CN110734855A

CN110734855A - electric shock tubes

Info

Publication number: CN110734855A
Application number: CN201810799258.0A
Authority: CN
Inventors: 戴晓兵; 朱士英; 高腾森
Original assignee: SUZHOU ETTA BIOTECH Co Ltd
Current assignee: SUZHOU ETTA BIOTECH Co Ltd
Priority date: 2018-07-20
Filing date: 2018-07-20
Publication date: 2020-01-31

Abstract

The invention provides electric shock tubes, which comprise a tube body (1), a tube cover (2) and a needle electrode array (3), wherein a cavity (5) for accommodating a target liquid sample is arranged in the tube body (1). The electric shock tube has the advantages that the direction of an electric field is vertical to the tube body, an effective electric field can be formed without filling liquid, no air bubble is formed in the cavity to cause poor electrode contact, and the influence of the poor electrode contact on the electrotransformation effect can be avoided.

Description

electric shock tubes

Technical Field

The invention belongs to the technical field of electrotransfection, and relates to electric shock tubes, in particular to electric shock tubes with needle electrode arrays.

Background

Since the 1970 s electroporation technology was used to introduce molecules into animal or plant cells, researchers have demonstrated that exposure of cells to brief, sustained, high voltage electric fields can create channels in the cell membrane through which macromolecules such as proteins and DNA can enter the cell.electroporation is a widely adopted and strongly recommended method for cell testing and gene therapy by .

The current apparatus for electrotransfection of cells is mainly an electric cuvette.

US20130052711a1 describes electric shock tubes, which are used to add liquid samples containing cells and substances to be injected into the cells into the tubes, and the upper and lower ends of the electric shock tubes are respectively provided with an upper electrode and a lower electrode, and the upper and lower electrodes are connected with a power supply of a cell electric rotating machine, so that an electric field is formed in the electric shock tubes, and the substances outside the cells enter the cells.

However, in use, the shock tube in the above patent has a high requirement on the manufacturing accuracy and the operation of experimenters is also high, if the manufacturing accuracy is not high enough, the poor sealing between the electrode and the upper edge of the cavity is easy to cause, the situation that the electrode is tilted up and then leaves the upper edge of the cavity again after the electrode is covered, so that the outside air enters the cavity again can occur, in addition, the residual air bubbles in the tube are easy to cause when the operator uses the device improperly, and the cell electrotransfection effect is affected, the reliability requirement of scientific research experiments on the instrument equipment is very high, and therefore, the shock tube needs to be designed by in the manufacturing process to improve the operation reliability of the experimenters.

Chinese patent CN201410722470 provides electric shock tubes and a cell electrotransfection instrument having the electric shock tubes, which belongs to the technical field of biomedical instruments and devices, the electric shock tube includes a tube body, a electrode, a second electrode and a stopper, a cavity for containing a target liquid sample is provided inside the tube body, the electrode is provided at end of the tube body, the second electrode is disposed in the tube cap, and the outer end of the second electrode can be electrically connected with the outside through an opening of the tube cap, an elastic member connected with the second electrode is provided in the tube cap, the outer side of the elastic member is connected with the tube cap, and the inner side of the elastic member is connected with the second electrode.

But the electric shock tube has the following technical problems when in use:

the throughput of each electrotransfection must be greater than the capacity of the chamber, resulting in waste, the cell fluid must overflow the chamber, and if the cell fluid is less than the volume of the chamber, the second electrode will not be in contact with the fluid and an electrical connection cannot be made.

Secondly, during electrotransfection, bubbles are formed in the cavity and cannot be removed in time, and the bubbles are attached to the second electrode to influence the electrotransfection effect

Thirdly, when the electric shock tube is used, high-voltage pulse treatment is required, so that electric arcs are easy to generate, and the safety of operators is affected.

Disclosure of Invention

In order to solve the technical problem, the invention provides electric shock tube devices with similar appearance structures, but high-density distributed stereoscopic electrodes are adopted to replace a th electrode and a second electrode.

The technical scheme includes that electric shock tubes comprise a tube body, a tube cover and a needle electrode array, wherein a cavity used for containing a target liquid sample is formed in the tube body, the electric shock tube is characterized in that the end of the tube body is sealed, the other end of the tube body is provided with an opening communicated with the cavity, the working part of the needle electrode array is arranged in the cavity, the edge of the opening is provided with an annular end face, the needle electrode array is arranged in the tube cover and can be electrically connected with the outside through an external opening of the tube cover, the inner end face of the tube cover can be fixedly connected with the edge of the opening of the tube body through the annular end face, and a printed circuit board is arranged outside the tube cover.

Preferably, the needle electrode array comprises a plurality of needle electrodes, each of the plurality of needle electrodes is applied with an electrical pulse of th polarity and an electrical pulse of second polarity according to a time cycle, wherein the electrodes corresponding to the electrical pulse of th polarity are distributed around the electrodes corresponding to the electrical pulse of second polarity, and the th polarity and the second polarity are opposite in polarity.

Preferably, the plurality of needle electrodes in the needle electrode array are arranged in a regular polygon, and the distances between two adjacent electrodes in the needle electrode array are equal.

Preferably, the needle electrode array is in the shape of a regular hexagon consisting of a plurality of equilateral triangles, and the electrodes are positioned on the vertexes of the equilateral triangles.

Preferably, the th polarity is a positive polarity and the second polarity is a negative polarity.

Preferably, the diameter of the electrodes is 0.01-1.2 mm, the distance between the central points of the two adjacent electrodes is 0.1-2.4 mm, the number of the electrodes is more than 19, and the material of the electrodes includes but is not limited to stainless steel.

Preferably, the length of the needle electrode array within the cavity is the same as the length of the cavity.

Preferably, the inner end surface of the pipe cover and the outer side of the opening edge of the pipe body, which is provided with an annular end surface, are respectively provided with a thread, and the pipe body is fixedly connected with the pipe cover through the thread.

Preferably, at least circles of grooves are formed in the outer side of the annular end face, corresponding protruding structures are arranged on the inner end face of the pipe cover, the protruding structures correspond to the grooves formed in the outer side of the annular end face, and fixed connection is formed.

Preferably, the pipe body is provided with a clamping hook, and the pipe cover is provided with a buckle which can be clamped with the clamping hook.

Preferably, the pipe cover is provided with a clamping hook, and the pipe body is provided with a buckle which can be clamped with the clamping hook.

Preferably, the inner diameter of the inner end surface of the opening of the pipe cover is smaller than the outer diameter of the annular end surface of the edge of the opening of the pipe body.

Preferably, the inner diameter of the inner end surface of the opening of the pipe cover is equal to the outer diameter of the annular end surface of the edge of the opening of the pipe body.

Preferably, the pipe cover is flexibly connected with the pipe body through a connecting rod.

Preferably, the tube body and the tube cover are both prepared by adopting a mode of molding bodies.

The invention has the beneficial technical effects that:

compared with the electric shock tube with a similar structure provided by CN201410722470, the electric shock tube has the advantages that the direction of an electric field is vertical to the tube body, an effective electric field can be formed without filling liquid, no bubbles are formed in the cavity, and the electric conversion effect can be prevented from being influenced by the fact that the electric connection between electrodes is lost or the electric connection contact is poor due to the fact that the liquid is not filled or the bubbles are not filled.

Drawings

FIG. 1 is a schematic view of the overall structure of the shock tube of the present invention;

FIG. 2 is a schematic view of the overall structure of the shock tube of the present invention;

the attached drawings are annotated: the electrode structure comprises a tube body 1, a tube cover 2, a pin electrode array 3, a printed circuit board 4, a cavity 5, a groove 6, a bulge structure 7, an external thread 8 and an internal thread 9.

Detailed description of the preferred embodiments

The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention easier to understand by those skilled in the art, and thus will clearly and clearly define the scope of the invention.

Example

As shown in figure 1, the electric shock tube provided by the invention comprises a tube body (1), a tube cover (2), a needle electrode array (3) and a printed circuit board (4).

The tube body (1) is internally provided with a cavity (5) for containing a target sample solution, the end of the tube body (1) is sealed, the other end of the tube body (1) is provided with an opening communicated with the cavity (5), the edge of the opening is provided with an annular end face, the tube cover (2) is made of a plastic material with fixed elasticity, the inner diameter of the tube cover (2) is smaller than or equal to the outer diameter of the annular end face of the opening edge of the tube body (1), circles of grooves (6) are formed in the outer side of the annular end face and used for fixing the tube cover (2), corresponding protruding structures (7) are arranged on the inner end face of the tube cover (2) and correspond to the grooves (6) formed in the outer side of the annular end face, when the tube cover (2) covers the tube body (1), the inner end face of the tube cover (2) can be slightly deformed, and is attached and sealed with the annular end face of.

The electrode structure comprises a printed circuit board (4), a plurality of electrode needles are welded on the printed circuit board (4) to form a needle electrode array (3), the printed circuit board (4) is located at an opening on the outer portion of a tube cover (2), the needle electrode array (3) penetrates through the tube cover (2), a pad is arranged on the top of the printed circuit board (4) and is electrically connected with electrical connection equipment, the needle electrode array (3) is in a regular hexagon formed by a plurality of equilateral triangles, the distance between the outer edges of the regular hexagon is equal to the inner diameter of the tube body (1), after the needle electrode array (3) is inserted into the tube body (1), the outer needle electrodes are attached to the inner wall of the tube body (1), the needle electrodes are located on the vertexes of the equilateral triangles, the diameter of the electrodes is 0.4 mm, the distance between the central points of two adjacent electrodes is 1.1 mm, the needle electrodes are divided into three groups, electrodes of which are the group of positive electrodes, the other two groups of negative electrodes are of negative electrodes, the periphery of each positive electrode.

When the electric shock tube is used, a mixed solution sample of cells and an electrotransfection substance is injected into a cavity, the tube cover (2) is fixed at the opening end of the tube body (1), the needle electrode array (3) is contacted with the bottom of the tube body (1), the mixed solution sample is uniformly distributed in the needle electrode array (3), and is connected with a pulse power supply through a pad at the top of the printed circuit board (4), an electric field is formed in the needle electrode array (3), and micropores are generated on the surface of a cell membrane by utilizing an electroporation phenomenon, so that a target substance in the mixed solution sample enters the cells through the micropores.

Example two

As shown in fig. 2, a groove (6) is formed on the outer side of an annular end surface in the shock tube structure of embodiment , and a corresponding protrusion structure (7) is formed on the inner end surface of the tube cap (2) and replaced by a thread structure, specifically, an external thread (8) is formed on the outer side of the annular end surface at the edge of the opening of the tube body (1), an internal thread (9) is formed on the inner end surface of the tube cap (2), and the tube cap (2) can be fixedly connected with the external thread (8) on the tube body (1) through the internal thread (9).

EXAMPLE III

On the basis of the shock tube structure of embodiment , set up the pothook on body (1), tube cap (2) sets up the buckle that can with the pothook looks joint.

Example four

On the basis of the electric shock tube structure of the second embodiment, the tube cover (2) is provided with the clamping hook, and the tube body (1) is provided with the buckle which can be clamped with the clamping hook.

EXAMPLE five

The electric shock tube in the second embodiment of the invention is connected to a host machine of an electrotransfection instrument, the electrotransfection condition voltage is set to be 270 volts, the pulse width is 100 microseconds, the pulse times are 6 times, and the pulse interval is 1 second.

According to the description of the electric shock tube of CN201410722470.9, the electric shock tube with a similar structure is made by self and is used as a comparison experiment, and is connected to a Celetrix electrotransformation host machine, and the HEK293F electrotransformation condition is selected.

The experimental procedure was as follows: HEK293F cells (human embryonic kidney cells) were collected in logarithmic growth phase at 1000 rpm, centrifuged for 5 min and the supernatant discarded. Resuspend the cells with electrotransfection buffer to a density of 2X 10⁷Adding the plasmid pcDNA3.1-GFP which needs to be transfected into cells to ensure that the concentration of the plasmid is 20 micrograms/ml, and gently blowing, beating and uniformly mixing. The prepared cell plasmid electrotransformation buffer liquid suspension is respectively added into an electric shock tube of the second embodiment of the invention and an electric shock tube of CN201410722470.9, and cell electrotransfection treatment is carried out by matching with an electrotransfection instrument.

After the end of the electrotransfection, the transfected cell suspension was placed in a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. Discarding supernatant, adding 293FreeStyle culture medium to resuspend cells, inoculating, and culturing in a triangular conical flask at a culture density of 2 × 10⁶And culturing the cells/ml on a shaking table at the rotating speed of 125 revolutions per minute under the culture conditions: the temperature is 37 ℃ and the carbon dioxide concentration is 5%. Electrotransfection efficiency was measured after 24 hours on a flow cytometer.

Through observation of the electrotransfection process, in the electroporation cuvette provided by the second embodiment of the invention, fewer bubbles are generated near the electrode in the electrotransfection process, and the surface of the electrode is not changed after the electrotransfection.

By comparing the electrotransfection results, according to the description of the electric shock tube of CN201410722470.9, the electrotransfection efficiency of the cells obtained by self-making the electric shock tube with a similar structure is about 67-73%; the electrotransfection efficiency of the cells obtained by using the electric shock tube of the second embodiment of the invention is about 83-85%.

While the invention has been described in detail, those skilled in the art will recognize that aspects of the invention and portions of the various embodiments and features described above and/or in the appended claims can be combined or interchanged either in whole or in part.

Claims

The electric shock tube comprises a tube body (1), a tube cover (2) and a needle electrode array (3), wherein a cavity (5) used for containing a target liquid sample is formed inside the tube body (1), the tube body is characterized in that the end of the tube body (1) is sealed, an opening communicated with the cavity (5) is formed in the other end of the tube body (1), the working part of the needle electrode array (3) is arranged in the cavity (5), the edge of the opening is provided with an annular end face, the needle electrode array (3) is arranged in the tube cover (2) and can be electrically connected with the outside through an external opening of the tube cover (2), and the inner end face of the tube cover (2) can be fixedly connected with the annular end face on the edge of the opening of the tube body (1).
2. The shock tube according to claim 1, characterized in that a printed circuit board (4) is provided on the outside of the tube cover (2).
3. The shock tube according to claim 2, wherein the inner end surface of the tube cap (2) and the outer side of the end surface of the tube body (1) with the annular opening are respectively provided with threads, and the tube body (1) is fixedly connected with the tube cap (2) through the threads.
4. The shock tube according to claim 2, characterized in that the outer side of the annular end surface is provided with at least circles of grooves (6), the inner end surface of the tube cover (2) is provided with corresponding protruding structures (7), and the protruding structures (7) correspond to the grooves (6) arranged on the outer side of the annular end surface and form a fixed connection.
5. The shock tube according to of any one of claims 1-4, wherein the tube body (1) is provided with a hook, and the tube cover (2) is provided with a buckle capable of being clamped with the hook, or the tube cover (2) is provided with a hook, and the tube body (1) is provided with a buckle capable of being clamped with the hook.
6. The shock tube according to claim 5, characterized in that the tube cover (2) is flexibly connected to the tube body (1) by a connecting rod.
7. The shock tube according to claim 3 or 4, characterized in that the inner end surface inner diameter of the tube cover (2) is equal to the end surface outer diameter of the opening edge ring of the tube body (1); or the inner diameter of the inner end surface of the pipe cover (2) is smaller than the outer diameter of the annular end surface of the opening edge of the pipe body (1).
8. The shock tube of claim 5, wherein the needle electrode array (3) comprises a plurality of needle electrodes, each of the plurality of needle electrodes is applied with an electrical pulse of th polarity and an electrical pulse of second polarity according to a time period, wherein the electrodes corresponding to the electrical pulse of th polarity are distributed around the electrodes corresponding to the electrical pulse of second polarity, and the th polarity and the second polarity are opposite.
9. The shock tube of claim 8, wherein the length of the needle electrode array (3) within the cavity (5) is the same as the length of the cavity (5).
10. The shock tube of any one of claims 1-9, wherein the tube cover (2) is made of a deformable organic polymer material, and the deformable organic polymer material is plastic, rubber or plastic.