CN103320319B - Alternative high-throughput electroporation device - Google Patents

Abstract

The invention discloses a selective high-throughput electroporation device. The electroporation device includes a substrate; the substrate is provided with several through holes; each of the through holes is provided with a pair of electrodes, and the electrodes are connected to the wires provided on the substrate; Electrodes can be single-point strobed. The electroporation device provided by the invention can be manufactured according to the PCB manufacturing method. The electroporation device provided by the present invention can be used for electroporation of cells, such as electroporation of animal cells or bacteria. In use, electroporation pulses are applied to the electroporation system suspended within the through-hole by electrodes at the through-hole location. The electroporation device described in the present invention can be manufactured by PCB technology, the technology is mature, the cost is low, and it can be mass-produced. The electroporation device made by PCB technology can be seamlessly connected with the PCB-based circuit control system, which is conducive to the realization of high-throughput and automated gene function screening based on electroporation.

Description

A selective high-throughput electroporation device

technical field

The invention relates to a selective high-flux electroporation device, which belongs to the field of electroporation.

Background technique

Electroporation is a cell transfection or bacterial transformation technology that uses an external pulsed electric field to generate a transmembrane voltage on the cell membrane. When the cell transmembrane voltage exceeds a certain threshold, the cell membrane undergoes reversible perforation to introduce foreign substances into the cell. Electroporation has the advantages of no cytotoxicity, low cost, and high transfection efficiency. At present, electroporation technology has been widely used in biological research. Several types of electroporation methods exist, such as placing cells between parallel electrodes a few millimeters apart (for example, U.S. Pat. 5389069), or allowing cells to flow between parallel electrodes (for example, U.S. Pat. 6773669; Chinese patent application, publication number: CN1195997A), all have the disadvantages of troublesome use and difficulty in processing multiple samples in parallel. High-throughput electroporation allows simultaneous or automated sequential electroporation of multiple samples, a technique suitable for many processes such as high-throughput gene function studies and therapeutic target screening.

The existing high-throughput electroporation devices are all based on a porous plate structure, and the electroporation system is added to the porous plate structure, and each hole of this porous plate structure contains a pair of electrodes for generating electric pulses, and the electric pulse is applied according to a certain procedure. The pulse can electroporate the cells in each well (eg patent: International Patent Application PCT Publication: WO2004/050866A1; Chinese Patent: CN101384697B). Although this type of device solves the problem of high-throughput electroporation, the process of sample preparation, transfer and device cleaning is cumbersome, and each reaction consumes more cells and transfected materials, which improves the efficiency of high-throughput screening. the cost of.

In 2008, G.Emmanuel of Stanford University in the United States reported a hanging drop electroporation device in the journal Nature Methods. The device consists of two finger-shaped metal sheets to form 96 pairs of metal electrodes corresponding to a 96-well plate. 10 to 20 microliters of cell suspension is added between the metal electrodes and will adhere to the gap between the electrodes due to surface tension. Applying an electric pulse between the two electrodes electroporates the cells adhered between the electrode pair, and the perforated cells are flushed by a large amount of cell culture medium into the multi-well plate below the electrodes. The sample handling process of the device is very simple, compatible with conventional multi-well plates and can be used with liquid workstations. However, there are differences in the optimal electroporation conditions between different combinations of cells and transfection objects, and the device has only two electrodes, so it is impossible to perform selective electroporation on different samples simultaneously, and the processing cost is high, which limits its application. Therefore, how to achieve low-cost, selective high-throughput electroporation has become a very important issue in large-scale screening. Printed circuit board (PCB) is a mature technology used in the production of electronic circuits. It can be mass-produced and the processing cost is low. There have been some reports on the application of PCB technology to cell manipulation. For example, PCB technology can be used to generate through holes with a diameter of about 100 microns. Through-hole capture single cell research) and electrical impedance measurement (in 2012, F.Andrea from the University of Bologna in Italy reported in Lab on a Chip magazine using through-holes on PCB to measure cell impedance), which are applied to single-cell Potential for cell manipulation and analysis. Likewise, PCB technology has the potential to be applied to cell electroporation.

Contents of the invention

The purpose of the present invention is to provide a convenient and selective high-throughput electroporation device.

The invention provides a selective high-throughput electroporation device, which includes a substrate;

The substrate is provided with several through holes; each of the through holes is provided with a pair of electrodes, and the electrodes are connected to the wires provided on the substrate;

The electrodes can be single-point strobed.

The "single-point gating" in the present invention means that each pair of electrodes on the substrate is connected in parallel, so that they can be individually controlled to achieve selective control.

In the above-mentioned electroporation device, the substrate can be made of insulating material, such as glass or insulating polymer.

In the above-mentioned electroporation device, the through holes may be circular, elliptical, square or rectangular.

In the above electroporation device, the electrodes are made of metal or conductive polymer.

When a cell is in a pulsed electric field, the cell membrane will generate a transmembrane voltage. When the transmembrane voltage exceeds a certain threshold, recoverable micropores will be formed on the surface of the cell membrane, thereby introducing foreign substances into the cell. Generating a suitable electric field is the key to efficient electroporation. Pulse signal introduction into the mixed system containing cells and material to be transfected is accomplished through conductive electrodes in contact with it. Therefore, for the electroporation device, the setting of electrodes will directly affect the effect of electroporation, and reasonable wiring arrangement is particularly important to reduce the complexity of the device and realize selective control. According to the above principles, in the present invention, the electrodes can be arranged on the side walls of the through holes; or, the electrodes can be arranged on the upper and lower sides of the substrate, and the middle section of the electrodes is in line with the The inner walls of the through holes are on the same vertical plane.

In the above-mentioned electroporation device, the distance between the two electrode plates of the electrodes is 0.2-2 mm.

In the above-mentioned electroporation device, the substrate may be a printed circuit board (PCB), which can be mass-produced compared with the disclosed electroporation device, and the processing cost is greatly reduced. In addition, because the cost of mass-produced PCB boards is very low and can be used once, it can reduce the cleaning process of the electroporation device after electroporation, reduce the operation steps, and avoid the pollution problem between different batches of experiments.

The electroporation device provided by the invention can be manufactured according to the PCB manufacturing method.

The electroporation device provided by the present invention can be used for electroporation of cells, such as electroporation of animal cells or bacteria. In use, electroporation pulses are applied to the electroporation system suspended within the through-hole by electrodes at the through-hole location.

When the present invention is in use, it is used in conjunction with a porous plate arranged under the electroporation device, such as a commercially available standard porous plate, which can be a 6, 8, 16, 24, 48, 96, or 384-hole plate.

Compared with the prior art, the present invention has the advantages of:

1. The process of sample addition, electroporation and pipetting is very convenient, and various optimized electroporation conditions corresponding to various samples can be applied simultaneously.

2. In the present invention, each electroporation system is relatively small, ranging from 1 microliter to 50 microliters. Fewer cells and material to be transfected are consumed per sample, facilitating large-scale screening.

3. The electroporation system is suspended between two parallel metal plates with a distance of 0.2 to 2 mm. Its large surface area is conducive to heat dissipation, thereby avoiding damage to the perforated cells caused by the temperature rise of the solution caused by electroporation.

4. The electroporation device of the present invention can be manufactured by PCB technology, the technology is mature, the cost is low, and it can be produced in batches. The electroporation device made by PCB technology can be seamlessly connected with the PCB-based circuit control system, which is conducive to the realization of high-throughput and automated gene function screening based on electroporation.

Description of drawings

Fig. 1 is a top view of the high-throughput electroporation device of the present invention.

FIG. 2 is a schematic structural view of one unit of the electroporation device shown in FIG. 1 .

Figure 3 is a physical picture of a high-throughput electroporation device based on printed circuit board technology (PCB).

FIG. 4 is a schematic diagram of the principle of breaking the metal layer inside the through hole of the PCB in FIG. 3 into two electrodes.

Fig. 5 is a schematic flow chart of the use of the electroporation device proposed by the present invention.

Figure 6 shows the process of adding samples using a row gun.

FIG. 7 is a schematic diagram of the principle of milling away the metal layer inside the circular PCB through hole.

Each mark in the figure is as follows: 1 substrate, 2 through hole, 3 electrode, 4 top layer wire, 5 bottom layer wire.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1. A high-throughput electroporation device based on a printed circuit board. The electroporation electrodes are metal parallel plates perpendicular to the substrate.

Fig. 1 is a top view of the electroporation device provided in this embodiment, which can be matched with a 96-well plate.

As shown in FIG. 1 , the electroporation device in this embodiment includes a substrate 1 on which 96 through holes 2 are arranged, and are matched with a 96-well plate. One electrode 3 is provided on the side wall of each through hole 2, that is, one pair of electrodes. The distance between the two electrode plates is 2mm.

In order to achieve selective control of each through hole with as few pads as possible, all electrodes on the upper side of a row of 12 holes are commonly connected to the top layer wires 4 distributed along each row on the top of the substrate 1, while all electrodes on the lower side of a row of 8 holes are connected to each other. The electrodes are commonly connected to the underlying wires 5 distributed along each column at the bottom of the substrate. Because the electroporation at position 2 of each through hole needs to be powered by electrodes on the upper and lower sides, 8×12 pads can be used here to achieve selective control of single-point gating for all 96 through holes. Here, the distribution of the top-layer wires 4 and the bottom-layer wires 5 on the substrate can be interchanged, and if the substrate 1 can be routed in multiple layers, the wires can also go through the middle layer of the substrate 1 .

Fig. 2 is an enlarged view of a unit of the electroporation device in Fig. 1, as can be seen from Fig. 2, the property of the through hole 2 in the present embodiment is a rectangle, and it can also be set as various shapes such as a circle, an ellipse, a square, etc. shape.

Fig. 3 is a physical picture of a high-throughput electroporation device using a printed circuit board (PCB) as a substrate.

Specifically, a PCB board with double-layer wiring is used, which contains 96 oval through holes. The metal layers above and below the long axis of each elliptical through hole are connected to 8×12 solder pads on the PCB through the above wiring method. The inner wall of the via hole made by the conventional PCB process is a whole piece of metal connected together. In order to form the electrode pair required for electroporation, an additional process is required. As shown in Figure 4, after the copper is poured on the via hole, use a drill bit for drilling the PCB to mill off the copper clad at both ends of the via hole. Through this operation, a pair of parallel metal electrodes is generated in each via hole on the PCB.

When using the electroporation device of the present invention, it can be carried out according to the method shown in FIG. 5 , which is specifically divided into three steps: 1. sample addition, 2. hanging drop electroporation, and 3. sample transfer.

The specific process is as follows: the constructed electroporation device is placed above the porous plate, and the through holes of the electroporation device correspond to the holes of the porous plate below. Pipette a small amount of the electroporation system containing the cells and material to be transfected into the wells of the electroporation device. The electroporation system is suspended in the through-holes of the electroporation device due to surface tension. The regime of electroporation can be controlled by the dimensioning of the through-holes of the electroporation device. Apply electrical pulses synchronously or rapidly sequentially to the electroporation system in all through holes to complete the cell electroporation process. Add a large volume of cell culture medium through the through hole of the electroporation device, and the cell culture medium will wash the perforated cell suspension into the lower porous plate to complete the transfer of the perforated cells. The multi-well plate is then placed in a cell culture incubator for subsequent experiments and analysis.

The operation of the entire electroporation is very simple, and the sample loading process can be completed with a row gun (as shown in Figure 6) or an automated manipulator, which is very suitable for high-throughput screening.

Embodiment 2, a high-throughput electroporation device based on a printed circuit board, the electroporation electrodes are parallel to the substrate

This embodiment is similar to Embodiment 1 in that the electroporation device is made of a PCB, and the difference is that this embodiment uses a circular PCB through hole to carry the electroporation system. Similar to Example 1, the inside of the PCB through-hole is covered with a metal layer. In order to generate the electrode pairs required for electroporation, the metal of the PCB passing through the inner wall can be milled off with a drill (as shown in Figure 7), so that the metal on the top and bottom of the PCB The layer becomes the electroporation electrode. An alternative processing method is to directly drill holes at the intersection of the upper and lower metal traces. As long as the diameter of the drill is smaller than the width of the metal traces, a structure similar to that shown in the right figure of Figure 7 can also be obtained. The distance between two electrode plates in the horizontal direction is 0.2mm.

The usage flow of the electroporation device generated by this method is the same as that in Example 1, and also includes three steps: 1. Sample addition, 2. Hanging drop electroporation, 3. Sample transfer. The specific operation process is as follows: the constructed electroporation device is placed above the porous plate, and the through holes of the electroporation device correspond to the holes of the porous plate below. The electroporation system containing the cells and the material to be transfected is pipetted into the through-wells of the electroporation device. The volume of the added system should be enough to fill the through holes on the PCB, that is, the solution can contact the electrodes on the upper and lower surfaces of the PCB at the same time. Apply electrical pulses synchronously or rapidly sequentially to the electroporation system in all through holes to complete the cell electroporation process. Add a larger volume of cell culture medium through the through hole of the electroporation device, and the cell culture medium will wash the perforated cell suspension into the lower porous plate to complete the transfer of the perforated cells. The multi-well plate is then placed in a cell culture incubator for subsequent experiments and analysis.

Compared with Example 1, in this example, the contact area between the electrode and the solution is smaller, which can reduce the factors that are not conducive to cell survival, such as the temperature rise of the solution caused by water electrolysis and the change of pH value on the surface of the electrode during electroporation. . In addition, the via hole made by this method is small, which is suitable for electroporation of a small number of cells or even a single cell. This method has the potential to be integrated with PCB-based cell electrical impedance measurement (reported in Lab on a Chip by F.Andrea et al. from the University of Bologna, Italy in 2012) to become a platform for single-cell transfection and real-time cell electrophysiological monitoring.

Embodiment 3. A high-throughput electroporation device based on a printed circuit board. The electroporation electrodes are metal parallel plates perpendicular to the substrate. The distance between the two electrode plates is 1mm. Its concrete experimental process is similar to embodiment 1. Compared with Example 1, the distance between electrodes is smaller, so the voltage required for electroporation is smaller.

Claims (6)

1. A selective high-throughput electroporation device, characterized in that: The electroporation device includes a substrate; The substrate is provided with several through holes; each of the through holes is provided with a pair of electrodes, and the electrodes are connected to the wires provided on the substrate; The electrodes are arranged on the upper and lower sides of the substrate, and the middle section of the electrodes is on the same vertical plane as the inner wall of the through hole; The electrodes can be single-point strobed; An electroporation system is suspended within the through holes. 2. The electroporation device according to claim 1, wherein the substrate is made of insulating material. 3. The electroporation device according to claim 1 or 2, wherein the through hole is circular, oval or rectangular. 4. The electroporation device according to claim 3, wherein the electrodes are made of metal or conductive polymer. 5 . The electroporation device according to claim 4 , wherein the distance between the two electrode plates of the electrode is 0.2-2 mm. 6. The electroporation device according to claim 5, wherein the substrate is a printed circuit board.