CN114752492A

CN114752492A - Cell electrotransformation chip, electrotransformation device and electrotransformation system

Info

Publication number: CN114752492A
Application number: CN202210350561.9A
Authority: CN
Inventors: 师传胤; 钱其军
Original assignee: Maxirna Shanghai Pharmaceutical Co Ltd; Maxirna Zhejiang Technology Co Ltd
Current assignee: Maxirna Shanghai Pharmaceutical Co Ltd; Maxirna Zhejiang Technology Co Ltd
Priority date: 2022-04-02
Filing date: 2022-04-02
Publication date: 2022-07-15

Abstract

The invention provides a cell electrotransfer chip, an electrotransfer device and an electrotransfer system. The electric rotating chip comprises: the electric-to-liquid conversion device comprises a closed body communicated with the outside through an electric-to-liquid inlet, an electric-to-liquid outlet and a pressurization gas port, wherein the closed body is internally provided with an electric-to-liquid inlet buffer area, an electric-to-liquid outlet buffer area, a pressurization gas port buffer area and an electric-to-liquid conversion cavity; electrode plates which are arranged on two opposite planes of the electrotransformation cavity in parallel; and a conductive electrode protruding from a surface of one side of the electrode plate facing away from the electrotransformation chamber. The invention has the advantages of small space volume, continuous electric conversion, stable electric field, automatic control, closed chip space, high biological safety and the like.

Description

Cell electrotransformation chip, electrotransformation device and electrotransformation system

Technical Field

The invention belongs to the technical field of biological cell sample electrotransformation, and particularly relates to an electrotransformation chip, an electrotransformation device and an electrotransformation system for various cells (including immune cells, stem cells, cell strains and the like).

Background

Cell electroporation (or electroporation) is a common technique in the field of cell transfection, and the principle is that a short-pulse high-voltage electric field is applied to a cell to generate a channel on the cell membrane for allowing molecules outside the membrane to enter in a short time. The cells are typically some cells of a human or animal; the molecule is typically a plasmid, DNA, RNA, or the like. Cell electrotransformation is applied to the fields of cell experiments, immune gene research, immune cell therapy and the like.

The cell electric conversion effect is related to the cell type, the condition of the electric field and the electric conversion container. The conditions of the high-voltage electric field required by different types of cells are different; the electric field conditions include pulse width, voltage amplitude, number of pulses, and the like; the electroporation vessel is a place where cells are electroporated and is the most central component. For immune cells or stem cells to be used in clinical therapeutic studies, cell electroporation also requires strict assurance of sterility of the sample. Most of the existing cell electric transfer devices are scientific research-grade equipment in laboratories, most of the existing cell electric transfer devices are only suitable for scientific research and exploration, and the electric transfer containers are repeatedly and openly added with sample or mixed during cell electric transfer, so that the sterility of samples is difficult to guarantee.

A conventional electric rotating vessel is a disposable electric rotating cup, which includes two parallel electrode plates. The electrode plates are all connected to an external strong electric field, and cell fluid to be electrified can be placed between the electrode plates. In the prior art, on one hand, water in the solution is easy to generate bubbles through electrolysis at the electrode end during electric conversion, and the bubbles can further influence the electric field environment of the electric conversion. On the other hand, the temperature of the electrode and the electrotransformation solution is gradually increased along with the progress of electrotransformation of the electrode in a short-frequency high-voltage electric field, and the cell is damaged by overhigh temperature, so that the state of the subsequent cell is influenced.

CN1965079B relates to a fiber type electroporation device, comprising a pulse generator, a long hollow sample filling member, a pressure device, a reservoir and a storage holding device. The reservoirs are respectively arranged at two ends of the pulse generator, and the electrodes are respectively placed in the reservoirs at the two ends; the long hollow sample filling piece is a hollow fiber filament, the interior of the long hollow sample filling piece can be used for filling mixed liquid to be electrically converted, and two ends of the fiber filament are respectively placed in the two containers; the container is filled with electrotransformation liquid. When the fiber is electrically switched, air bubbles are generated in the reservoirs at the two ends, and the electrical switching of cells in the fiber is not influenced. The pressure device is used to maintain a suitable pressure to achieve complete filling of the sample inside. However, the electric field of the device is not uniform, and the electric effect on the cells in the hollow fibers is inconsistent.

CN104357323B relates to a cell electrotransformation appearance, including casing, power module, electric shock pipe and micro-control device. The cell electrotransformation instrument is internally provided with a fixed seat, the fixed seat is provided with a jack for fixing the electric shock tube, the shell is also internally provided with a first electrode and a second electrode which are connected with the power module, the first electrode and the second electrode are respectively used for applying an electrotransformation electric field against the two electrodes of the electric shock tube, and the micro control device controls the power module. The device compresses tightly two electrodes of the electric shock tube through the spring, enhances the tightness of the electric shock tube, and compresses air bubbles possibly generated by electrochemistry to a certain extent. However, the sealing pressure that can be provided by this device is limited and does not completely prevent the generation of electrolytic bubbles.

CN102268425A relates to a related method and apparatus for a large volume electrohydrodynamic flow electroporation chamber, comprising an electroporation chamber with a manifold to regulate sample flow. The electroporation chamber includes a circular fluid flow path that allows conditions to be varied to allow the sample therein to be uniformly processed in individual components or in individual volumes within a completely sterile sealed system. The device can circularly electrolyze a large volume of sample, but does not mention air bubble and temperature control during electric conversion.

Disclosure of Invention

Aiming at the defects of the existing cell electrotransfer method and device, the invention provides an electrotransfer chip which has simple structure and convenient operation and is suitable for various cells, especially immune cells and an electrotransfer device containing the electrotransfer chip, and the invention takes the cell engineering in the cell treatment field into consideration as the trend of the future clinical application of the cell engineering into consideration, and exogenous molecules can be translated in the cells (DNA and mRNA) to generate a plurality of substances such as chemotactic factors, cytokines, antibodies, surface marks and the like to improve the curative effect of the cell treatment. The electrotransfer chip can realize a new cell electrotransfer scheme and can independently process the cell sample of each clinical patient sample. A certain amount of cells, particularly immune cells, are taken out from a patient body and are mixed with certain specific molecular liquid, and then stable electric conversion is carried out in the electric conversion system, and finally specific molecules can be successfully introduced into the cells through cell membranes. The cell electrotransfer chip establishes a stable environment capable of meeting continuous electrotransfer by pressurizing and controlling temperature in an electrotransfer environment, reduces damage to cells, and is suitable for electrotransfer of various cells in cell clinical application.

Specifically, the present invention provides an electrotransfer chip, comprising:

the electric-to-liquid conversion device comprises a closed body communicated with the outside through an electric-to-liquid conversion inlet, an electric-to-liquid conversion outlet and a pressurization gas port, wherein the closed body is internally provided with an electric-to-liquid conversion inlet buffer area, an electric-to-liquid conversion outlet buffer area, a pressurization gas port buffer area and an electric-to-liquid conversion cavity; electrode plates which are arranged on two opposite planes of the electrotransformation cavity in parallel; and

and the conductive electrode protrudes from the surface of one side of the electrode plate, which is away from the electrotransformation cavity.

In one or more embodiments, the electrotransfer chamber is a flat-type cavity.

In one or more embodiments, the cavity parallel cross-section includes a circular middle portion contoured to have a circular arc smooth transition to the electric transfer fluid inlet buffer zone and the electric transfer fluid outlet buffer zone on one side and to a boost gas port buffer zone on the other side.

In one or more embodiments, the electrotransfer liquid inlet buffer and the electrotransfer liquid outlet buffer are covered by electrode plates and the boost gas port buffer is covered by electrode plates.

In one or more embodiments, the electric rotary chip comprises a sealing stopper for separating and forming the electric rotary fluid inlet buffer zone, the electric rotary fluid outlet buffer zone, the pressurizing air port buffer zone and the electric rotary fluid cavity.

In one or more embodiments, the side of the sealing stop adjacent to the electrotransfer chamber is curved.

In one or more embodiments, the closed body comprises a body upper layer, a body middle layer and a body lower layer which are closely attached, wherein the body middle layer is provided with a cavity which comprises an electric rotating liquid inlet buffer zone, an electric rotating liquid outlet buffer zone, a pressurizing air port buffer zone and an electric rotating liquid cavity; the electrode plates are respectively embedded in the upper layer and the lower layer of the body; the conductive electrodes are respectively embedded in the conductive electrode fixing holes in the upper layer and the lower layer of the body and are tightly attached to the electrode fixing holes.

In one or more embodiments, the gap between the electrode plates is 0.5 to 2mm, preferably 0.8 to 1.5mm, and more preferably 1 mm.

In one or more embodiments, one side of the conductive electrode is in contact with the electrode plate, and the other side of the conductive electrode is parallel to or protrudes from the outer surface of the closed body and is used for being connected with an external electric-to-electric-field generating device.

In certain embodiments, the volume of the electrotransport fluid chamber is 1 to 5 ml.

The invention also provides a cell electrotransformation device, which comprises an electric field generating device and an electrotransformation chip arranged in the electric field generating device and provided with any one of the embodiments of the invention.

In one or more embodiments, the electric field generating device comprises a pulse power supply, an electrode column, a cavity for accommodating the electric conversion chip, and a detachable latch, wherein the latch is arranged on one side surface of the electric field generating device for closely fixing the electric conversion chip; the cavity is formed by the lock catch and the rest side surfaces of the electric field generating device together to form a cavity space for fixedly accommodating the electric conversion chip.

In one or more embodiments, the electric converter further comprises a support base for supporting and placing the electric field generator and the electric converter chip. After the electric transfer device with the electric transfer chip mounted therein is placed on the support base, the pressurizing air port side of the electric transfer chip in the horizontal direction is higher than the electric transfer liquid inlet and the electric transfer liquid outlet side of the electric transfer chip, preferably, the included angle between the electric transfer liquid inlet and the electric transfer liquid outlet side of the electric transfer chip in the horizontal direction and the horizontal line is 2 to 90 degrees, preferably 60 to 90 degrees, and more preferably 60 degrees.

In one or more embodiments, the electric rotating device further comprises a position adjusting device for adjusting an elevation angle between the electric rotating chip and a horizontal line.

The invention also provides a cell electrotransformation system, which comprises an electrotransformation unit, wherein the electrotransformation unit comprises the cell electrotransformation chip in any embodiment of the invention.

In one or more embodiments, the electrical conversion unit further comprises an electric field generating device.

In one or more embodiments, the electrical conversion chip is disposed inside the electrical field generating device.

In one or more embodiments, the electric field generating device includes a pulse power supply, an electrode column, a cavity for accommodating the electric conversion chip, and a detachable latch, wherein the latch is arranged on one side surface of the electric field generating device, which is closely fixed to the electric conversion chip, and forms the cavity for fixedly accommodating the electric conversion chip together with the other side surfaces of the electric field generating device.

In one or more embodiments, the electrotransfer system further comprises:

a sample supply unit, a buffer unit, a sample collection unit, a pressurization unit and a temperature control unit,

a pipeline communicated with the sample supply unit, the buffer unit, the sample collection unit, the pressurization unit and the temperature control unit,

a driving unit for driving the material to flow among the sample supply unit, the buffer unit, the sample collection unit, the pressurizing unit, the temperature control unit and the pipeline, an

And the control unit is used for controlling the communication among the sample supply unit, the buffer unit, the sample collection unit, the pressurization unit and the temperature control unit.

Drawings

Fig. 1 shows a schematic structural diagram of an electric power conversion system of the present invention.

Fig. 2 shows a schematic structure of an electrotransfer chip of the present invention.

Fig. 3 shows a schematic cross-sectional view of a structure of an electric conversion chip of the present invention after being combined with an electric conversion device.

Fig. 4 is a schematic rear view showing a structure of an electric conversion chip of the present invention combined with an electric conversion device.

Fig. 5 shows a schematic structural view of an electric conversion apparatus of the present invention.

Fig. 6 shows a schematic configuration of a pressure-increasing holding device of the pressure-increasing unit of the electric conversion apparatus of the present invention.

The drawings of the invention are illustrated below:

the device comprises a buffer sample introduction flow path A, a liquid inlet flow path B, a liquid outlet flow path C and a pressurization gas path D.

1. A sample supply unit; 10. a sample supply unit access device; 101. liquid stopping clips;

2. a buffer unit; 20. the buffer unit is connected with the liquid inlet device; 201. liquid stopping clips; 21. the buffer unit is provided with a liquid outlet access device; 211. liquid stopping clips; 22. a gas filtering device;

3. an electric conversion unit; 30. electrically converting the chip; 300. a middle layer of the body; 301. sealing the stop block; 3010. an electrode plate fixing groove; 302. the upper layer of the body; 303. a lower layer of the body; 304. an electrotransfer chamber; 305. an electrotransfer liquid inlet buffer zone; 3050. an electrotransfer liquid inlet; 306. an electrotransfer liquid outlet buffer zone; 3060. an electrotransformation liquid outlet; 307. a pressurized gas buffer region; 3070. a booster air port; 308. a conductive electrode; 3080. a conductive electrode fixing hole; 309. electrode plate

31. A temperature control layer; 32. a supporting seat; 33. an electric field generating device; 330. a device slot; 34. fixing the slot; 35. a lock; 350. a lock groove; 36. an electrode column; 37. a position adjustment device;

4. a sample collection unit; 401. liquid stopping clips;

5. a pressurizing unit; 50. a gas plunger tube; 51. a plunger rod; 52. a stroke pushing piece; 53. a filter member; 54. an air valve;

6. a temperature control unit; 60. a chip temperature control device; 61. a pressurizing unit temperature control device;

7. a control device; 70. a peristaltic pump; 71. a peristaltic pump; 72. a peristaltic pump.

Detailed Description

To make the features and effects of the invention comprehensible to those skilled in the art, general description and definitions shall be provided below with respect to terms and words mentioned in the specification and claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the description of the present invention, the orientation or positional relationship referred to with the related terms "front", "rear", "left", "right", "upper", "lower", "horizontal", "vertical", etc., is based on the orientation or positional relationship shown in the drawings, and is only for convenience of description and simplification of description, and does not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.

The cell electric transfer system has the working principle that a stable environment capable of meeting continuous electric transfer is established by pressurizing and controlling temperature in an electric transfer environment, so that the damage to cells is reduced.

The cell electrotransformation system comprises a sample supply unit, a buffer unit, an electrotransfer unit, a sample collection unit, a pressurization unit, a temperature control unit, a pipeline communicated with the sample supply unit, the buffer unit, the electrotransfer unit, the sample collection unit, the pressurization unit and the temperature control unit, a driving unit for driving liquid to flow among the sample supply unit, the buffer unit, the electrotransfer unit, the sample collection unit, the pressurization unit and the pipeline, and a control unit for controlling the communication among the sample supply unit, the buffer unit, the electrotransfer unit, the sample collection unit and the pressurization unit.

The sample supply unit and the sample collection unit comprise a container for containing a sample which is not electrically converted (a mixed solution of a cellular fluid to be electrically converted, a macromolecule to be electrically converted and an electrical conversion fluid), a container for completing the electrically converted sample, and an optional access device for accessing the container for containing the sample which is not electrically converted and the container for completing the electrically converted sample into a pipeline. The container for holding the sample that has not been electrically transferred, the container for the sample that has been electrically transferred and the access device thereof may be conventional containers and access devices in the art, for example, the container for holding the sample that has not been electrically transferred, the container for the sample that has been electrically transferred are reagent bags that have been packaged in advance, and the access device may be an access needle.

The buffer unit is arranged between the sample supply unit and the electric conversion unit, is a buffer area of a sample which is not subjected to electric conversion before entering the electric conversion unit, can be used for buffering the sample introduction speed of the sample supply unit, can also discharge bubbles in a sample liquid, and prevents the bubbles in the sample liquid from directly entering the electric conversion unit. The buffer unit comprises a container for containing a sample to be electrified, an optional access device for accessing the container for containing the sample to be electrified into a pipeline respectively communicated with the sample supply unit, a pipeline communicated with the electrified conversion unit, and an air filtering device communicated with the outside. The pipeline communicated with the sample supply unit and the access device communicated with the pipeline of the electric conversion unit are positioned on the same side of a container for accommodating a sample to be converted, and the air filter device is positioned on the other side of the container. The container for receiving the sample to be electrically transferred and the access device thereof may be conventional in the art, for example, the container for receiving the sample to be electrically transferred is a cylindrical tube, PVC material. The pipeline communicated with the sample supply unit and the pipeline communicated with the electric conversion unit are connected in a connecting device at the bottom of the cylindrical pipe, the air filtering device is positioned at the top of the cylindrical pipe, and the air filtering device is provided with a filter element which is air-permeable and water-impermeable.

An electrotransfer unit (also referred to herein as a "cell electrotransfer device") is used to introduce a specific molecule into a cell (e.g., an immune cell) through the cell membrane under the action of a stable electrotransfer electric field. The electric transfer unit is disposed between the buffer unit and the sample collection unit, and generally includes an electric field generating device and a container (also referred to herein as a "cell electric transfer chip") for accommodating and transferring a sample, such as a chip-type structure. The electric field generating device is used for providing a stable electric field with short pulse high voltage and comprises a pulse power supply, an electrode column, a cavity for accommodating the electric conversion chip and a detachable lock catch. The pulse power supply is a power supply device capable of forming a short pulse high voltage. The positive and negative electrodes of the pulse power supply are respectively connected to the positive and negative electrode columns respectively. The electrode posts are fixed on the opposite surfaces of the cavities through the electrode slots of the cavities respectively, and the top ends of the electrode posts are provided with contact spring pieces. The lock catch is arranged on one side surface of the electric field generating device, which is tightly attached and fixed with the electric conversion chip, and forms a cavity for fixedly accommodating the electric conversion chip together with the other side surfaces of the electric field generating device. Generally, the space thickness of the cavity for accommodating the electro-conversion chip is 10-20 mm, preferably 10 mm. The shape of the concave cavity is matched with that of the electric rotating chip, and the contact spring pieces at the top ends of the electrode columns are respectively connected with the corresponding conductive electrodes of the electric rotating chip. Preferably, the cavity and the electrical rotating chip are both flat.

The electric conversion unit also comprises a supporting seat for supporting and placing the electric field generating device and the electric conversion chip. Preferably, the electric field generating device is positioned on the support at an angle such that the electric field generating device with the electric conversion chip mounted therein is placed on the support at an angle of 2 ° to 90 °, preferably 60 ° to 90 °, and more preferably 60 ° to the horizontal, with respect to the horizontal, at which the sample flows into the electric conversion chip, i.e., the pressurizing gas port side of the electric conversion chip in the horizontal direction is higher than the electric conversion liquid inlet and the electric conversion liquid outlet side of the electric conversion chip, and preferably, the electric conversion liquid inlet and the electric conversion liquid outlet side of the electric conversion chip in the horizontal direction form an angle of 60 ° to 90 °, and more preferably 60 °, with respect to the horizontal.

The electric rotating unit may further comprise a position adjusting device for adjusting the relative position of the electric field generating device placed on the supporting base and the supporting base. The position adjusting means may be formed integrally with the electric field generating means or the support base, or may be formed separately. The position adjusting device is used for changing the inclination angle of the electric field generating device. The position adjusting means may be a rotating means including a rotating shaft and a support member supporting the rotating shaft, for rotating the electric field generating means about the rotating shaft, thereby changing an elevation angle of the electric field generating means. Preferably, the rotating device is arranged such that after the electric field generating device with the electric conversion chip mounted therein is placed on the support base, the pressurizing air port side of the electric conversion chip in the horizontal direction is higher than the electric conversion liquid inlet and the electric conversion liquid outlet side of the electric conversion chip, and the included angle between the electric conversion liquid inlet and the electric conversion liquid outlet side of the electric conversion chip and the horizontal line is 60 to 90 degrees, more preferably 60 degrees.

Through the arrangement of the supporting seat of the electric conversion unit, the sample liquid flows into the electric conversion chip and is firstly concentrated at the bottom, the liquid level slowly rises upwards and gradually fills the whole electric conversion liquid cavity, and air in the electric conversion liquid cavity is discharged. After the electric liquid transfer cavity is filled, the sample liquid level is in the pressurizing air port buffer zone, and after the gas is pressurized, the sample liquid level is still in the pressurizing air port buffer zone, so that the distribution of the electric field is guaranteed to be influenced by no bubbles in the electric liquid transfer cavity.

The cell electrotransformation chip provided by the invention is provided with a closed body, an electrotransformation liquid inlet, an electrotransformation liquid outlet, a pressurization gas port, a sealing stop block, an electrode plate and a conductive electrode. The closed body is communicated with the outside only through the electric liquid transfer inlet, the electric liquid transfer outlet and the pressurization air port. The electric conversion liquid inlet is communicated with the buffer unit; the electrotransformation liquid outlet is communicated with the sample collection unit; the booster port is for communication with a booster unit. Sealed dog is used for forming electric commentaries on classics liquid import buffer, electric commentaries on classics liquid export buffer, pressure boost gas port buffer and electric commentaries on classics liquid chamber with the inside space separation of airtight body, and electric commentaries on classics liquid import buffer, electric commentaries on classics liquid export buffer and pressure boost gas port buffer all only communicate each other through electric commentaries on classics liquid chamber. One side of the sealing stop block close to the electric liquid transfer cavity is a curved surface, so that the electric liquid transfer inlet buffer area, the electric liquid transfer outlet buffer area and the electric liquid transfer cavity are in smooth transition.

The electric conversion liquid inlet buffer area is an area to be subjected to electric conversion in the electric conversion chip and can buffer the flow speed of the electric conversion liquid inlet liquid; the electric conversion liquid inlet is communicated with the electric conversion liquid inlet buffer zone. The structure of the electric liquid transfer inlet buffer zone can be smoothly transited to the electric liquid transfer cavity from a liquid inlet flow path. In some embodiments, there is a separation column between the electrotransfer inlet buffer zone and the electrotransfer chamber.

The electric conversion liquid outlet buffer area is an area to be flowed out in the electric conversion chip and can buffer the flow velocity of the electric conversion liquid outlet, and the electric conversion liquid outlet is communicated with the electric conversion liquid outlet buffer area. The structure of the electric liquid transfer outlet buffer zone can be smoothly transited to the electric liquid transfer cavity from the liquid outlet flow path. In some embodiments, there is a separation column between the electrotransfer outlet buffer and the electrotransfer chamber. In order to avoid multiple electric conversion, the electric conversion liquid inlet buffer area and the electric conversion liquid outlet buffer area are not covered by electrode plates.

The pressurizing air port buffer zone is a buffer zone of gas after the electric-to-liquid cavity is pressurized, liquid and gas are contained in the zone, the electric-to-liquid cavity is pressurized during electric rotation, no bubble is formed in the electric-to-liquid cavity, and the pressurizing air port is communicated with the pressurizing air port buffer zone. The structure of the pressurization gas port buffer zone can be smoothly transited to the pressurization gas circuit from the electric liquid transfer cavity. The pressurizing air port buffer area is covered by an electrode.

The conductive electrodes comprise at least two conductive electrodes which are respectively and oppositely embedded on the closed body. In some embodiments, the conductive electrode is tightly embedded in the through hole (also called conductive electrode fixing hole) of the conductive electrode on the sealing body, and the conductive electrode is tightly attached and sealed with the conductive electrode fixing hole. One side of the conductive electrode is contacted with the electrode plate, and the other side of the conductive electrode is parallel or convex with the outer surface of the body and is connected with an electrode column of an external electric field generating device. The electrode plate comprises two parallel conductive thin plates which are respectively arranged on two opposite planes of the electrotransformation liquid cavity in parallel; the gap between the electrode plates is 0.5-2 mm, preferably 0.8-1.5 mm, and more preferably 1 mm.

The electric transfer liquid cavity on the electric transfer chip is a flat cavity, the electric transfer liquid cavity is respectively connected with an electric transfer liquid inlet buffer area, an electric transfer liquid outlet buffer area and a pressurizing air port buffer area, the electric transfer liquid inlet buffer area and the electric transfer liquid outlet buffer area are arranged on the same side of the electric transfer liquid cavity, and the pressurizing air port buffer area is positioned on the other side of the electric transfer liquid cavity. In some embodiments, the electrical transfer chip is flat, and the electrical transfer liquid inlet, the electrical transfer liquid outlet and the pressurization gas port are all arranged at the position in the thickness direction of the electrical transfer chip. The electric rotating chip provided by the invention is provided with the pressurizing air port which can pressurize sample liquid in electric rotating, so that the sample liquid is prevented from generating bubbles in an electric rotating field. Optionally, the sealing body may be composed of an upper layer, a middle layer and a lower layer, i.e., the sealing body includes an upper layer (simply referred to as the upper layer), a middle layer (simply referred to as the middle layer) and a lower layer (simply referred to as the lower layer). The upper and lower layers of the body may be collectively referred to as an outer layer of the body (outer layer for short). The bulk-middle layer may be referred to as a bulk-inner layer (inner layer for short). The upper layer and the lower layer of the body are respectively used for embedding the electrode plates and the conductive electrodes, and the two electrode plates are parallel conductive thin plates and are respectively arranged on two opposite planes of the electrotransformation liquid cavity in parallel. The two conductive electrodes are embedded in the conductive electrode fixing holes on the upper layer and the lower layer of the body, and the conductive electrodes are tightly attached and sealed with the through holes. One side of the conductive electrode is contacted with the electrode plate, and the other side of the conductive electrode is flush with the outer surface of the outer layer of the body or protrudes from the outer surface and is connected with an electrode column of an external electric field generating device. An electric conversion liquid inlet, an electric conversion liquid outlet and a pressurizing air port are arranged on the thickness side surface of the middle layer and communicated with the outside. The upper layer and the middle layer, and the middle layer and the lower layer are respectively connected together to form a closed body. The middle layer has a cavity. A plurality of sealing check blocks can be arranged in the space of the middle layer, and the sealing check blocks and the upper layer and the lower layer are respectively sealed and bonded at intervals to form an electric conversion liquid inlet buffer zone, an electric conversion liquid outlet buffer zone, a pressurizing air port buffer zone and an electric conversion liquid cavity. Optionally, the electric rotating chip can further have a fixing structure for fixing the placement position of the electric rotating chip, such as a positioning pin. Preferably, the pressurizing air port side of the electric conversion chip is higher than the electric conversion liquid inlet and the electric conversion liquid outlet side of the electric conversion chip. The electrotransfer chip can be directly applied to continuous immunocyte electrotransfer with a certain volume, and the volume of the electrotransfer cavity is 1-5 ml, so that the problem of pollution caused by multiple open sample adding operations of a sample is avoided.

The material of the sealed body of the electrical conversion chip is usually plastic, and preferably plastic with good compatibility. The size of the electric conversion chip is matched with the size of the concave cavity of the electric conversion unit, so that after the electric conversion chip is combined with the electric conversion device, the conductive electrode of the electric conversion chip can be in good contact with the electrode column of the electric conversion device.

Therefore, the invention also provides a cell electric conversion device, which comprises an electric field generating device and an electric conversion chip arranged in the electric field generating device, and an optional supporting seat. Preferably, the electric field generating device comprises a pulse power supply, an electrode column, a cavity for accommodating the electric conversion chip and a detachable latch, wherein the latch is arranged on one side surface of the electric field generating device, which is closely fixed to the electric conversion chip, and forms the cavity for fixedly accommodating the electric conversion chip together with the other side surfaces of the electric field generating device.

The pressurizing unit comprises a pressurizing and maintaining device, an optional connecting device, an air filtering device for blocking liquid, a pipeline and a connecting device, wherein the pressurizing and maintaining device is used for injecting or releasing gas into the electrotransformation cavity and can also generate air pressure by compressing the volume of the gas, and the connecting device is used for connecting the pressurizing and maintaining device into the pipeline communicated with the pressurizing unit. The pressurized holding device comprises a gas plunger tube, a plunger rod and a stroke pushing piece. The gas plunger tube is a cylindrical gas cavity and is tightly matched with the plunger rod, wherein one side surface of the gas cavity is provided with a gas outlet communicated with the electric conversion chip, and the other side surface of the gas cavity slides along with the stroke of the plunger rod. The stroke pushing piece is used for controlling the sliding stroke of the plunger rod. The pressurizing unit also comprises a filtering device with a ventilating and liquid-blocking filter membrane, which is arranged in front of the gas plunger pipe and is used for blocking the liquid of the electric rotating unit from entering the gas plunger pipe.

The temperature control unit comprises a chip temperature control device for controlling the temperature around the electric rotary chip, and the chip temperature control device comprises a temperature control layer arranged in a supporting seat of the electric rotary unit and a temperature controller (such as a temperature control semiconductor) suitable for the temperature control layer. The temperature control layer is arranged in the supporting seat and can be tightly attached to the electric conversion chip or keep a certain distance with the electric conversion chip so as to establish a good temperature environment for controlling the temperature rise of the electric conversion. Optionally, the temperature control unit further comprises a pressurizing unit temperature control device, which is disposed around the gas plunger tube of the pressurizing unit and the pipeline communicated with the pressurizing unit, and is used for controlling the temperature of the gas in the pressurizing unit, and when the cooling gas in the pressurizing unit fills the electric liquid transfer cavity, the gas contacts and cools the electrode plate of the electric liquid transfer cavity. The temperature control device of the pressurizing unit can effectively cool the electrode plate particularly during continuous electric rotation of a large-volume sample.

The pipeline that intercommunication sample supply unit, buffer unit, electricity change unit, sample collection unit, pressure boost unit and temperature control unit, including each trunk line. The inner diameter of the pipe is not particularly limited, but may be usually 0.2mm to 10 mm. The main pipeline comprises a buffer sample introduction flow path, a liquid inlet flow path, a liquid outlet flow path and a pressurization gas path. One end of the buffer sample feeding flow path is communicated with a liquid outlet of the sample supply unit for accommodating sample containers which are not subjected to electric conversion, and the other end of the buffer sample feeding flow path is communicated with a liquid inlet of the buffer unit for accommodating sample containers to be subjected to electric conversion; one end of the liquid inlet flow path is communicated with a liquid outlet of the buffer unit for accommodating a sample container to be electrotransferred, and the other end of the liquid inlet flow path is communicated with an electrotransferred liquid inlet of the electrotransferred chip; one end of the liquid outlet flow path is communicated with an electrotransformation liquid outlet of the electrotransformation chip, and the other end is communicated with a liquid inlet of a container of the sample collection unit for accommodating a sample which completes electrotransformation; one end of the pressurization gas circuit is communicated with the pressurization gas port of the electric conversion chip, and the other end of the pressurization gas circuit is communicated with the gas outlet of the pressurization unit.

The driving unit is used for driving the liquid to flow in the pipeline. The position of the driving unit is not particularly limited as long as the sample to be electrically transferred of the sample supply unit can flow into the container containing the sample to be electrically transferred of the buffer unit through the pipeline, the sample to be electrically transferred of the buffer unit flows into the electric liquid transfer cavity of the electric liquid transfer chip through the pipeline, and the sample to be electrically transferred flows into the container containing the sample to be electrically transferred of the sample collection unit through the pipeline. The drive unit may be a drive unit conventional in the art, such as a pump; preferably, the pump is contacted and clamped with the outer surface of the liquid path pipe, such as a peristaltic pump and an infusion pump which are commonly available in the market, and the inside of the pump is not contacted with any liquid so as to avoid cross contamination among multiple samples. The number of drive units may be determined according to actual needs. In some embodiments, the buffer sample flow path, the liquid inlet flow path and the liquid outlet flow path are respectively provided with peristaltic pump pipelines which are connected with peristaltic pumps, so that the flow rates of the buffer sample flow path, the liquid inlet flow path and the liquid outlet flow path can be accurately controlled.

The control unit for controlling the communication between the sample supply unit, the buffer unit, the electrical transfer unit, the sample collection unit and the pressurizing unit may be a switch for controlling the liquid to flow into and out of each unit, which is conventional in the art, and for example, the control unit may be a liquid stop clamp and a gas valve. The number of control units can be determined according to actual needs. In some embodiments, the control unit comprises: the first control device is used for controlling the smoothness and the closure of the buffer sample injection flow path; the second control device is used for controlling the smoothness and the closure of the liquid inlet flow path; the third control device is used for controlling the liquid outlet flow path to be unblocked and closed; and the fourth control device is used for controlling the smoothness and the closing of the pressurization gas path.

Optionally, some or all of the units and pipes of the electrical conversion system of the present invention may be arranged, mounted or housed within or on a housing or rack that constitutes the electrical conversion system, and may be mounted or secured using means conventional in the art.

The cells that can be used for cell electroporation using the electroporation system, the electroporation chip or the electroporation apparatus of the present invention may be various cells known in the art, including but not limited to immune cells, stem cells, cell lines, and the like. The cell is preferably a cell of mammalian origin, more preferably a human cell. In some embodiments, the cell is selected from the group consisting of a peripheral blood cell, a hematopoietic cell, a neural stem cell, and a tumor cell. In some embodiments, the cell is selected from the group consisting of a red blood cell, a neutrophil, an eosinophil, a basophil, a peripheral blood monocyte, or a lymphocyte, and the like.

The invention also includes a method for cell electrotransformation by using the electrotransformation system, the electrotransformation chip or the electrotransformation device of the invention, comprising the following steps:

(1) preparing, including: filling mixed macromolecule and sample liquid of electric conversion liquid which needs to be electrically converted into a sample container without electric conversion, filling a gas plunger tube into a stroke pushing piece, placing an electric conversion chip in an electric field generating device, adjusting the included angle of the electric conversion device to a certain angle such as 60 degrees by a position adjusting device, starting a temperature control unit, and controlling a temperature control layer in a supporting seat to a preset temperature;

(2) a feed liquor comprising: the buffer sample introduction flow path is in a smooth state, the liquid inlet flow path, the liquid outlet flow path and the pressurization gas path are in a closed state, a sample is required to flow into a container of the sample to be electrically converted of the buffer unit under the driving of the driving unit, and the buffer unit is kept stand for a period of time if necessary to dissipate bubbles in the sample liquid and then the next step is carried out;

(3) an electro-conversion fluid comprising: the liquid inlet flow path and the pressurization gas path are in a smooth state, the buffer sample introduction flow path and the liquid outlet flow path are in a closed state, under the driving of the driving unit, sample liquid to be electrically converted flows into the liquid conversion cavity and slowly fills the liquid conversion cavity from bottom to top, and gas in the liquid conversion cavity is discharged into a gas plunger pipe of the pressurization unit through the pressurization gas path;

(4) electro-rotating, comprising: only the pressurizing gas circuit is in a smooth state, the buffer sample introduction flow path, the liquid inlet flow path and the liquid outlet flow path are in a closed state, and the stroke pushing piece of the pressurizing unit moves towards the volume direction of the compressed gas to increase and maintain the gas pressure of the electric liquid transfer cavity communicated with the gas plunger tube; at the moment, the electric field is switched on, and cell electric conversion is completed in the short-pulse high-voltage pulse electric field;

(5) collecting, comprising: the pressurizing gas circuit and the liquid outlet flow path are in a smooth state, the buffer sample introduction flow path and the liquid inlet flow path are in a closed state, and under the driving of the driving unit and the pressurizing gas circuit stroke pushing piece, sample liquid which completes electric conversion flows into a container which is used for accommodating a sample which completes electric conversion and is arranged in the sample collecting unit; the gas passing through the temperature control unit fills the liquid transferring cavity again, contacts and cools the electrode plate of the electric transferring chip;

(6) repeating the electric transfer, and subsequently repeating the steps 3-5 until the electric transfer of the required sample liquid is completed;

the sample collection unit can be cut by heat sealing with a pipeline heat sealing device.

In certain embodiments, each container of the sample supply unit, the buffer unit, the electric conversion unit, the sample collection unit, and the pressurization unit, and one or more pairs of the corresponding buffer sample introduction flow path, the liquid inlet flow path, the liquid outlet flow path, and the pressurization gas path are detachable, and the preparation step further comprises connecting the detachable container and the corresponding pipeline thereof.

In some embodiments, the electric conversion chip is detachable from the liquid inlet flow path, the liquid outlet flow path and the pressurization gas path, and the step further comprises connecting the electric conversion chip with the liquid inlet flow path, the liquid outlet flow path and the pressurization gas path.

In the present disclosure, the conventional electric rotary cup is converted into an electric rotary chip, and the electric rotary is performed in a closed state, so that continuous electric rotary, automatic control, chip space sealing and high biological safety are realized.

In this disclosure, have pressure boost gas port buffers in the electricity commentaries on classics chip, to the pressurization of electricity commentaries on classics liquid intracavity promptly, avoid the electricity to change the time and have the bubble to produce, realize promptly that the electricity changes the electric field stability, improve the electricity and change the effect.

In this disclosure, the electric rotating chip overall structure is the platykurtic and wraps up and be convenient for control the temperature in the electric rotating device, avoids the problem of plate electrode accuse temperature when a large amount of electricity changes promptly.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Unless otherwise specified, various materials of the present invention are commercially available; or prepared according to conventional methods in the art. Any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.

This embodiment describes the structures of the electric transfer system, the electric transfer unit, and the electric transfer chip of the present invention with reference to fig. 1 to 6.

As shown in fig. 1, the electric rotating system includes a sample supply unit 1, a sample supply unit access device 10, a buffer unit 2, a buffer unit liquid inlet access device 20, a buffer unit liquid outlet access device 21, an electric rotating unit 3, an electric rotating chip 30, a sample collection unit 4, a pressurizing unit 5, a filter 53, an air valve 54, a temperature control unit 6, a control device 7, a peristaltic pump 70, a peristaltic pump 71, a peristaltic pump 72, a liquid stopping clamp 101, a liquid stopping clamp 201, a liquid stopping clamp 211, a liquid stopping clamp 401, and a pipeline connecting the units. The pipeline comprises a buffer sample introduction flow path A, a liquid inlet flow path B, a liquid outlet flow path C and a pressurization gas path D. One end of the buffer sample injection flow path A is communicated with a liquid outlet of the sample supply unit 1, and the other end of the buffer sample injection flow path A is communicated with a liquid inlet of the buffer unit 2; one end of the liquid inlet flow path B is communicated with a liquid outlet of the buffer unit 2, and the other end of the liquid inlet flow path B is connected with an electric conversion liquid inlet of the electric conversion unit 3; one end of the liquid outlet flow path C is connected with the electric conversion liquid outlet of the electric conversion unit 3, and the other end is communicated with the liquid inlet of the sample collection unit 4; one end of the pressurization gas path D is connected with the pressurization gas port of the electric rotating unit 3, and the other end is connected with the gas outlet of the pressurization unit 4.

The sample supply unit 1 is a reagent bag that is packaged in advance, and has an insertable interface. The sample supply unit access device 10 can penetrate through the interface of the sample supply unit 1 to communicate the sample to the buffer sample injection flow path a. The outlet of the sample supply unit access device 10 is provided with a liquid stopping clamp 101 for controlling the smoothness and the closing of the buffer sample introduction flow path a.

The buffer unit 2 is a buffer area of the sample liquid which is not electrically converted before entering the electric conversion unit 3, and can be used for buffering the sample introduction speed of the sample supply unit 1, and also can discharge bubbles in the sample liquid, thereby preventing the bubbles in the sample liquid from directly entering the electric conversion unit 3. The buffer unit 2 is a reagent bag interposed between the sample supply unit 1 and the electrophoresis unit 3, and has an insertable interface. The buffer unit liquid inlet and

outlet device

20 and 21 can penetrate the inlet and outlet of the buffer unit 2. The buffer sample introduction flow path A and the liquid inlet flow path B connected with the buffer unit 2 are the inflow and outflow channels of the liquid in the reagent bag of the buffer unit 2. The buffer unit 2 is also provided with an air filter 22 positioned at the top of the reagent bag, and the air filter 22 is internally provided with a filter element and keeps air pressure balance with the external atmosphere.

As shown in fig. 1 and 6, the pressurizing unit 5 includes a pressurizing holding device for injecting or releasing gas into or from the electric liquid transfer chamber to generate air pressure, a filter 53 for blocking liquid, and a gas valve 54 for controlling the pressurizing gas path D to be open or closed. The pressurizing unit 5 communicates with the pressurizing gas port 3070 of the pressurizing gas buffer area 307 of the electric power conversion unit 3 through the pressurizing gas path D. The pressurizing and maintaining device comprises a gas plunger tube 50, a plunger rod 51 and a stroke pushing piece 52, wherein the gas plunger tube 50 is a cylindrical gas cavity and is tightly matched with the plunger rod 51, a gas outlet communicated with the electric conversion chip is formed in one side surface of the gas cavity, and the other side surface of the gas cavity slides along the stroke of the plunger rod. The stroke pusher 52 is used to control the sliding stroke of the plunger rod 51. The filter member 53 comprises a gas impermeable filter membrane disposed in front of the pressurized retaining means for blocking the liquid in the electric rotor unit 3 from entering the gas plunger tube 50.

The sample collecting unit 4 is used for collecting sample liquid after completing electric conversion, and liquid stopping clamps 401 are respectively arranged at the inlets of the sample collecting unit 4 and are switches for controlling the opening and closing of the liquid outlet flow path C. The sample collection unit 4 may also have an insertable interface for subsequent sampling operations.

The temperature control unit 6 includes a chip temperature control device 60 for controlling the temperature around the electric rotating chip, and the chip temperature control device 60 includes a temperature control layer disposed in the supporting base 32 of the electric rotating unit 3 and a temperature controller (such as a temperature control semiconductor) adapted to the temperature control layer. The temperature control layer is arranged in the supporting seat and can be tightly attached to the electric conversion chip or keep a certain distance with the electric conversion chip so as to establish a good temperature environment for controlling the temperature rise of the electric conversion. The temperature control unit 6 further comprises a pressurizing unit temperature control device 61 which is disposed around the gas plunger tube 50 of the pressurizing unit 5 and the pipeline communicating with the pressurizing unit, and is used for controlling the temperature of the gas in the pressurizing unit, and when the cooling gas in the pressurizing unit fills the electric liquid transfer chamber, the gas contacts and cools the electrode plate of the electric liquid transfer chamber. The temperature control device of the pressurizing unit can effectively cool the electrode plate particularly during continuous electric rotation of a large-volume sample.

The peristaltic pump pipeline is arranged on the buffer sample injection flow path A, so that the peristaltic pump 70 can be conveniently connected, and the peristaltic pump 70 can be used for accurately controlling the flow speed of the buffer sample injection flow path A. The peristaltic pump pipeline is arranged on the liquid inlet flow path B, so that the connection of the peristaltic pump 71 is facilitated, and the peristaltic pump 71 can be used for accurately controlling the flow rate of the liquid inlet flow path B. The liquid outlet flow path C is provided with a peristaltic pump pipeline, so that the peristaltic pump 72 can be conveniently connected, and the peristaltic pump 72 can be used for accurately controlling the flow rate of the liquid outlet flow path C. Peristaltic pumps 70, 71 and 72 are commercially available peristaltic pumps that do not contact any liquid to avoid cross-contamination between multiple samples.

As shown in fig. 2, the electrical rotating chip 30 includes an upper layer 302, a middle layer 300, and a lower layer 303, and the upper layer 302 and the lower layer 303 each have an electrode plate fixing slot 3010 for mounting an electrode plate and a conductive electrode fixing hole 3080 for mounting a conductive electrode, respectively. The electric rotating chip 30 further includes two electrode plates 309 and two conductive electrodes 308. The electrode plates 309 are parallel conductive thin plates, and are closely attached in parallel to the electrode plate fixing grooves 3010 of the upper layer 302 and the lower layer 303, respectively. The gap between the two electrode plates 308 is 0.5-2 mm, preferably 0.8-1.5 mm, and more preferably 1 mm. The conductive electrode 308 is tightly embedded in the conductive electrode fixing holes 3080 of the upper layer 302 and the lower layer 303, respectively, and the conductive electrode 308 is tightly attached and sealed with the conductive electrode fixing holes 3080. The conductive electrode 308 is generally cylindrical, and the cylindrical surface is wrapped by an insulating layer, and the upper and lower circular surfaces are conductive contact surfaces, one side of the conductive contact surface is in contact with the electrode plate, and the other side is parallel to or protrudes from the outer surface of the outer layer of the body and is connected with the electrode column 36 of the external electric field generating device 33. The middle layer 300 is a hollow structure, and the upper layer 302 and the middle layer 300, and the middle layer 300 and the lower layer 303 are tightly connected together to form a closed body. A plurality of sealing blocks 301 can be arranged in the space of the middle layer 300, and the sealing blocks 301 are respectively sealed and bonded with the upper layer and the lower layer at intervals to form an electric transfer liquid inlet buffer zone 305, an electric transfer liquid outlet buffer zone 306, a pressurizing air port buffer zone 307 and an electric transfer liquid cavity 304. The electric transfer liquid inlet buffer zone 305, the electric transfer liquid outlet buffer zone 306, and the pressurizing air port buffer zone 307 are all communicated with each other only through the electric transfer liquid chamber 304. The side surface of the middle layer 300 is further provided with an electric conversion liquid inlet 3050, an electric conversion liquid outlet 3060 and a pressurizing air port 3070 which are respectively communicated with the electric conversion liquid inlet buffer area 305, the electric conversion liquid outlet buffer area 306 and the pressurizing air port buffer area 307. The sealed body is communicated with the outside only through the electric liquid transfer inlet 3050, the electric liquid transfer outlet 3060 and the pressurizing air port 3070. The electric conversion liquid inlet buffer area 305 is an area to be electrically converted in the electric conversion chip 30 and can buffer the flow rate of the liquid at the electric conversion liquid inlet 3050; the electric conversion liquid outlet buffer zone 306 is a zone to be flowed out in the electric conversion chip 30 and can buffer the flow speed of the liquid at the electric conversion liquid outlet 3060; the pressurizing gas port buffer zone 307 is a buffer zone for gas after pressurization of the electrotransformation liquid cavity 304, and the buffer zone is provided with liquid and gas, so that the sample liquid in the electrotransformation liquid cavity 304 is pressurized, and the sample liquid is prevented from generating bubbles in an electrotransformation electric field. The electrotransfer chip 30 can be directly applied to continuous cell electrotransfer with a certain volume, and the volume of the electrotransfer cavity 304 is 1-5 ml, so that the problem of pollution caused by multiple open sample adding operations of a sample is avoided.

As shown in fig. 3 and 4, the electrical conversion chip 30 is placed in the electric field generating device 33 to perform electrical conversion. The locking member 35 is matched with the fixing slot 34 at the corresponding position of the electric field generating device 33 for fixing the electric rotating chip 30. The conductive electrodes 308 on both sides of the electro-rotating chip 30 are respectively in contact with the corresponding electrode posts 36 and conduct electricity.

The electric conversion unit 3 further comprises a temperature control layer 31, a support base 32, an electric field generating device 33, a locking piece 35, an electrode column 36 and a position adjusting device 37. The electric field generating means 33 comprises a cavity, the inner surface of which is attached to the temperature control layer 31. The temperature control layer 31 is provided with a chip temperature control device 60 of the temperature control unit 6 therein, and is wrapped around the electro-rotation chip 30. The cavity in the temperature control layer 31 can be used for placing the electric rotating chip 30. The electric field generating device 33 has two device notches 330 and a fixing slot 34. The device slot 330 is used for passing through the electric transfer liquid inlet 3050 and the electric transfer liquid outlet 3060. The locking member 35 is inserted into a side of the electric rotating chip 30 from the insertion groove 34, and the locking member 35 and the remaining side of the electric field generating device 33 together form a cavity for fixedly receiving the electric rotating chip 30. The locking member 35 has a locking member slot 350 formed therein for passing through the upper plenum port 3070. The flat surface of the electric field generator 33 further includes a pair of electrode posts 36, one end of which is connected to the conductive electrode 308 of the electric conversion chip 30, and the other end of which is connected to the power supply. Except for the conductive contact points, the electrode posts 36 are wrapped with an insulating layer. The support base 32 is a support fixture of the entire electric rotating unit 3. The position adjusting device 37 is arranged on the supporting seat 32, the electric field generating device 33 can rotate around the transmission shaft of the position adjusting device 37, the liquid enters the electric transfer liquid inlet buffer area 305 and the electric transfer liquid outlet buffer area 306 of the electric transfer chip 30, and then the liquid is filled into the electric transfer liquid cavity 304 from bottom to top and exhausts the air in the electric transfer liquid cavity 304, finally the liquid level rises to the pressurizing air port buffer area 307, the pressurizing unit 5 increases the air pressure, exhausts the air possibly remaining in the electric transfer liquid cavity, and maintains a certain pressure of the electric transfer liquid, thereby avoiding the generation of bubbles during electric transfer.

The electric conversion unit also comprises a supporting seat for supporting and placing the electric field generating device and the electric conversion chip. Preferably, the electric field generating device is positioned on the support at an angle such that the electric field generating device with the electric conversion chip mounted therein is placed on the support at an angle of 2 ° to 90 °, preferably 60 ° to 90 °, more preferably 60 ° to the horizontal, with respect to the horizontal, at which the sample flows into the electric conversion chip at an angle of 2 ° to 90 °, more preferably 60 °, i.e., the pressure-increasing gas port side of the electric conversion chip in the horizontal direction is higher than the electric conversion liquid inlet and the electric conversion liquid outlet side of the electric conversion chip, and preferably, the electric conversion liquid inlet and the electric conversion liquid outlet side of the electric conversion chip in the horizontal direction form an angle of 60 ° to 90 °, more preferably 60 ° to the horizontal

This example describes the process of cell electroporation using the electroporation system, the electroporation chip or the electroporation apparatus of the present invention with reference to FIGS. 1 to 6.

(1) And (4) sample preparation. The macromolecule to be electrotransferred and the sample solution of the electrotransferred solution are prepared in advance, mixed and then loaded into the sample supply unit 1. Closing the liquid stopping clamp 101, the liquid stopping clamp 201, the liquid stopping clamp 211 and the liquid stopping clamp 401, namely closing the buffer sample introduction flow path A, the liquid inlet flow path B and the liquid outlet flow path C of the pipeline at the moment; the air valve 54 is closed, that is, the pressurization air path D is in a closed state. The sample supply unit access device 10, the buffer unit liquid inlet access device 20 and the buffer unit liquid outlet access device 21 are respectively communicated with the inflow port and the outflow port of the sample supply unit 1 and the buffer unit 2 in a puncture way. Peristaltic pump tubing is placed into peristaltic pump 70, peristaltic pump 71, and peristaltic pump 72. The gas plunger tube 50 is fitted into the stroke pusher 52; the electric rotating chip 30 is placed in the electric field generating device 33, the position adjusting device 37 is arranged to enable the elevation angle of the direction of the sample flowing into the electric rotating chip and the horizontal line to be 60 degrees, the temperature control unit is started, and the temperature control layer in the supporting seat is controlled to be at the preset temperature;

(2) and (4) pre-feeding liquid. Firstly, opening the liquid stopping clamp 101 and the liquid stopping clamp 201, closing the liquid stopping clamp 211 and the liquid stopping clamp 401, closing the air valve 54, only keeping the buffer sample introduction flow path A in a smooth state, starting the peristaltic pump 70, enabling the to-be-electrotransformed liquid in the sample supply unit 1 to flow into a container of the to-be-electrotransformed sample in the buffer unit 2, after the liquid inlet is completed, closing the liquid stopping clamp 101 and the liquid stopping clamp 201, and closing the peristaltic pump 70. If necessary, the buffer unit is kept stand for a period of time, and then the next step is carried out after the bubbles in the sample liquid are dissipated;

(3) and electrically converting into liquid. And closing the liquid stopping clamp 101, the liquid stopping clamp 201 and the liquid stopping clamp 401, opening the liquid stopping clamp 211, opening the air valve 54, keeping the buffer sample introduction flow path A and the liquid outlet flow path C in a closed state, and keeping the liquid inlet flow path B and the pressurization air path D in a smooth state. Starting the peristaltic pump 71, the sample liquid to be electrically converted flows into the liquid conversion cavity 304 from the container of the sample to be electrically converted of the buffer unit 2 and slowly fills the liquid conversion cavity from bottom to top, and the gas in the liquid conversion cavity 304 is discharged into the gas plunger tube 50 of the pressurizing unit 5 through the pressurizing gas circuit D;

(4) and (6) electrically turning. And closing the liquid stopping clamp 101, the liquid stopping clamp 201, the liquid stopping clamp 211 and the liquid stopping clamp 401, and opening the air valve 54, namely, only the pressurization air path D is in a smooth state. The stroke pushing piece 52 of the pressurizing unit 5 moves towards the volume direction of the compressed gas, and the gas pressure of the electric liquid transfer cavity communicated with the gas plunger pipe 50 is increased and maintained; at the moment, the electric field is switched on, and cell electric transfer is completed in the short-pulse high-voltage pulse electric field;

(5) and (6) collecting. And closing the liquid stopping clamp 101, the liquid stopping clamp 201 and the liquid stopping clamp 211, opening the liquid stopping clamp 401, and opening the air valve 54, namely the liquid outlet flow path C and the pressurization air path D are in a smooth state. Starting the peristaltic pump 72, driving the stroke pushing piece 52 of the pressurization gas circuit to discharge gas, and allowing the sample liquid which completes electric conversion to flow into a container which contains the sample which completes electric conversion and is arranged in the sample collection unit 4; the gas passing through the temperature control unit 6 fills the charge liquid transfer cavity 304 again, contacts and cools the electrode plate of the electric transfer chip;

(6) repeating the electric transfer, and subsequently repeating the steps 3-5 until the electric transfer of the required sample liquid is completed; and finally, respectively carrying out heat sealing and intercepting on the pipelines of the sample collection unit 4 by adopting a heat sealing machine, and completing electrotransformation.

Claims

1. An electrotransfer chip, comprising:

the electric-to-liquid conversion device comprises a closed body communicated with the outside through an electric-to-liquid conversion inlet, an electric-to-liquid conversion outlet and a pressurization gas port, wherein the closed body is internally provided with an electric-to-liquid conversion inlet buffer area, an electric-to-liquid conversion outlet buffer area, a pressurization gas port buffer area and an electric-to-liquid conversion cavity;

electrode plates which are arranged on two opposite planes of the electrotransformation cavity in parallel; and

2. The chip according to claim 1, wherein the cavity is a flat cavity, and preferably the volume of the cavity is 1-5 ml; preferably, the parallel cross section of the cavity comprises a circular middle part, one side of the contour of the circular middle part is connected to the electric conversion liquid inlet buffer area and the electric conversion liquid outlet buffer area in a circular arc smooth transition mode, and the other side of the contour of the circular middle part is connected to the pressurizing air port buffer area in a circular arc smooth transition mode.

3. An electrotransfer chip according to claim 1 or claim 2, comprising a sealing barrier for separating the electrotransfer inlet buffer zone, the electrotransfer outlet buffer zone, the pressurising gas port buffer zone and the electrotransfer chamber.

4. An electrical conversion chip according to any of claims 1 to 3, wherein the gap between the electrode plates is 0.5 to 2mm, preferably 0.8 to 1.5mm, more preferably 1 mm.

5. The electric conversion chip according to any one of claims 1 to 4, wherein the closed body comprises a body upper layer, a body middle layer and a body lower layer which are closely attached, wherein the body middle layer is provided with a cavity comprising an electric conversion liquid inlet buffer zone, an electric conversion liquid outlet buffer zone, a pressurizing air port buffer zone and an electric conversion liquid cavity; the electrode plate is inlayed respectively in body upper strata and body lower floor, the conducting electrode inserts respectively the conducting electrode in body upper strata and the body lower floor is fixed and with the electrode fixed orifices closely laminates, preferably, a conducting electrode side and electrode plate contact, and parallel or protruding from airtight body surface between opposite side and the airtight body surface for be connected with outside electric field generating device that changes.

6. A cell electroporation device comprising an electric field generating device and the electroporation chip according to any one of claims 1 to 4 disposed inside the electric field generating device.

7. The cell electric conversion device according to claim 6, wherein the electric field generating device comprises a pulse power supply, an electrode column, a cavity for accommodating the electric conversion chip and a detachable lock catch, and the lock catch is arranged on one side surface of the electric field generating device for closely fixing the electric conversion chip; the cavity is formed by the lock catch and the rest side surfaces of the electric field generating device together to form a cavity space for fixedly accommodating the electric conversion chip.

8. The cell electro-transformation device according to claim 6 or 7, further comprising a support base for supporting and placing the electric field generation device and the electro-transformation chip, and optionally a position adjustment device for adjusting an elevation angle between the electro-transformation chip and a horizontal line; preferably, after the electric relay device with the electric relay chip mounted therein is placed on the support base, the pressurizing air port side of the electric relay chip in the horizontal direction is higher than the electric relay liquid inlet and the electric relay liquid outlet side of the electric relay chip, and preferably, the electric relay liquid inlet and the electric relay liquid outlet side of the electric relay chip in the horizontal direction form an angle of 2 ° to 90 °, preferably 60 ° to 90 °, and more preferably 60 ° to the horizontal line.

9. A cell electroporation system comprising an electroporation unit comprising an electroporation chip according to any one of claims 1 to 5 or the cell electroporation device according to any one of claims 6 to 8.

10. The cell electroporation system of claim 9, further comprising: