CN220079090U - Immune cell electrotransfection micro-fluidic chip based on electrode array - Google Patents

Abstract

The utility model relates to an immune cell electrotransfection micro-fluidic chip based on an electrode array, which comprises the following components: the upper surface of the substrate is provided with an electrode group, the electrode group comprises a plurality of columns of sub-electrodes, and the sub-electrodes comprise an upper excitation electrode, a plurality of electric suspension electrodes and a lower excitation electrode; the two ends of the cover plate are respectively provided with an inlet and an outlet which penetrate through the wall thickness of the cover plate, the lower surface of the cover plate is provided with a recessed micro-channel, and the micro-channel is communicated with the inlet and the outlet; the micro channel comprises a main channel and a channel group, the channel group comprises a branch channel and a plurality of rows of sub-channels, the sub-channels comprise a plurality of micro grooves which are distributed at intervals, and the micro grooves are respectively connected with the main channel through the branch channel; the lower surface of the cover plate is contacted with the upper surface of the substrate, and the sub-electrodes are arranged in one-to-one correspondence with the sub-channels; the two ends of the micro-groove are respectively positioned above the adjacent upper excitation electrode and the adjacent electric suspension electrode, above the adjacent lower excitation electrode and the adjacent electric suspension electrode or above the adjacent two electric suspension electrodes. The utility model can realize high-efficiency and controllable cell electrotransfection.

Description

Immune cell electrotransfection micro-fluidic chip based on electrode array

Technical Field

The utility model relates to the technical field of cell transfection, in particular to an immune cell electrotransfection micro-fluidic chip based on an electrode array.

Background

Gene therapy and immune cell therapy are the current hot research directions in the biomedical field, and the core is to carry out gene modification or drug treatment on cells so as to improve or cure diseases.

Electrotransfection, also known as electroporation, is a commonly used technique for instantaneously breaking down cell membranes by generating high-intensity electrical pulses to allow foreign molecules in the environment to enter the cell. The electrotransfection method has the advantages of simple operation, high repeatability, high transfection efficiency and the like. In bioengineering, cell perforation can be used to introduce anticancer drugs and genes into tumor cells for the treatment of tumors. In the aspect of genetic engineering, the technology can be applied to obtain transgenic new quality animals and plants. Therefore, the electrotransfection technology has excellent application prospect in biology, medicine and the like.

Traditional electrotransfection methods for cells mainly include flow electrotransfection techniques and single cell electrotransfection techniques. In the flow electrotransfection technology, the permeability of a cell membrane is enhanced by utilizing proper electric field strength under the flowing state of cell fluid, and exogenous molecules are introduced into cells through pores on the surface of the cell membrane. However, the method needs to continuously electric shock to cells, is easy to cause irreversible damage to cell membranes, and has high cell death rate. Single cell electrotransfection techniques are techniques that are more precisely controlled for electroporation of single cells. Compared with the electrotransfection of cells in a population, the single-cell electrotransfection technology does not need to continuously shock the cells, and simultaneously the perforation voltage can be greatly reduced to reduce the damage to the cells, but the electrotransfection efficiency is lower and the possibility of off-target is also caused.

In recent years, microfluidic chip technology has been widely used, and the advantages of high integration, small volume, high throughput, low cost and the like make it a powerful tool for cell manipulation and cell research. The micro-fluidic system is a micro device and a micro-fluidic system capable of processing fluid, integrates operation functional units such as separation, mixing, filtration, purification and reaction on biological samples in the prior art on a chip, generates and operates liquid in a closed micro-channel network, can control micro-liter of fluid to construct an integrated, miniaturized and automated chemical biological platform, and has the advantages of small volume, portability, small reagent usage and measurement, quick reaction, parallel processing and automation and the like.

The electric force is the main form of current microfluid distribution force driving, the microfluid chip passes through an alternating current electric field, so that polarized dielectric particles can generate dielectrophoresis motion under the action of dipole moment to capture cells, alternating current electric heating flow vortex is formed by utilizing the alternating current electric heating coupling effect, nucleic acid is wrapped in the vortex, and the nucleic acid enters the cells through electroporation, so that the electric transfection of the cells is realized. The single channel power-up used by the traditional microfluidic chip has the defects that the occupied volume of the electrode is too large, the multichannel operation cannot be realized, the channel is large, the transfection rate is too low, and the single channel utilization rate provided by the single chip and the electrode is relatively low.

However, the microfluidic chips currently on the market have the following problems in cell capture and electrotransfection: cell transfection is inefficient and uncontrollable. Therefore, it is necessary to develop a novel immune cell electrotransfection micro-fluidic chip based on an electrode array to realize efficient and controllable cell transfection operation.

Disclosure of Invention

Therefore, the utility model aims to solve the technical problems of low cell transfection efficiency and uncontrollable cell transfection in the prior art.

In order to solve the technical problems, in one aspect, the utility model provides an immune cell electrotransfection micro-fluidic chip based on an electrode array, comprising:

the upper surface of the substrate is provided with an electrode group, the electrode group comprises a plurality of columns of sub-electrodes, and the sub-electrodes comprise an upper excitation electrode, a plurality of electric suspension electrodes and a lower excitation electrode; the upper excitation electrode, the plurality of electric suspension electrodes and the lower excitation electrode are sequentially arranged at intervals in the connecting line direction of the upper excitation electrode and the lower excitation electrode;

the cover plate is arranged at the top of the substrate, two ends of the cover plate are respectively provided with an inlet and an outlet which penetrate through the wall thickness of the cover plate, the lower surface of the cover plate is provided with a recessed micro-channel, and the micro-channel is communicated with the inlet and the outlet; the micro channel comprises a main channel and a channel group, the channel group comprises a branch channel and a plurality of rows of sub-channels, the sub-channels comprise a plurality of micro grooves which are distributed at intervals, and the micro grooves are respectively connected with the main channel through the branch channel;

the lower surface of the cover plate is contacted with the upper surface of the substrate, and the sub-electrodes are arranged in one-to-one correspondence with the sub-channels; a micro groove positioned at one end of the sub-channel, and two ends of the micro groove are respectively positioned above the adjacent upper excitation electrode and the electric suspension electrode; the micro groove is positioned at the other end of the sub-channel, and the two ends of the micro groove are respectively positioned above the adjacent lower excitation electrode and the electric suspension electrode; and the two ends of the micro groove are respectively positioned above the two adjacent electric suspension electrodes.

In one embodiment of the utility model, the electrode group is a plurality of sub-electrodes which are arranged at intervals in the X direction, and the X direction is parallel to the connecting line of the inlet and the outlet;

the channel groups are also a plurality of, the channel groups are arranged in one-to-one correspondence with the electrode groups, and the channel groups are communicated through the main channels.

In one embodiment of the utility model, a plurality of electrically suspended electrodes in a plurality of columns of sub-electrodes are arranged in a rectangular array;

the micro grooves in the multiple rows of sub-channels are arranged in a rectangular array.

In one embodiment of the utility model, upper excitation electrodes of the multiple columns of sub-electrodes are electrically connected; the lower excitation electrodes of the multiple columns of sub-electrodes are electrically connected.

In one embodiment of the utility model, the upper and lower excitation electrodes in the sub-electrodes are electrically connected to an external signal source through wires, respectively.

In one embodiment of the utility model, the utility model further comprises a microinjection pump in communication with the inlet through a hose.

In one embodiment of the utility model, the utility model further comprises a collecting member, which communicates with the outlet through a hose.

In one embodiment of the utility model, the main channel, the branch channel and the micro-groove are the same depth.

In one embodiment of the utility model, the widths of the main channel, the branch channel, and the micro-groove are the same.

In one embodiment of the utility model, the cover sheet is made of PDMS; and/or the number of the groups of groups,

the substrate is made of glass.

Compared with the prior art, the technical scheme of the utility model has the following advantages:

the utility model relates to an immune cell electrotransfection micro-fluidic chip based on an electrode array, which is characterized in that a plurality of columns of sub-electrodes are arranged on the upper surface of a substrate, and each column of sub-electrodes comprises an upper excitation electrode, a plurality of electric suspension electrodes and a lower excitation electrode. Therefore, the upper excitation electrode, the plurality of electric suspension electrodes and the lower excitation electrode are sequentially arranged at intervals, and more uniform electric field distribution can be formed between the upper excitation electrode and the lower excitation electrode after the pulse signal source is introduced into the upper excitation electrode and the lower excitation electrode, so that in the electric transfection, the cell survival rate is improved, and the electric transfection efficiency is further improved. In this embodiment, a plurality of columns of sub-channels are disposed on the lower surface of the cover plate, each column of sub-channels is provided with a plurality of micro-grooves arranged at intervals, and two ends of each micro-groove are located above the adjacent upper excitation electrode and the adjacent electric suspension electrode, above the adjacent two electric suspension electrodes or above the adjacent lower excitation electrode and the adjacent electric suspension electrode, after the high-frequency signal source is introduced into the upper excitation electrode and the lower excitation electrode, the cell sample flowing in the micro-channel is captured into the micro-groove and positioned into the micro-groove. This enables a controlled electrotransfection of cells by capturing and positioning the cell sample into the microwell prior to electrotransfection.

Drawings

In order that the utility model may be more readily understood, a more particular description of the utility model will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings, in which

FIG. 1 is a schematic diagram of an immune cell electrotransfection micro-fluidic chip based on an electrode array in a preferred embodiment of the utility model;

FIG. 2 is an exploded view of an immunocyte electrotransfection microfluidic chip based on an electrode array in a preferred embodiment of the utility model;

FIG. 3 is a front view of an immune cell electrotransfection microfluidic chip of FIG. 1 based on an electrode array;

FIG. 4 is a B-B cross-sectional view of an immune cell electrotransfection microfluidic chip based on the electrode array shown in FIG. 3;

FIG. 5 is a schematic diagram of a plurality of electrode sets in an electrode array-based immune cell electrotransfection microfluidic chip of FIG. 1;

FIG. 6 is a top view of a cover sheet in the electrode array-based immune cell electrotransfection microfluidic chip of FIG. 1;

FIG. 7 is a schematic view of a projection of a plurality of electrode sets and micro-channels on a cover plate in the immunocyte electrotransfection microfluidic chip shown in FIG. 1 based on an electrode array;

FIG. 8 is a three-dimensional schematic diagram of an immune cell electrotransfection microfluidic chip based on the electrode array shown in FIG. 7;

FIG. 9 is a schematic diagram showing steps of an immunocyte electrotransfection method according to a preferred embodiment of the present utility model.

Description of the specification reference numerals: 100. a substrate; s, an electrode group; 110. a sub-electrode; 111. an upper excitation electrode; 112. an electrically suspended electrode; 113. a lower excitation electrode;

200. a cover plate; 210. an inlet; 220. an outlet; 230. a main channel; m, a channel group; 240. a branch channel; 250. a sub-channel; 251. a micro-groove;

300. and (5) a line.

Detailed Description

The utility model will be further described in connection with the accompanying drawings and specific examples which are set forth so that those skilled in the art will better understand the utility model and will be able to practice it, but the examples are not intended to be limiting of the utility model.

Referring to fig. 1 to 8, in one aspect, the utility model provides an immune cell electrotransfection micro-fluidic chip based on an electrode array, comprising:

the upper surface of the substrate 100 is provided with an electrode set S, the electrode set S comprises a plurality of columns of sub-electrodes 110, and the sub-electrodes 110 comprise an upper excitation electrode 111, a plurality of electric suspension electrodes 112 and a lower excitation electrode 113; the upper excitation electrode 111, the plurality of electric suspension electrodes 112 and the lower excitation electrode 113 are sequentially arranged at intervals in the connection direction of the upper excitation electrode 111 and the lower excitation electrode 113; the connection line between the upper excitation electrode 111 and the lower excitation electrode 113 may be a straight line or a curved line; the upper excitation electrode 111 and the lower excitation electrode 113 are energized to form an electric field in which the plurality of electrically suspended electrodes 112 are located;

the cover plate 200 is arranged at the top of the substrate 100, two ends of the cover plate 200 are respectively provided with an inlet 210 and an outlet 220 penetrating through the wall thickness of the cover plate 200, the lower surface of the cover plate 200 is provided with a recessed micro-channel, and the micro-channel is communicated with the inlet 210 and the outlet 220; the micro channel comprises a main channel 230 and a channel group M, wherein the channel group M comprises a branch channel 240 and a plurality of columns of sub-channels 250, the sub-channels 250 comprise a plurality of micro grooves 251 which are arranged at intervals, and the micro grooves 251 are respectively connected with the main channel 230 through the branch channels 240;

wherein, the lower surface of the cover plate 200 contacts with the upper surface of the substrate 100, and the sub-electrodes 110 are arranged in one-to-one correspondence with the sub-channels 250; a micro groove 251 at one end of the sub-channel 250, both ends of which are respectively located above the adjacent upper excitation electrode 111 and electric suspension electrode 112; a micro groove 251 at the other end of the sub-channel 250, both ends of which are respectively located above the adjacent lower excitation electrode 113 and electric suspension electrode 112; the micro-groove 251 located in the middle of the sub-channel 250 has two ends respectively located above the adjacent two electrically suspended electrodes 112.

In the present embodiment, during use, the cell sample (including cells and cell suspension, including nucleic acid therein) is injected into the inlet 210 by the microinjection pump. As the microinjection pump continues to inject the cell sample into the inlet 210, the cell sample flows in the microchannel and out the outlet 220.

It should be noted that, in this embodiment, a plurality of columns of sub-channels 250 are disposed on the lower surface of the cover 200, each column of sub-channels 250 is provided with a plurality of micro-grooves 251 arranged at intervals, and a plurality of columns of sub-electrodes 110 are disposed on the upper surface of the substrate 100, each column of sub-electrodes 110 includes an upper excitation electrode 111, a plurality of electrically floating electrodes 112 and a lower excitation electrode 113. The sub-electrodes 110 are arranged in one-to-one correspondence with the sub-channels 250; a micro groove 251 at one end of the sub-channel 250, both ends of which are respectively located above the adjacent upper excitation electrode 111 and electric suspension electrode 112; a micro groove 251 at the other end of the sub-channel 250, both ends of which are respectively located above the adjacent lower excitation electrode 113 and electric suspension electrode 112; a micro groove 251 positioned in the middle of the sub-channel 250, two ends of which are respectively positioned above two adjacent electric suspension electrodes 112; that is, the cell sample located in each micro-groove 251 is set up between the adjacent upper excitation electrode 111 and the electric suspension electrode 112, between the adjacent electric suspension electrodes 112, or between the adjacent electric suspension electrodes 112 and the lower excitation electrode 113. During the flow of the cell sample in the microchannel, the upper excitation electrode 111 and the lower excitation electrode 113 are connected with high-frequency signals, and cells in the cell sample are captured to the electric suspension electrode 112 and positioned in the micro-groove 251. Then, the high-frequency signal is changed to a pulse signal source, and an electric field distribution is formed between the upper excitation electrode 111 and the lower excitation electrode 113, and a plurality of electric suspension electrodes 112 are positioned in the electric field distribution, so that electric potentials exist between the adjacent upper excitation electrode 111 and the electric suspension electrodes 112, between the adjacent electric suspension electrodes 112 and the lower excitation electrode 113, and thus the permeability of the cytoplasmic membrane captured and positioned in each micro groove 251 is changed, the surface of the cytoplasmic membrane is perforated, and nucleic acid in the cell suspension is introduced into the cell, thereby realizing the electric transfection of the cell.

Specifically, the present embodiment provides a plurality of columns of sub-electrodes 110 on the upper surface of the substrate 100, each column of sub-electrodes 110 including an upper excitation electrode 111, a plurality of electrically floating electrodes 112, and a lower excitation electrode 113. In this way, the upper excitation electrode 111, the plurality of electric suspension electrodes 112 and the lower excitation electrode 113 are sequentially arranged at intervals, and a more uniform electric field distribution is formed between the upper excitation electrode 111 and the lower excitation electrode 113 after the pulse signal source is introduced into the upper excitation electrode 111 and the lower excitation electrode 113, so that in the electric transfection, the cell survival rate is improved, and the electric transfection efficiency is further improved. In this embodiment, a plurality of columns of subchannels 250 are disposed on the lower surface of the cover 200, each column of subchannels 250 is provided with a plurality of micro-grooves 251 arranged at intervals, and two ends of each micro-groove 251 are located above the adjacent upper excitation electrode 111 and the adjacent electric suspension electrode 112, above the adjacent two electric suspension electrodes 112, or above the adjacent lower excitation electrode 113 and the adjacent electric suspension electrode 112, after the high-frequency signal source is introduced into the upper excitation electrode 111 and the lower excitation electrode 113, the cell sample flowing in the micro-channel is captured into the micro-groove 251 and positioned into the micro-groove 251. This enables a controlled electrotransfection of cells by capturing the cell sample into the micro-well 251 and positioning it into the micro-well 251 prior to electrotransfection.

Therefore, the embodiment can realize efficient and controllable cell electrotransfection, and has important significance for researching biological processes in cells, developing gene therapy and the like.

Further, the electrode group S is plural, the plural sub-electrodes 110 are arranged at intervals in the X direction, and the X direction is parallel to the line connecting the inlet 210 and the outlet 220;

the number of the channel groups M is also plural, the channel groups M are arranged in one-to-one correspondence with the electrode groups S, and the channel groups M are communicated through the main channel 230.

Specifically, the electrode set S of this embodiment is plural, so that capturing and positioning of cells and electrotransfection can be performed on the cell sample plural times; further improving the efficiency of electrotransfection.

Further, the plurality of electrically floating electrodes 112 in the plurality of columns of sub-electrodes 110 are arranged in a rectangular array;

the plurality of micro grooves 251 in the plurality of columns of sub-channels 250 are arranged in a rectangular array.

Specifically, in this embodiment, the plurality of electric suspension electrodes 112 are arranged in a rectangular array, and the plurality of micro-grooves 251 are arranged in a rectangular array, so that the non-uniformity of the electric field can be reduced, the transfection efficiency can be improved, and the damage and toxicity to cells can be reduced.

Further, the upper excitation electrodes 111 of the plurality of columns of sub-electrodes 110 are electrically connected; the lower excitation electrodes 113 of the columns of sub-electrodes 110 are electrically connected.

Further, the upper and lower excitation electrodes 111 and 113 in the sub-electrode 110 are electrically connected to external signal sources through lines 300, respectively.

Specifically, in this embodiment, the upper excitation electrode 111 and the lower excitation electrode 113 are electrically connected to an external signal source through the lines 300, respectively, so that it is convenient to control the on-off of the lines 300 and the external signal source, thereby realizing whether the upper excitation electrode 111 and the lower excitation electrode 113 are connected to a power supply.

Further, the depth of the main channel 230, the sub channel 240, and the micro grooves 251 is the same.

Specifically, the depths of the main channel 230, the sub channel 240 and the micro groove 251 are the same in this embodiment, which facilitates the processing of the main channel 230, the sub channel 240 and the micro groove 251.

Further, the depth of the main channel 230, the sub channel 240, and the micro grooves 251 is 30 to 60 μm. Alternatively, the depth of the main channel 230, the sub channel 240, and the micro grooves 251 is 40 μm.

Further, the widths of the main channel 230, the sub channel 240, and the micro grooves 251 are the same.

Specifically, the widths of the main channel 230, the sub channel 240 and the micro groove 251 in the present embodiment are the same, which facilitates the processing of the main channel 230, the sub channel 240 and the micro groove 251.

Further, the width of the main channel 230, the sub channel 240, and the micro grooves 251 is 30 to 100 μm. Alternatively, the width of the main channel 230, the sub channel 240, and the micro grooves 251 is 40 μm.

Specifically, the width of the main channel 230, the branch channel 240 and the micro groove 251 in this embodiment is 30-100 μm, which facilitates the flow of cells in the micro channel.

The thickness of the cover 200 is 2mm.

The thickness of the substrate 100 is 1.1mm. The thickness of the electrode set S is negligible.

Further, the cover 200 is made of PDMS.

Further, the substrate 100 is made of glass.

Further, the cover 200 is made of PDMS; the substrate 100 is made of glass.

Further, the present utility model also includes a microinjection pump, which communicates with the inlet 210 through a hose.

Specifically, the present embodiment provides a micro syringe pump connected to the inlet 210, so that a full-automatic micro experiment can be realized. Namely, the shell can control the cells by controlling the input speed of the microinjection pump, the cells and the nucleic acid are captured to react in the same area during transfection, the transfection efficiency and the nucleic acid utilization rate are improved, and the cells can be observed through the WIFI digital microscope.

Further, the present utility model also includes a collection member in communication with the outlet 220 via a hose.

In particular, the present embodiment provides a collection member in communication with the outlet 220, which facilitates collection of the cell sample after electrotransfection is completed.

Referring to fig. 9, in another aspect, the utility model provides an immune cell electrotransfection method, which uses the immune cell electrotransfection micro-fluidic chip based on the electrode array in the above embodiment to perform electrotransfection, the steps include:

capturing and positioning cells, injecting a cell sample (the cell sample comprises cells and cell suspension) into an inlet 210 of the microfluidic chip, connecting a high-frequency signal source to an upper excitation electrode 111 and a lower excitation electrode 113 of the sub-electrode 110, capturing the cells to the electric suspension electrode 112 and positioning the cells into the micro-groove 251;

electric transfection, switching a high-frequency signal into a pulse signal source, setting the voltage of the pulse signal source to a preset value, and forming electric field distribution among the upper excitation electrode 111, the plurality of electric suspension electrodes 112 and the lower excitation electrode 113 of the sub-electrode 110; under the electric field distribution, the permeability of the cytoplasmic membrane positioned in the micro groove 251 is changed, the surface of the cytoplasmic membrane is perforated, and the nucleic acid in the cell suspension is introduced into the cell, so that the electrotransfection of the cell is realized.

Specifically, in this embodiment, the upper excitation electrode 111 and the lower excitation electrode 113 of the sub-electrode 110 are connected to a high-frequency signal source, so as to capture cells to the electric suspension electrode 112 and position the cells in the micro-groove 251, thereby realizing dynamic capture of cells; the high-frequency signal is switched to a pulse signal source, and electric field distribution is formed among the upper excitation electrode 111, the plurality of electric suspension electrodes 112 and the lower excitation electrode 113 of the sub-electrode 110, so that static electric transfection is realized. Therefore, the embodiment combines dynamic capture cells and static electrotransfection, and improves the controllability and the survival rate of cell transfection; and the synchronous capturing and transfection of cells and nucleic acid are realized by utilizing the principles of dielectrophoresis, fluid vortex generated by alternating-current electric heating coupling effect and the like. In the embodiment, the capturing and positioning steps of the cells go from the electrotransfection step to the gradual conversion of the electric signals from high-frequency signals to pulse signals, so that the frequency, amplitude and pulse width of the electric field are sequentially reduced to adapt to the specific requirements of the cells; thereby further improving the controllability and the survival rate of cell transfection.

The utility model realizes synchronous capturing and transfection of cells and nucleic acid, and fills the blank of the prior art. The utility model is suitable for biomedical fields such as basic medical research, immunotherapy, gene modification, biopharmaceuticals and the like.

Further, during the capture and localization of the cells, the voltage of the high frequency signal is changed (e.g., increased), or the flow rate of the cell sample is reduced. For example, the cell sample is injected into the inlet 210 by the microinjection pump, and the flow rate of the cell sample can be controlled by controlling the injection speed of the microinjection pump. According to the embodiment, the cells can be controlled through the adjusted voltage and the flow rate, the cells and the nucleic acid are captured to react in the same area during transfection, the transfection efficiency and the nucleic acid utilization rate are improved, the cells can be observed through a WIFI digital microscope, the cells can be released through adjusting the voltage after the successful transfection, and the controllability is high.

In particular, this allows to modulate the electrical conductivity of the cell suspension in which the cells of the cell sample are immersed, thus increasing the activity of the cells and thus the efficiency of cell capture.

Further, during the electrotransfection process, the width, amplitude and frequency of the pulse signal may be adjusted.

Specifically, the width, amplitude and frequency of the pulse signal in this embodiment can be adjusted, so that different widths, amplitudes and frequencies can be selected for different cells, thereby increasing the transfection activity of the cells and improving the cell transfection survival rate (because the electrical characteristics of different cells are different).

Further, preprocessing the microfluidic chip before capturing and positioning the cells;

the microfluidic chip pretreatment comprises: firstly, sterilizing and cleaning a microfluidic chip; in some embodiments, the microfluidic chip is sterilized with 70% ethanol solution, the surface of the microfluidic chip is washed with sterile physiological saline, then a physiological saline solution containing 2% BSA is added to the microfluidic chip for incubation for 30 minutes, and the remaining BSA solution is removed; washing the microfluidic chip with physiological saline; then, a high-frequency signal source and a pulse signal source are connected to the micro-fluidic chip, the frequency of the high-frequency signal is 1MHz, and the voltage of the high-frequency signal is 10V; the frequency of the pulse signal source is 1kHz, and the voltage of the pulse signal source is 1.5V.

Specifically, the purpose of the pretreatment of the microfluidic chip in this embodiment is to form a stable electrochemical interface on the surface of the electrode set S and remove the oxide on the surface, so as to ensure the electrotransfection effect.

Further, cell sample preparation is performed before the microfluidic chip pretreatment, and the cell sample preparation includes: culturing the cell sample to 70% -80% density, collecting cells, centrifuging to remove supernatant; then, the cells were resuspended in physiological saline to a cell density of 1X 10≡7cells/mL.

Specifically, cell sample preparation is performed to ensure the activity of the cell sample, thereby further improving the capturing efficiency of the cells.

Further, the isolation and analysis of cells after electrotransfection is performed, and includes: the microfluidic chip was washed with physiological saline, and cells were collected and centrifuged and analyzed. For example, cell morphology changes are observed, gene expression levels are detected, and the like.

In summary, in some comparative examples, cell electrotransfection has problems of low transfection efficiency, cell damage, etc. due to the non-uniformity of electric field distribution, so that the production cost is high and the utilization rate is poor. Therefore, the utility model provides an electrotransfection micro-fluidic chip, which utilizes the characteristic that the array of the electric suspension electrodes 112 can generate induced potential through external electric field polarization, and the purpose of combining the advantages of the flow electrotransfection and single cell electrotransfection is achieved by increasing the number of the micro-grooves 251.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present utility model will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious changes and modifications which are extended therefrom are still within the scope of the utility model.

Claims (10)

1. An immune cell electrotransfection micro-fluidic chip based on an electrode array is characterized in that: comprising the following steps:

the electrode group comprises a plurality of columns of sub-electrodes, wherein each sub-electrode comprises an upper excitation electrode, a plurality of electric suspension electrodes and a lower excitation electrode; the upper excitation electrode, the plurality of electric suspension electrodes and the lower excitation electrode are sequentially arranged at intervals in the connection line direction of the upper excitation electrode and the lower excitation electrode;

the cover plate is arranged at the top of the substrate, an inlet and an outlet which penetrate through the wall thickness of the cover plate are respectively arranged at two ends of the cover plate, a recessed micro-channel is arranged on the lower surface of the cover plate, and the micro-channel is communicated with the inlet and the outlet; the micro channel comprises a main channel and a channel group, the channel group comprises a branch channel and a plurality of rows of sub-channels, the sub-channels comprise a plurality of micro grooves which are distributed at intervals, and the micro grooves are respectively connected with the main channel through the branch channel;

the lower surface of the cover plate is in contact with the upper surface of the substrate, and the sub-electrodes are arranged in one-to-one correspondence with the sub-channels; a micro groove positioned at one end of the sub-channel, wherein two ends of the micro groove are respectively positioned above the upper excitation electrode and the electric suspension electrode which are adjacent; the micro groove is positioned at the other end of the sub-channel, and two ends of the micro groove are respectively positioned above the lower excitation electrode and the electric suspension electrode which are adjacent; and the two ends of the micro groove are respectively positioned above the two adjacent electric suspension electrodes.

2. The electrode array-based immune cell electrotransfection microfluidic chip of claim 1, wherein: the electrode groups are multiple, the multiple sub-electrodes are arranged at intervals in the X direction, and the X direction is parallel to the connecting line of the inlet and the outlet;

the number of the channel groups is also multiple, the channel groups are arranged in one-to-one correspondence with the electrode groups, and the channel groups are communicated through the main channel.

3. The electrode array-based immune cell electrotransfection microfluidic chip of claim 2, wherein:

the plurality of electric suspension electrodes in the plurality of columns of sub-electrodes are arranged in a rectangular array;

the micro grooves in the multiple rows of sub-channels are arranged in a rectangular array.

4. The electrode array-based immune cell electrotransfection microfluidic chip of claim 3, wherein: the upper excitation electrodes of the multiple columns of sub-electrodes are electrically connected; the lower excitation electrodes of the multiple columns of sub-electrodes are electrically connected.

5. The electrode array-based immune cell electrotransfection microfluidic chip of claim 4, wherein: the upper excitation electrode and the lower excitation electrode in the sub-electrodes are respectively and electrically connected with an external signal source through circuits.

6. The electrode array-based immune cell electrotransfection microfluidic chip of claim 1, wherein: and the micro-injection pump is connected with the inlet through a hose.

7. The electrode array-based immune cell electrotransfection microfluidic chip of claim 6, wherein: and a collecting member which communicates with the outlet through a hose.

8. The electrode array-based immune cell electrotransfection microfluidic chip of claim 1, wherein: the depths of the main channel, the branch channel and the micro groove are the same.

9. The electrode array-based immune cell electrotransfection microfluidic chip of claim 1, wherein: the widths of the main channel, the branch channel and the micro groove are the same.

10. The electrode array-based immune cell electrotransfection microfluidic chip of claim 1, wherein: the cover plate is made of PDMS; and/or the number of the groups of groups,

the substrate is made of glass.