of the Y-axis module 22 and the bracket 23, respectively. The drive end of the Y-axis module 22 drives the first slider 221 to move the bracket 23 along the Y-axis, thereby driving the perforated plate 10 to move along the Y-axis. The first slider 221 and the Y-axis guide rail 222 cooperate to play a guiding and supporting role, improve the Y-axis displacement accuracy and stability, and reduce the overturning moment.

[0069] Furthermore, such as Figure 2 , Figure 3 As shown, the X-axis module 21 is equipped with a matching second slider 211 and an X-axis guide rail 212. The second slider 211 is connected to both the X-axis module drive end and the Y-axis module. The X-axis module drive end drives the second slider 211 to move the Y-axis module along the X-axis, thereby driving the perforated plate 10 to move along the X-axis. The second slider 211 and the X-axis guide rail 212 cooperate to provide guidance and support, improve the X-axis displacement accuracy and stability, and reduce the overturning moment.

[0070] In one specific embodiment, the X-axis module 21 and / or the Y-axis module 22 are mechanisms formed by the cooperation of a stepper motor and a lead screw and nut pair. The stepper motor drives the lead screw to rotate, so that the nut moves along the lead screw. Figure 2 As shown, the device body 100 is provided with a displacement drive circuit 91 to drive the stepper motors of the X-axis module 21 and the Y-axis module 22.

[0071] In one embodiment, such as Figure 2 , Figure 3 As shown, the bottom of one end of the Y-axis module 22 is connected to the drive end of the X-axis module 21, and the bottom of the other end is supported by the guide component 24; the guide component 24 includes a matching linear guide rail 241 and a third slider 242, which improves the Y-axis displacement accuracy and stability.

[0072] In one embodiment, such as Figure 2 , Figure 3 As shown, the device body 100 also includes a base plate 80 for forming a support structure, such as for supporting the drive mechanism 20 and the water tank 31.

[0073] Furthermore, the base plate 80 is made of steel, resulting in high structural strength.

[0074] In one embodiment, such as Figure 2 , Figure 3 As shown, the bottom of the X-axis module 21 is fixed to the base plate 80 via the first base 81.

[0075] In one embodiment, such as Figure 2 , Figure 3 As shown, the bottom of the guide assembly 24 is fixed to the base plate 80 via the second base 82.

[0076] In one embodiment, such as Figure 1 , Figure 2As shown, the base plate 80 is provided with several threaded mounting holes, the bottom of the water tank 31 is provided with several first clearance holes 311, and the insulation layer 33 is provided with several second clearance holes 331; the bottom of the ultrasonic transducer is screwed into the threaded mounting hole, and the upper part of the ultrasonic transducer passes through the corresponding second clearance hole 331 and first clearance hole 311 in sequence before being immersed in the deionized water in the water tank 31.

[0077] In one embodiment, such as Figures 1 to 3 As shown, the outer surface of the water tank 31 is covered with a heat insulation layer 33 to reduce heat transfer and help maintain a constant temperature in the reaction system.

[0078] In one embodiment, the water tank 31 is made of 304 stainless steel.

[0079] In one embodiment, the insulation layer 33 is a polyurethane layer.

[0080] In one embodiment, the heating element 32 is a resistance heating tube located in the lower part of the water tank 31, used to regulate the temperature at the bottom of the receiving hole 11. Preferably, the resistance heating tube is used to ensure that the temperature at the bottom of the receiving hole 11 is (37±0.5)℃. Within this temperature range, the cells are in good condition, which is conducive to the recovery after cell membrane perforation and improves transfection efficiency and cell survival rate.

[0081] In one embodiment, a temperature sensor is also included to obtain temperature information at the bottom of the receiving hole 11. Specifically, the device body 100 is provided with a temperature feedback circuit to collect feedback signals from the temperature sensor and control the power of the heating element 32 to keep the temperature at the bottom of the orifice plate holder constant at the target temperature. Further, as... Figure 2 As shown, the temperature feedback circuit and the first drive circuit for driving the ultrasonic transducer are integrated on the circuit board 92.

[0082] Furthermore, the temperature sensor is fixed on bracket 23 for easy installation.

[0083] Furthermore, such as Figure 2 As shown, the displacement drive circuit 91 and the circuit board 92 are mounted on the base plate 80.

[0084] In one embodiment, such as Figure 1 , Figure 4As shown, it also includes an outer shell 50 and a drain pipe 34. The outer shell 50 includes a hinged box body 51 and a box cover 52, which together form a closed cavity. The perforated plate 10, the drive mechanism 20, the water tank 31, the heating element 32, and the ultrasonic transducer are disposed within the cavity. One end of the drain pipe 34 is connected to the interior of the water tank 31, and the other end extends outside the outer shell 50. The drain pipe 34 facilitates the discharge of deionized water from the water tank 31 for replacement. The water tank 31, the heating element 32, the insulation layer 33, and the drain pipe 34 together form a water bath structure to provide a suitable temperature environment for cell transfection. A base plate 80 is installed on the bottom wall inside the box body 51 to enhance support.

[0085] Furthermore, the lid 52 is made of polyethylene.

[0086] Furthermore, the drain pipe 34 is fixed to the base plate 80.

[0087] Furthermore, the power connector hole of the device body 100 is located on the back of the housing 50.

[0088] In one embodiment, such as Figure 1 , Figure 4 As shown, it also includes:

[0089] A control panel 60 is disposed on one side of the housing 50; the control panel 60 is electrically connected to the displacement drive circuit 91 and the circuit board 92.

[0090] Several adjusting feet 70 are provided at the bottom of the housing 50 to level the housing 50.

[0091] In one embodiment, such as Figure 1 , Figure 2 As shown, the heating element 32 is a resistance heating tube, and the resistance heating tube is U-shaped; the resistance heating tube is fixed to the base plate 30. Specifically, as... Figure 2 , Figure 3 As shown, the base plate 80 is provided with two mounting seats 83, one side arm of the water tank 31 is provided with two first through holes 312, and the insulation layer 33 is provided with two second through holes 332; the resistance heating tube is located inside the water tank 31 and its two ends pass through the corresponding second through holes 332 and first through holes 312 respectively and are fixed to the two mounting seats 83 respectively.

[0092] The transfection operation using the device body 100 includes the following steps:

[0093] (1) Prepare the mixture to be transfected;

[0094] (2) Close the drain pipe 34, open the box cover 52, and inject deionized water into the water tank 31 to the water level line; connect the device body 100 to the power supply, operate the control panel 60, set the temperature to 37℃ for 5 minutes of preheating; select the ultrasonic transducer of the corresponding frequency and make it work for 2 minutes to be in a stable resonance state.

[0095] (3) Open the box cover 52, install the well plate 10 containing the mixture to be transfected, and close the box cover 52;

[0096] (4) Operate the control panel 60 to perform ultrasonic transfection; select an ultrasonic irradiation time of 10s-3min per well, an ultrasonic intensity of 0-4W / cm2 (spatial mean time peak), and a duty cycle of 5% to 100%.

[0097] (5) After all the mixture to be transfected in all the wells has been irradiated, take out the well plate 10 and place it in a carbon dioxide incubator. After culturing at 37°C and 5% carbon dioxide atmosphere for 4 hours, replace with fresh culture medium. After culturing for another 24 hours, observe the transfection efficiency under a fluorescence microscope.

[0098] like Figure 5 As shown, the measured sound field intensity distribution of a 1MHz ultrasonic transducer at a far field (200mm) under a driving voltage of 2Vpp, 1MHz frequency, 1kHz pulse repetition frequency, and 50% duty cycle is relatively uniform and varies little with spatial position.

[0099] The present invention provides an ultrasonic perforation device for in vitro transfection and transformation of eukaryotic and prokaryotic cells. The device body 100 has a simple structure and the sound intensity of the sound field changes little with spatial position, which is conducive to ensuring a relatively stable ultrasonic cavitation intensity and ultrasonic perforation effect. The repairable ultrasonic perforation of the cell membrane results in high cell infection efficiency and high cell survival rate.

[0100] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Those skilled in the art can readily implement the present invention based on the accompanying drawings and the description above. However, any modifications, alterations, or variations made by those skilled in the art without departing from the scope of the present invention, utilizing the disclosed technical content, are equivalent embodiments of the present invention. Furthermore, any equivalent changes, alterations, or variations made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the present invention.

Claims

1. An ultrasonically perforated eukaryotic and prokaryotic cell in vitro transfection and transformation device, characterized in that, include: The well plate (10) is provided with a plurality of receiving holes (11); the receiving holes (11) are used to hold cell suspension, ultrasound contrast agent, and plasmid mixture; A drive mechanism (20) is used to drive the perforated plate (10) to move in at least one direction in the horizontal plane; A water tank (31) is used to hold deionized water; the bottom of the perforated plate (10) is immersed in the deionized water. Heating element (32) is used to heat deionized water; At least two ultrasonic transducers of different frequencies are disposed below the orifice plate (10) and their emitting ends are immersed in deionized water; The driving mechanism (20) drives the orifice plate (10) to move in the horizontal plane so that the receiving hole (11) to be tested is located in the far field region directly above the corresponding ultrasonic transducer for transfection or conversion; the vertical distance between the center of the bottom surface of the receiving hole (11) and the upper surface of the ultrasonic transducer is 160mm-200mm. The transmitting end of the ultrasonic transducer is placed in deionized water. The ultrasonic transducer resonates and transmits ultrasonic waves through the deionized water to the mixed liquid in the containment hole (11). Under the action of the ultrasonic contrast agent, the cells undergo ultrasonic perforation effect to achieve transfection.

2. The ultrasonic perforation eukaryotic and prokaryotic cell in vitro transfection and transformation device according to claim 1, characterized in that, The ultrasonic transducer is selected from at least two of the following: a first ultrasonic transducer (41) with a frequency of 800±80kHz, a second ultrasonic transducer (42) with a frequency of 1±0.1MHz, a third ultrasonic transducer (43) with a frequency of 2±0.2MHz, and a fourth ultrasonic transducer with a frequency of 40±4kHz.

3. The ultrasonic perforation eukaryotic and prokaryotic cell in vitro transfection and transformation device according to claim 2, characterized in that, The perforated plate (10) is a six-hole plate or a twelve-hole plate.

4. The ultrasonic perforation eukaryotic and prokaryotic cell in vitro transfection and transformation device according to any one of claims 1-3, characterized in that, The aforementioned receiving holes (11) are distributed in multiple rows and columns.

5. The ultrasonic perforation eukaryotic and prokaryotic cell in vitro transfection and transformation device according to claim 4, characterized in that, The drive mechanism (20) includes an X-axis module (21) and a Y-axis module (22).

6. The ultrasonic perforation eukaryotic and prokaryotic cell in vitro transfection and transformation device according to any one of claims 1-3, characterized in that, The outer surface of the water tank (31) is covered with an insulation layer (33).

7. The ultrasonic perforation eukaryotic and prokaryotic cell in vitro transfection and transformation device according to any one of claims 1-3, characterized in that, The heating element (32) is a resistance heating tube, which is located in the lower receiving hole inside the water tank (31).

8. The ultrasonic perforation eukaryotic and prokaryotic cell in vitro transfection and transformation device according to any one of claims 1-3, characterized in that, It also includes a temperature sensor to obtain temperature information at the bottom of the receiving hole (11).

9. The ultrasonic perforation device for in vitro transfection and transformation of eukaryotic and prokaryotic cells according to any one of claims 1-3, characterized in that, It also includes an outer shell (50) and a drain pipe (34); the outer shell (50) includes a hinged box body (51) and a box cover (52); one end of the drain pipe (34) is connected to the inside of the water tank (31), and the other end extends out of the outer shell (50).

10. The ultrasonic perforation eukaryotic and prokaryotic cell in vitro transfection and transformation device according to claim 9, characterized in that, Also includes: A control panel (60) is disposed on one side of the housing (50); Several adjustable feet (70) are provided at the bottom of the housing (50).

Images