US20160137966A1

US20160137966A1 - A High Density Distributed Three-dimensional Electrode Device

Info

Publication number: US20160137966A1
Application number: US14/896,902
Authority: US
Inventors: Edward Dai
Original assignee: Etta Biotech Co Ltd
Current assignee: Etta Biotech Co Ltd
Priority date: 2013-06-08
Filing date: 2014-06-06
Publication date: 2016-05-19
Also published as: CN103275874A; US20190136175A1; EP3006552A4; US11421196B2; WO2014194859A1; CN103275874B; EP3006552A1; EP3006552B1

Abstract

A high-density distributed three-dimensional electrode device, comprising an electrode array and an electrode fixing assembly on which the electrode array is fixed, the electrode array comprising a plurality of electrodes respectively applied with an electric pulse of a first polarity and an electric pulse of a second polarity according to a time period, wherein the electrodes corresponding to the electric pulse of the second polarity are distributed around the electrodes corresponding to the electric pulse of the first polarity.

Description

TECHNICAL FIELD

The present invention relates to a cell electroporation technique, in particular a three-dimensional electrode device for electroporation.

BACKGROUND

Since 1970s, the electroporation technique was used to insert molecules into animal cells or plant cells. It is proved by researchers that exposing a cell to a short-lasted high-voltage electric field may enable formation of pathways through the cell membrane, and macromolecules such as proteins and DNAs may enter into the cell through those pathways. Those pathways are reffered as electric pores which are permeability increased zone caused by a local fracture of cell membrance resulted from high voltage electric field. Although the existing times of the pores are brief, it is enough to satisfy the requirement of the macromolecules such as plasmid DNA molecules entering into the cell. The cell may tolerate the formation of the pores, however, the cell may be killed by the processes of the formation and the molecules introduced thereby if the formed pores are too much and overlarge.
At the earliest, the electroporation is carried out by using the simplest capacitor with parallel-plate, and a substantially homogeneous electric field may be formed between the electrodes opposite to each other. The cell suspension prepared for electroporation and the molecules which the operator wants to introduce are mixed and placed between the two electrodes, and a short-time high voltage electric field pulse is applied to the electrodes by one or more times such that the result of introducing the molecules into cells by electroporation can be achieved. However, the distance between the parallel-plate electrodes is large, the required voltage is usually up to several thousands volts, thus generating of cathode effect is inevasible, which has a huge damage to the cells.
Although the planar electrodes arose later solve the negative effect brought by the overhigh voltage, they are not suitable for high throughput experiment operations due to that the planer electrodes can process a very small amount of cells every time. Three-dimensional electrodes easily penetrate into tissues and living bodies, and usually use for electroporations in clinic for tumour tissues or living tissues, the electroporation efficiency of which is not high, and there is no related report of extracorporeal cell electroporation such as electroporation aimed at suspended cells or attached cells via three-dimensional electrodes.

SUMMARY

The present invention provides a high-density distributed three-dimensional electrode device, which has a simple structure and is easy to manufacture.
To solve the above-mentioned technical problems, the present invention provides a high-density distributed three-dimensional electrode device, comprising an electrode array and an electrode fixing assembly on which the electrode array is fixed, the electrode array comprising a plurality of electrodes divided into at least two groups, an electric pulse of a first polarity and an electric pulse of a second polarity are respectively applied on the at least two groups according to a time period, wherein the first polarity and the second polarity are different, and the electrodes corresponding to the electric pulse of the second polarity are distributed around the electrodes corresponding to the electric pulse of the first polarity.
In a preferred embodiment of the present invention, the plurality of electrodes in the electrode array is arranged according to an equilateral polygon, and the distances between every two adjacent electrodes in the electrode array are equal.
In a preferred embodiment of the present invention, a shape of the electrode array is an equilateral hexagon formed by several equilateral triangles, and the electrodes are located at the vertexes of the equilateral triangles.
In a preferred embodiment of the present invention, the first polarity is positive polarity and the second polarity is negative polarity.
In a preferred embodiment of the present invention, the first polarity is negative polarity, and the second polarity is positive polarity.
In a preferred embodiment of the present invention, the diameters of the electrodes are 0.01-1.2 mm, the distance between the center points of the two adjacent electrodes is 0.1-2.4 mm, the number of the electrodes is more than 5, and the number is preferred to be more than 19. The material of the electrodes is preferably stainless steel.
In a more preferred embodiment of the present invention, the diameters of the electrodes are 0.1-0.4 mm, the distance between the center points of the two adjacent electrodes is 0.2-1.5 mm, and the number of the electrodes is more than 36. The diameters of the electrodes are preferably 0.3 mm, the distance between the center points of the two adjacent electrodes is preferably 1 mm, and the number of the electrodes is preferably 37.
In a preferred embodiment of the present invention, the electrode fixing assembly comprises an electrode connecting circuit board and an electrode positioning board, the electrode connecting circuit board connecting the electrodes applied with the electric pulse of the same polarity together via a line thereon, and the electrodes being inserted into the electrode positioning board.
In a preferred embodiment of the present invention, the electrodes in the electrode array are divided into several groups, and the electrodes in the same group are only applied with the electric pulse of the same polarity, wherein one of the groups is applied with the electric pulse as a positive electrode, the rest groups are applied with the electric pulse as a negative electrode, and then another one of the groups is applied with the electric pulse as the positive electrode, the rest groups are applied with the electric pulse as the negative electrode, and alternately in this way, the obtained electric field may achieve a homogeneous electric field by superposition.
The beneficial effects of the present invention are that: the high density distributed three-dimentional electrode device of the present invention employs grouped reused electrodes; can compensate for the unevenness of the electric field caused by the three-dimentional electrode at the greatest extent; can process milliliter level of cells once; can be used in both pore plate devices and flow devices; requires a small electroporation voltage due to a small distance between the electrodes, avoiding the damage to the cells from the high voltage; low cost; is a cell electroporation device with high throughput and high efficiency.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a structure schematic diagram of a preferable embodiment of a high-density distributed three-dimentional electrode device according to the present invention.

FIG. 2 is a front view of the high-density distributed three-dimentional electrode device shown in FIG. 1.

FIG. 3 is a top view of the high-density distributed three-dimentional electrode device shown in FIG. 1.

FIG. 4 is a topological structure diagram of an electrode array in the high-density distributed three-dimentional electrode device shown in FIG. 1.

Wherein reference numbers and corresponding parts are as follow: 1.electrode array, 2.electrode positioning plate, 3.electrode connecting circuit board.

DETAIL DESCRIPTION OF EMBODIMENTS

In the following, the preferable embodiments of the present invention are explained in detail combining with the accompanying drawings so that the advantages and features of the present invention can be easily understood by the skilled persons in the art, and thus it is clear to define the protective scope of the present invention.

Embodiment 1

Referring to FIG. 1, FIG. 2, FIG. 3 and FIG. 4, the present invention provides a high-density distributed electrode device, comprising an electrode array 1 and an electrode fixing assembly on which the electrode array 1 is fixed.
The electrode array 1 is formed by 37 solid cylinder electrodes, and the arrangement rules are as follow: all the electrodes are arranged to be an equilateral hexagon shaped structure, the distances between two adjacent electrodes are equal, the inside of the equilateral hexagon shape is divided into several small equilateral triangle units by taking the distances between two adjacent electrodes as side length, and one electrode is placed at each vertex of the equilateral triangle units, that is, all the electrodes are divided into three groups, Group I, II and III, and vertexes of each equilateral triangle unit respectively belong to Group I, II and III.
The inner diameter of the equilateral hexagon of the electrode array 1 is matched up with the plate holes of a perforated plate, and a distance between the center points of two adjacent electrodes is 1 mm. The distance between two electrodes may affect the voltage during electroporation, and can be adjusted as required. The electrodes are inserted into the perforated plate and have a distance of 0.1 mm-1 mm from the bottom of the perforated plate.
The diameter of the electrodes is 0.3 mm, and both too large and too small diameters of the electrodes may affect the effect of the electroporation. When the diameters are too large, the effective area of electric field may be reduced, resulting in a decrease on the number of cells dealed with by electroporation, and going against highthroughput of cell electroporation. When the diameters are too small, the electrodes are easily bended resulting in a large increase of the manufacturing cost.
The material of the electrodes may be optionally selected from electric conductive metals and other electric conductive materials, wherein the stainless steel is an excellent material for electrodes. The stain steel material possesses favorable bio-compatibility, is easy to clean and not easy to be oxidized, easy to form relatively long electrodes, and is able to be mass produced and be reused for multiple times without affecting the conduction properties thereof.
The electrode fixing assembly comprises an electrode positioning board 2 and an electrode connecting circuit board 3, the electrodes are positioned by going through the electrode positioning board 2 and connected to the electrode connecting circuit board 3, and the electrode connecting circuit board 3 connects the electrodes applied with the electric pulse of the same polarity together via a line thereon. Due to that the electrodes are relatively long and the electroporation has a very high accuracy requirement for the distance between the electrodes, the electrode positioning board 2 is employed to position the electrodes in electroporation experiments. The electrode positioning board 2 can confine and position the electrodes along a long distance due to a thickness thereof is about 1 cm, and therefore, the electrodes can be controlled accurately to reach the bottom of the perforated plate. A support structure of the high density distributed three-dimentional electrode device may be easily extended to form electrode networks of any combination array such as 2*2, 1*4, 12*8 etc., and such flexible and varied conbinations may be compatible with the perforated plate structure at the greatest extent, being conveniet for user.
The electrode connecting circuit board 3 connects the electrodes in the same group together by welding connection, conducting adhesive or other electrical connecting manner such as printed circuit board and anyother componet which can connect regulation lines. The electrode positioning board 2 is used for positioning the electrodes which are vimineous and easily bended. Therefore, in the embodiments of FIGS. 1-2, the electrode positioning board 2 is arranged approximately at the middle of the electrodes for positioning. The use of the electrode positioning board 2 can reduce the inconformity of the distances between the electrodes and thus improve the homogeneity of the electric field.
The high-density distributed three-dimensional electrode device may be a monoporate device, and 4, 96 or more of this device can be used to form a group to cooperate with a perforated plate structure commonly used in biology.
The electroporation method of the high-density distributed three-dimensional electrode device in the present embodiment is: during the electroporation, firstly by taking Group I as a positive electrode and Group II, III as a negative electrode the electric pulse is applied, then by taking Group II as a positive electrode and Group I, III as a negative electrode the electric pulse is applied, and then by taking Group III as a positive electrode and Group I, II as a negative electrode the electric pulse is applied.
For certain kind of cells, pores may appear on the cell membranes when the electric field is higher than a certain threshold. The death rate of the cells may arise as the electric field gradually increases. To ensure a high electroporation rate and low death rate of the cells, it is desired to accurately control the electric field to be the threshold electric field of the electroporation.
As long as the homogeneous electric field intensity is controlled to be the optimal electroporation voltage of the electroporation, cells in the whole effective area may experience electroporation at the greatest extent. It can be seen that grouping and reusing compensate for the unevenness of the electric field caused by single group, increases the electroporation efficiency, and thus it can be determined that such kind of combination of electric fields has a much higher electroporation efficiency than the conventional electroporation.
The high-density distributed three-dimensional electrode device may carry out electroporation for many cell lines in suspension or in adherence. 12 kinds of cells, 7 HEK-293A, Hela, MCF-7, A-375, Neuro-2A, U251, C2C12, 3T3-L1, CHO, MDCK, HL-60, HUVEC, are choosen to undergo electroporation, and GFP molecules are used as marker. The GFP molecules may enter into the cells and synthesize fluorescent substances in the cells if the cells are electroporated, and the synthesized fluorescent substances may glow green fluorescence under a fluorescent field such that the electroporation rate of the cells may be obtained from the number of cells in the fluorescent field divided by the total number of cells, that is to say, the higher the fluorescence intensity of the same density of cells is, the higher the efficiency of the electroporation is.
The high-density distributed three-dimensional electrode device according to the present invention may be applied in a flow device. By placing the high-density distributed three-dimensional electrode device in the flow device, the cells evenly distributed and flowing along with the flowing fluid may accept an optimal electroporation stimulation conditions when the cells experience the control of the flowing rate and pulse stimulation during passing through the electrode array, the electroporation stimulation conditions comprising the voltage amplitude value of pulse, pulse width, pulse interval, pulse number and electrode swapping control. In a continuous flowing system, except that the time when the pulses are applied to the electrodes and the time when the cells begin to flow among the electrodes need to be harmonized, the pulses and the flowing of the cell do not need a harmonization of “duration handling” between them.
The high-density distributed three-dimensional electrode device according to the present invention both has a advantage of continuous flow electroporation, in particularly being able to carry out a high-throughput electroporation for cells in a sterile closed system, and is also able to ensure that each of the cells is subjected with optimal number of pulses and most even electric field to improve the electroporation efficiency and low down the death rate.

Embodiment 2

The high-density distributed three-dimensional electrode device according to the present embodiment is similar to that of Emobodiment 1 differing in the arrangement of the electrode array. In the present embodiment, the arrangement rule of the electrode array is as follow: all the electrodes are arranged to form an equilateral quadrangle shaped structure, the distances between the adjacent two electrodes are equal, the inside of the equilateral quadrangle is divided into several smaller square units by taking the distances between the adjacent two electrodes as side length, and one electrode is placed at each vertex of the squares, i.e. all the electrodes are divided into four groups, Group I, II, III, and IV, and vertexes of each square respectively belong to
Group I, II, III, and IV, and these groups take turns to be applied with the electric pulse by taking one of the groups as a positive electrode and the rest groups as a negative electrode. The arrangement of the electrode array of this embodiment may be represented by slightly modifying the circumstances of Embodiment 1 shown in FIG. 4, and therefore no more figures are shown.

Embodiment 3

The high-density distributed three-dimensional electrode device provided by the present embodiment is similar to that of Emobodiment 1 differing in that the arrangement of the electrode array is as follow: all the electrodes are arranged to form an equilateral hexagon structure, the distances between the adjacent two electrodes are equal, the inside of the equilateral hexagon is divided into several smaller equilateral hexagon units by taking the distances between the adjacent two electrodes as side length, and one electrode is placed at each vertex of the hexagon units, i.e. all the electrodes are divided into six groups, Group I, II, III, IV, V, and VI, and vertexes of each hexagon unit respectively belong to Group I, II, III, IV, V, and VI, and these groups take turns to be applied with the electric pulse by taking one of the groups as a positive electrode and the rest groups as a negative electrode. The arrangement of the electrode array of the present embodiment may be represented by slightly modifying the circumstances of Embodiment 1 shown in FIG. 4, and therefore no more figures are shown.
The above are only embodiments of the present invention, and are no way to limit the scope of the present invention. Any equivalent structures or process changes, or direct or indirect application on other relative technical fields by taking advantage of the content of the present invention should be covered by the scope of the present invention.

Claims

1. A high-density distributed three-dimensional electrode device characterized in that:

the device comprises an electrode array and an electrode fixing assembly on which the electrode array is fixed,

the electrode array comprising a plurality of electrodes divided into at least two groups, an electric pulse of a first polarity and an electric pulse of a second polarity are respectively applied on the at least two groups according to a time period,

wherein the electrodes corresponding to the electric pulse of the second polarity are distributed around the electrodes corresponding to the electric pulse of the first polarity, and the first polarity and the second polarity are opposite.

2. The device as claimed in claim 1, wherein the diameters of the electrodes are 0.01-1.2 mm, the distance between the center points of two adjacent electrodes is 0.1-2.4 mm, and the number of the electrodes is more than 5.

3. The device as claimed in claim 2, wherein the diameters of the electrodes are 0.1-0.4 mm, the distance between the center points of two adjacent electrodes is 0.2-1.5 mm, and the number of the electrodes is more than 36.

4. The device as claimed in claim 3, wherein the diameters of the electrodes are 0.3 mm, the distance between the center points of two adjacent electrodes is 1 mm, and the number of the electrodes is 37.

5. The device as claimed in claim 1, wherein the plurality of electrodes in the electrode array is arranged according to an equilateral polygon, and the distances between every two adjacent electrodes in the electrode array are equal.

6. The device as claimed in claim 5, wherein a shape of the electrode array is an equilateral hexagon formed by several equilateral triangles, and the electrodes are located at the vertexes of the equilateral triangles.

7. The device as claimed in claim 1, wherein the material of the electrodes is stainless steel.

8. The device as claimed in claim 1, wherein the electrode fixing assembly comprises an electrode connecting circuit board and an electrode positioning board, and the electrode connecting circuit board connects the electrodes applied with the same electric pulse together, the electrodes are inserted into the electrode positioning board.

9. The device as claimed in claim 1, wherein the electrodes in the electrode array are divided into three or more groups, the electrodes in the same group are applied with the electric pulse of the same polarity, and the groups of electrodes are applied with an electric pulse according to a time period to achieve a homogeneous electric field by superposition of the resulted electric fields in the following alternate way: one of the groups is applied with the electric pulse as a positive electrode, the rest groups are applied with the electric pulse as a negative electrode, and then another one of the groups is applied with the electric pulse as the positive electrode, the rest groups are applied with the electric pulse as the negative electrode.