CN217973229U - Ultrasonic perforation eukaryotic and prokaryotic cell in-vitro transfection and transformation device - Google Patents

Abstract

The utility model provides an ultrasonic perforation eukaryotic and prokaryotic cell in vitro transfection conversion equipment, include: the orifice plate is provided with a plurality of accommodating holes; a drive mechanism; a water tank for containing deionized water; the bottom of the pore plate is immersed in deionized water; a heating element; at least two ultrasonic transducers with different frequencies, which are arranged below the pore plate and the emitting ends of which are immersed in the deionized water; the driving mechanism drives the orifice plate to displace in the horizontal plane, so that the accommodating hole to be measured is positioned in a far field region right above the corresponding ultrasonic transducer. The containing hole is positioned in a far field area of a sound field of the ultrasonic transducer, during transfection, the sound intensity of the sound field of the ultrasonic transducer changes slightly along with the space position, the space characteristic of the sound field is stable, relatively stable ultrasonic cavitation intensity and ultrasonic perforation effect can be guaranteed, and cell membrane repairability ultrasonic perforation enables cell transfection efficiency to be high and cell survival rate to be high. The transmitting end of the ultrasonic transducer is arranged in the deionized water, so that the pollution risk caused by direct contact with the cell suspension can be avoided.

Description

Ultrasonic perforation eukaryotic and prokaryotic cell in-vitro transfection and transformation device

Technical Field

The utility model relates to a transfection equipment technical field especially relates to an ultrasonic perforation eukaryotic and prokaryotic cell transfection conversion equipment in vitro.

Background

In the application of bioengineering, the target gene is often introduced into a recipient cell for the convenience of detecting the expression of the target gene or researching other functions of an exogenous gene, and the biological function of the target gene is researched through transient expression or stable expression of the target gene in vitro cultured cells, which is one of the basic links of subjects or industries such as cell engineering, genetic engineering and the like, and techniques such as gene therapy, cell factories and the like are also vigorously developed.

Various methods are used to introduce foreign genes (or drugs and proteins) into cells. For the mode of introduction into prokaryotic cells, generally referred to as transformation, the mode of introduction into eukaryotic cells with viral vectors is referred to as transduction, and the technique of introduction into eukaryotic cells with non-viral vectors is referred to as transfection. Non-viral gene transfer can be roughly classified into chemical methods and physical methods. The principle of the chemical introduction method is that a complex is formed by a polymer, liposome microbubbles, cations and other substances and nucleic acid with negative charges, the complex is combined on the surface of a cell membrane and enters the cell through endocytosis or membrane fusion. The physical introduction method is mainly to temporarily destroy the cell membrane by mechanical force or thermal effect to introduce the target gene into the eukaryotic cell, and mainly includes microinjection, particle gun method, laser perforation, electrotransfection, ultrasonic transfection and the like. The virus transfer method has high efficiency, but can express exogenous genes only by integrating the virus into chromosomes of transfected cells, has the risk of carcinogenesis and teratogenesis, has poor safety, small system, can only introduce DNA, RNA and other nucleic acid molecules, and has longer design and test period. The liposome method is more mature in commercialization in chemical methods, but has the defects of high cost, high cytotoxicity, easy generation of inflammatory reaction and the like because only nucleic acid molecules can be introduced. The electrotransfection method in the physical transfection method is more popular in vitro experiments, but the further development of the electrotransfection method is limited by the problems of low cell survival rate, complicated pretreatment, difficult expansion of a transfection system and the like.

The ultrasonic transfection method has the advantages of simple operation, high safety, small damage to cells, low cost, easy expansion of a reaction system and the like, can be used for introducing various molecules such as nucleic acid, medicaments, proteins and the like into receptor cells, and has wide application prospect. The main principle of ultrasonic transfection is sonoporation, which refers to the process of creating reversible holes in the cell membrane by using the cavitation effect of ultrasound. The cavitation effect of the ultrasonic waves refers to the vibration, growth and collapse of micro-gas nuclei in the liquid caused by the ultrasonic waves reaching the intensity threshold. This process is usually very rapid and violent, with the instantaneous local high temperature and pressure and microjets generated by the collapse of the bubbles. These effects can temporarily destroy cell membrane and generate several tens to one hundred nm pores, so that exogenous gene can enter target cell to reach the aim of gene introduction. In addition, the combined use of ultrasonic transfection and an ultrasonic contrast agent can achieve higher transfection efficiency. The addition of the ultrasound contrast agent provides a large number of cavitation nuclei, which can significantly enhance the cavitation effect, and the bubble size of a few microns is also very suitable for the ultrasound transfection experiment of megahertz ultrasound.

However, the conventional ultrasonic transfection instrument mainly adopts a handheld ultrasonic probe, and when the ultrasonic probe is directly immersed in a cell culture medium, cell pollution, metal ion pollution and corrosion of the ultrasonic probe are easily caused. The spatial position of the non-contact ultrasonic probe and the cell to be transfected can not be relatively fixed, and the ultrasonic parameters of the position of the cell can not be fixed. In the near field area of the ultrasonic transducer, the sound field parameters generated by the ultrasonic probe (ultrasonic transducer) change very violently with the space position, and in the sound field generated by the ultrasonic transducer at megahertz level, the peak negative pressure of the sound field in the space with the axial distance of several micrometers changes violently by more than 20 times of the amplitude between the minimum value and the maximum value, so that the ultrasonic transducer is not beneficial to providing stable experimental conditions. In addition, the environmental temperature of the whole transfection system is not stably ensured during the ultrasonic action, and the cells are not favorable for being in the optimal physiological activity and transfection state.

Aiming at the technical bottleneck existing in the transfection method and the equipment, the main object of the utility model is to research and develop a new ultrasonic perforation in vitro cell transfection special equipment aiming at the technical defect existing at present in the key step of transfection in cell engineering, and form a mature transfection operation flow with high transfection efficiency and low cost suitable for different cell systems.

SUMMERY OF THE UTILITY MODEL

In order to achieve the above purpose, the utility model is realized by the following technical scheme.

The utility model provides an ultrasonic perforation eukaryotic and prokaryotic cell in vitro transfection conversion equipment, include:

the orifice plate is provided with a plurality of accommodating holes; the accommodating hole is used for accommodating cell suspension, an ultrasonic contrast agent and a plasmid mixture;

a drive mechanism to drive movement of the orifice plate in at least one direction within a horizontal plane;

a water tank for containing deionized water; the bottom of the pore plate is immersed in deionized water;

a heating member for heating deionized water;

at least two ultrasonic transducers with different frequencies, which are arranged below the pore plate and the emitting ends of which are immersed in deionized water;

the driving mechanism drives the orifice plate to displace in a horizontal plane, so that the accommodating hole to be measured is positioned in a far field region right above the corresponding ultrasonic transducer to perform transfection or conversion.

Preferably, the ultrasonic transducers are selected from at least two of a first ultrasonic transducer having a frequency of (800 ± 80) kHz, a second ultrasonic transducer having a frequency of (1 ± 0.1) MHz, a third ultrasonic transducer having a frequency of (2 ± 0.2) MHz, and a fourth ultrasonic transducer having a frequency of (40 ± 4) kHz.

Preferably, the orifice plate is a six-orifice plate or a twelve-orifice plate; the vertical distance between the central part of the bottom surface of the accommodating hole and the upper surface of the ultrasonic transducer is 160-200 mm.

Preferably, the plurality of receiving holes are distributed in a plurality of rows and a plurality of columns.

Preferably, the driving mechanism comprises an X-direction module and a Y-direction module.

Preferably, the outer surface of the water tank is wrapped with an insulating layer.

Preferably, the heating element is a resistance heating pipe which is positioned in a lower accommodating hole in the water tank.

Preferably, the temperature sensor is further included to acquire temperature information of the bottom of the accommodating hole.

Preferably, the water purifier also comprises a shell and a drain pipe; the shell comprises a box body and a box cover which are hinged with each other; one end of the drain pipe is communicated with the inside of the water tank, and the other end of the drain pipe extends out of the shell.

Preferably, the method further comprises the following steps:

a control panel disposed at one side of the housing;

and the adjusting foot pads are arranged at the bottom of the shell.

Compared with the prior art, the beneficial effects of the utility model reside in that:

the utility model provides an external transfection conversion equipment of supersound perforation eukaryotic and prokaryotic cell, the accommodation hole is located the far-field region of ultrasonic transducer's sound field, and during the transfection, the sound intensity of ultrasonic transducer sound field is less along with spatial position's change, and sound field spatial characteristic is stable, does benefit to the supersound cavitation intensity and the supersound effect of perforating of guaranteeing relatively stable, and the supersound perforation of cell membrane repairability makes cell transfection efficient, and cell survival rate is high. In addition, ultrasonic transducer's transmitting end is put in the deionized water, and ultrasonic transducer produces the resonance to in spreading the mixed liquid in the accommodation hole with the ultrasonic wave through the deionized water, under the effect of ultrasonic contrast agent, thereby the cell takes place the supersound perforation effect and realizes the transfection, and this scheme can avoid ultrasonic transducer direct and the culture medium contact in the cell suspension and bring the pollution risk, and ultrasonic transducer is convenient for wash and the disinfection.

The above description is only an overview of the technical solution of the present invention, and in order to make the technical means of the present invention clearer and can be implemented according to the content of the specification, the following detailed description is made with reference to the preferred embodiments of the present invention and accompanying drawings. The following examples and the accompanying drawings illustrate specific embodiments of the present invention.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention. In the drawings:

fig. 1 is a first schematic view of a three-dimensional structure of a device body when a box cover is in an open state;

fig. 2 is an exploded view of a partial structure of the device body of the present invention;

fig. 3 is a schematic view of a partial three-dimensional structure of the device body of the present invention;

fig. 4 is a schematic view of a three-dimensional structure of the device body when the box cover is in an open state;

fig. 5 is a sound field intensity distribution diagram of the 1MHz ultrasonic transducer in an embodiment of the present invention.

In the figure: 100. a device body;

10. an orifice plate; 11. an accommodation hole;

20. a drive mechanism; 21. an X-direction module; 211. a second slider; 212. an X-direction guide rail; 22. a Y-direction module; 221. a first slider; 222. a Y-direction guide rail; 23. a support; 231. a support table; 232. a connecting plate; 24. a guide assembly; 241. a linear guide rail; 242. a third slider;

31. a water tank; 311. a first abdicating hole; 312. a first through hole; 32. a heating element; 33. a heat-insulating layer; 331. a second abdicating hole; 34. a drain pipe;

41. a first ultrasonic transducer; 42. a second ultrasonic transducer; 43. a third ultrasonic transducer;

50. a housing; 51. a box body; 52. a box cover;

60. a control panel;

70. adjusting the foot pad;

80. a base plate; 81. a first base; 82. a second base; 83. a mounting base;

91. a displacement drive circuit; 92. a circuit board.

Detailed Description

The foregoing and other objects, features, aspects and advantages of the present invention will become more apparent to those skilled in the art from the following detailed description, which, taken in conjunction with the annexed drawings, discloses a preferred embodiment of the present invention. In the drawings, the shape and size may be exaggerated for clarity, and the same reference numerals will be used throughout the drawings to designate the same or similar components. In the following description, terms such as center, thickness, height, length, front, back, rear, left, right, top, bottom, upper, lower, and the like 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 convenience of description and are not generally intended to require a particular orientation. Terms concerning attachments, coupling and the like (e.g., "connected" and "attached") refer to a relationship wherein structures are secured or attached to one another either directly or indirectly through intervening structures, as well as both movable or rigid attachments or relationships, unless expressly described otherwise.

The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that the embodiments or technical features described below can be arbitrarily combined to form a new embodiment without conflict.

Example 1

The utility model provides an ultrasonic perforation eukaryotic and prokaryotic cell external transfection conversion equipment, as shown in figure 1, figure 2, including device body 100, device body 100 includes:

the orifice plate 10 is provided with a plurality of accommodating holes 11; the accommodating hole 11 is used for accommodating a mixture of cell suspension, an ultrasonic contrast agent and plasmids;

a driving mechanism 20 for driving the movement of the orifice plate 10 in at least one direction in a horizontal plane;

a water tank 31 for containing deionized water; the bottom of the pore plate 10 is immersed in deionized water;

the heating element 32 is used for heating deionized water, and further regulating and controlling the temperature of the bottom of the pore plate 10 to provide a proper temperature for cells, so that the cell state is good, and the cell transfection efficiency is improved;

at least two ultrasonic transducers with different frequencies, which are arranged below the pore plate 10 and the emitting ends of which are immersed in deionized water;

wherein, the driving mechanism 20 drives the orifice plate 10 to displace in the horizontal plane, so that the receiving hole 11 to be measured is located in the far field region right above the corresponding ultrasonic transducer for transfection or transformation.

In this embodiment, the device body 100 is provided with a well plate 10 for containing different mixtures to be transfected. Before transfection, the cells are simply digested and resuspended, and the cell suspension, the ultrasonic contrast agent and the plasmids are added into the corresponding containing holes 11, so that the ultrasonic perforation in-vitro transfection process can be automatically completed in the device body 100. During transfection, the accommodating hole is positioned in a far field area of a sound field of the ultrasonic transducer, the sound intensity of the sound field changes little along with the space position, the space characteristic of the sound field is stable, relatively stable ultrasonic cavitation intensity and ultrasonic perforation effect can be guaranteed, and cell transfection efficiency and cell survival rate are high due to ultrasonic perforation of cell membrane repairability. In addition, the transmitting end of the ultrasonic transducer is arranged in the deionized water, the ultrasonic transducer generates resonance and transmits ultrasonic waves to mixed liquid in the accommodating hole 11 through the deionized water, cells generate an ultrasonic perforation effect under the action of an ultrasonic contrast agent so as to realize transfection, the scheme can avoid pollution risk caused by direct contact of the ultrasonic transducer with a culture medium in a cell suspension, and the ultrasonic transducer is convenient to clean and disinfect. The device body 100 is provided with a first drive circuit to obtain an average stable ultrasonic intensity in a far field region thereof with different ultrasonic transducers.

In particular, the preparation of the mixture to be transfected comprises the steps of:

(1) Culturing HeLa cells to 60% -70% adherence by using a carbon dioxide incubator at 37 ℃ and under the atmosphere of 5% carbon dioxide by using a pore plate 10, wherein the culture medium is a DMEM culture medium (Gibco company) containing 1% of double antibody (Gibco company) and 10% of fetal bovine serum (Gibco company); wherein the orifice plate 10 is a six-orifice plate;

(2) After washing the cells with PBS buffer for 2 times, the cells were digested with pancreatin-EDTA for 1min, the adherent cells were gently blown down by adding the medium, and the resulting cell suspension was centrifuged at 1000rpm for 5min at room temperature. The centrifugation waste liquid is aspirated, 2mL of culture medium is added to gently blow and beat the cells to resuspend the cells, and the cells are added into the pore plate 10;

(3) An ultrasound contrast agent (SonoVue, bracco Co.) was prepared with 5mL of PBS buffer, pBOBi-EGFP enhanced green fluorescent plasmid was added to a final concentration of 20. Mu.g/mL in an ultrasound contrast agent well plate 10 obtained at a concentration of 100. Mu.L/well, and the well plate 10 was gently shaken to mix well to obtain a mixture to be transfected, to prepare for transfection.

In one embodiment, as shown in fig. 1 and fig. 2, the ultrasonic transducers are selected from at least two of 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, so as to match transfection operations with different frequency requirements. Wherein, the first ultrasonic transducer 41 with the frequency of (800 +/-80) kHz, the second ultrasonic transducer 42 with the frequency of (1 +/-0.1) MHz and the third ultrasonic transducer 43 with the frequency of (2 +/-0.2) MHz are suitable for the transfection of eukaryotic cells, and the fourth ultrasonic transducer with the frequency of (40 +/-4) kHz is suitable for the transformation of prokaryotic cells.

Preferably, the frequency of the first ultrasonic transducer 41 is 800kHz, the frequency of the second ultrasonic transducer 42 is 1MHz, and the frequency of the third ultrasonic transducer 43 is 2MHz. Specifically, the first drive circuit drives the three ultrasonic transducers to obtain average stable ultrasonic intensity in the far field region, the irradiation time is 10s-3min, and the ultrasonic intensity is 0-4W/cm ² (space mean time peak value), the high-frequency signal can obtain 5% -100% duty ratio after pulse width modulation, meet different germ line cells, different cell states and experimental conditions such as density.

Further, the orifice plate 10 is a six-orifice plate or a twelve-orifice plate; the vertical distance between the central part of the bottom surface of the accommodating hole 11 and the upper surface of the ultrasonic transducer is 160mm-200mm. Specifically, the size of the orifice plate 10 and the vertical distance between the orifice plate 10 and the ultrasonic transducer are designed to match the far field regions of the three ultrasonic transducers, so that the mixture to be transfected in the accommodating hole 11 can be displaced into the far field region of the ultrasonic transducer under the driving of the driving mechanism 20, the ultrasonic cavitation intensity and the ultrasonic perforation effect are stabilized, and the cell transfection efficiency is improved. Preferably, the orifice plate 10 is a six-orifice plate, and the vertical distance between the central part of the bottom surface of the receiving hole 11 and the upper surface of the ultrasonic transducer is 200mm, so that the six-orifice plate is always positioned in the far field region of the ultrasonic transducer; each receiving hole 11 on the aperture plate 10 can be adjusted to a far field region directly above the corresponding ultrasonic transducer by driving of the driving mechanism 20.

In one embodiment, the receiving holes 11 are distributed in multiple rows and multiple columns, and the arrangement is compact.

Further, as shown in fig. 2, the driving mechanism 20 includes an X-direction module 21, a Y-direction module 22; the X-direction module 21 is configured to drive the orifice plate 10 to move along the X direction, and the Y-direction module 22 is configured to drive the orifice plate 10 to move along the Y direction, so as to implement two-dimensional movement, so as to adjust the orifice plate 10 to be located at different rows or different columns of the receiving holes 11 to be located at a transfection area, where the transfection area represents a far field area directly above a corresponding ultrasonic transducer.

In one embodiment, as shown in fig. 1-3, the orifice plate 10 is connected to the driving end of the Y-direction module 22 by a bracket 23, and the Y-direction module 22 is fixed to the driving end of the X-direction module 21.

Further, the bracket 23 is made of 6061 aluminum, and is light and has certain strength.

Furthermore, a support base 231 and a connection plate 232 are respectively formed by bending two ends of the bracket 23, the orifice plate 10 is mounted on the upper surface of the support base 231, and the connection plate 232 is fixed at the driving end of the Y-direction module 22.

Further, as shown in fig. 2 and 3, the Y-direction module 22 is provided with a first slider 221 and a Y-direction rail 222, the first slider 221 is connected to the driving end of the Y-direction module 22 and the support 23, respectively, the driving end of the Y-direction module 22 drives the first slider 221 to drive the support 23 to move along the Y-direction, and further drives the orifice plate 10 to move along the Y-direction. The first sliding block 221 and the Y-direction guide rail 222 are matched to play a role in guiding and supporting, so that the Y-direction displacement precision and stability are improved, and the overturning moment is reduced.

Further, as shown in fig. 2 and 3, the X-direction module 21 is provided with a second sliding block 211 and an X-direction guide rail 212, which are matched with each other, the second sliding block 211 is connected to the driving end of the X-direction module and the Y-direction module, respectively, and the driving end of the X-direction module drives the second sliding block 211 to drive the Y-direction module to move along the X direction, so as to drive the orifice plate 10 to move along the X direction. The second sliding block 211 is matched with the X-direction guide rail 212 to play a role in guiding and supporting, so that the X-direction displacement precision and stability are improved, and the overturning moment is reduced.

In one embodiment, the X-direction module 21 and/or the Y-direction module 22 is a mechanism formed by a stepping motor and a screw nut pair, and the stepping motor drives the screw to rotate so as to move the nut along the screw. As shown in fig. 2, the apparatus body 100 is provided with a displacement driving circuit 91 for driving the stepping motors of the X-direction module 21 and the Y-direction module 22.

In one embodiment, as shown in fig. 2 and 3, the bottom of one end of the Y-direction module 22 is connected to the driving end of the X-direction module 21, and the bottom of the other end is supported by the guiding component 24; the guide assembly 24 includes a linear guide 241 and a third slider 242, which are matched with each other, so as to improve the precision and stability of the Y-direction displacement.

In one embodiment, as shown in fig. 2 and 3, the apparatus body 100 further includes a bottom plate 80 for forming a support structure, such as supporting the driving mechanism 20 and the water tank 31.

Further, the bottom plate 80 is a steel member, and has high structural strength.

In one embodiment, as shown in fig. 2 and 3, the bottom of the X-direction module 21 is fixed to the bottom plate 80 by a first base 81.

In one embodiment, as shown in fig. 2 and 3, the bottom of the guide assembly 24 is secured to the base plate 80 by a second base 82.

In an embodiment, as shown in fig. 1 and fig. 2, the bottom plate 80 is provided with a plurality of threaded mounting holes, the bottom of the water tank 31 is provided with a plurality of first abdicating holes 311, and the insulating layer 33 is provided with a plurality of second abdicating holes 331; the bottom of the ultrasonic transducer is screwed in the threaded mounting hole, and the upper part of the ultrasonic transducer penetrates through the corresponding second abdicating hole 331 and the corresponding first abdicating hole 311 in sequence and then is immersed in the deionized water in the water tank 31.

In one embodiment, as shown in fig. 1 to 3, the water tank 31 is wrapped with an insulation layer 33 on the outer surface thereof for reducing heat transfer, which is beneficial to maintain the temperature of the reaction system constant.

In one embodiment, the water tank 31 is 304 stainless steel.

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

In one embodiment, the heating element 32 is a resistance heating tube, which is located at the lower portion of the water tank 31 for regulating the temperature of the bottom of the accommodating hole 11. Preferably, the resistance heating tube is used for enabling the temperature at the bottom of the accommodating hole 11 to be (37 +/-0.5) DEG C, and the temperature range is favorable for cell state, so that recovery after cell membrane perforation is facilitated, and transfection efficiency and cell survival rate are improved.

In an embodiment, a temperature sensor is further included to obtain information about the temperature at the bottom of the accommodating hole 11. Specifically, the device body 100 is provided with a temperature feedback circuit, collects feedback signals of the temperature sensor, and controls the power of the heating element 32 to keep the temperature of the bottom of the orifice plate frame constant at a target temperature. Further, as shown in fig. 2, the temperature feedback circuit and the first driving circuit for driving the ultrasonic transducer are integrated on the circuit board 92.

Further, the temperature sensor is fixed on the bracket 23, which is convenient for installation.

Further, as shown in fig. 2, the displacement drive circuit 91 and the circuit board 92 are disposed on the base plate 80.

In one embodiment, as shown in fig. 1 and 4, the water draining device further comprises a shell 50 and a water draining pipe 34; the shell 50 comprises a box body 51 and a box cover 52 which are hinged with each other, the box body 51 and the box cover 52 form a closed cavity together, and the orifice plate 10, the driving mechanism 20, the water tank 31, the heating element 32 and the ultrasonic transducer are arranged in the cavity; one end of the drain pipe 34 is communicated with the inside of the water tank 31, and the other end extends out of the housing 50, so that the deionized water in the water tank 31 can be drained through the drain pipe 34 to match the replacement of the deionized water. The water tank 31, the heating element 32, the insulating layer 33 and the drain pipe 34 form a water bath structure together to provide a temperature environment for good cell transfection. The bottom plate 80 is mounted on the bottom wall in the case body 51 to enhance the supporting force.

Further, the box cover 52 is of a polyethylene structure.

Further, the drain pipe 34 is fixed to the bottom plate 80.

Further, a power supply connector hole of the apparatus body 100 is provided on the back surface of the case 50.

In an embodiment, as shown in fig. 1 and 4, the method further includes:

a control panel 60 provided on one side of the housing 50; the control panel 60 is electrically connected to the displacement driving circuit 91 and the circuit board 92;

and a plurality of adjusting foot pads 70 arranged at the bottom of the housing 50 for leveling the housing 50.

In one embodiment, as shown in fig. 1 and 2, 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 shown in fig. 2 and 3, the bottom 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 heat insulation layer 33 is provided with two second through holes 332; the resistance heating pipe is located in the water tank 31, and two ends of the resistance heating pipe respectively penetrate through the corresponding second through hole 332 and the corresponding first through hole 312 in sequence and then are respectively fixed on the two mounting seats 83.

The operation of performing transfection using the device body 100 includes the steps of:

(1) Preparing a mixture to be transfected;

(2) Closing the drain pipe 34, opening the box cover 52, and injecting deionized water into the water tank 31 to reach a water level line; the device body 100 is connected with a power supply, the control panel 60 is operated, and preheating is carried out for 5min at the set temperature of 37 ℃; selecting an ultrasonic transducer with corresponding frequency, and enabling the ultrasonic transducer to work for 2min to be in a stable resonance state;

(3) Opening the lid 52, loading the well plate 10 containing the mixture to be transfected, and closing the lid 52;

(4) Operating the control panel 60 to perform ultrasonic transfection; if the ultrasonic irradiation time of each hole is selected to be 10s-3min, the ultrasonic intensity is 0-4W/cm < 2 > (space mean time peak value), and the duty ratio is 5-100%;

(5) After all the to-be-transfected mixtures in all the accommodating holes are irradiated, taking out the pore plate 10, placing the pore plate in a carbon dioxide incubator, culturing for 4 hours at 37 ℃ under the atmosphere of 5% carbon dioxide, and then replacing a fresh culture medium; after further incubation for 24h, transfection efficiency was observed under a fluorescent microscope.

As shown in fig. 5, the measured sound field intensity distribution diagram of the 1MHz ultrasonic transducer obtained at the far field (200 mm) under the driving voltage of 2Vpp driving voltage, 1MHz frequency, 1kHz pulse repetition frequency, and 50% duty ratio, the visible sound field intensity distribution is relatively uniform, and the variation with the spatial position is relatively small.

The utility model provides a pair of in vitro transfection conversion equipment of ultrasonic perforation eukaryotic and prokaryotic cell, device body 100 simple structure, the sound intensity of sound field is less along with spatial position's change, does benefit to the ultrasonic cavitation intensity and the ultrasonic perforation effect of guaranteeing relatively stable, and the ultrasonic perforation of cell membrane repairability makes cell infection efficiency high, and cell survival rate is high.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way; the utility model can be smoothly implemented by the ordinary technicians in the industry according to the drawings and the above description; however, those skilled in the art should understand that the equivalent embodiments of the present invention are equivalent embodiments of the present invention, and that the changes, modifications and evolutions made by the above-disclosed technical contents are not departed from the technical scope of the present invention; meanwhile, any changes, modifications, evolutions, etc. of the above embodiments, which are equivalent to the actual techniques of the present invention, still belong to the protection scope of the technical solution of the present invention.

Claims (10)

1. An ultrasonic perforation eukaryotic and prokaryotic cell in-vitro transfection and transformation device is characterized by comprising:

the orifice plate (10) is provided with a plurality of accommodating holes (11); the accommodating hole (11) is used for accommodating cell suspension, an ultrasonic contrast agent and a plasmid mixture;

a drive mechanism (20) for driving the movement of the orifice plate (10) in at least one direction in a horizontal plane;

a water tank (31) for containing deionized water; the bottom of the pore plate (10) is immersed in deionized water;

a heating member (32) for heating deionized water;

at least two ultrasonic transducers with different frequencies, which are arranged below the pore plate (10) and the emitting ends of which are immersed in deionized water;

the driving mechanism (20) drives the orifice plate (10) to displace in a horizontal plane, so that the accommodating hole (11) to be measured is positioned in a far field region right above the corresponding ultrasonic transducer to perform transfection or transformation.

2. The device for the in vitro transfection and transformation of eukaryotic and prokaryotic cells by ultrasonic perforation according to claim 1, wherein the ultrasonic transducers are selected from at least two of the first ultrasonic transducer (41) with frequency of (800 ± 80) kHz, the second ultrasonic transducer (42) with frequency of (1 ± 0.1) MHz, the third ultrasonic transducer (43) with frequency of (2 ± 0.2) MHz, and the fourth ultrasonic transducer with frequency of (40 ± 4) kHz.

3. The device for the in vitro transfection and transformation of eukaryotic and prokaryotic cells by ultrasonic perforation according to claim 2, characterized in that the well plate (10) is a six-well plate or a twelve-well plate; the vertical distance between the central part of the bottom surface of the accommodating hole (11) and the upper surface of the ultrasonic transducer is 160-200 mm.

4. The device for the in vitro transfection and transformation of eukaryotic and prokaryotic cells according to any one of claims 1 to 3, wherein a plurality of said receiving holes (11) are arranged in a plurality of rows and columns.

5. The device for the in vitro transfection and transformation of eukaryotic and prokaryotic cells by ultrasonic perforation according to claim 4, characterized in that said driving mechanism (20) comprises an X-direction module (21) and a Y-direction module (22).

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

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

8. The device for the in vitro transfection and transformation of eukaryotic and prokaryotic cells according to any one of claims 1 to 3, characterized by further comprising a temperature sensor for acquiring the temperature information at the bottom of the containing hole (11).

9. The device for the in vitro transfection and transformation of eukaryotic and prokaryotic cells by ultrasonic perforation according to any one of claims 1 to 3, characterized by further comprising a housing (50), a drain (34); the shell (50) comprises a box body (51) and a box cover (52) which are hinged with each other; one end of the drain pipe (34) is communicated with the inside of the water tank (31), and the other end of the drain pipe extends out of the shell (50).

10. The device of claim 9, further comprising:

a control panel (60) provided on one side of the housing (50);

a plurality of adjusting foot pads (70) arranged at the bottom of the shell (50).

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