CN116606733A - Electrode-array-based immune cell electrotransfection microfluidic chip and electrotransfection method - Google Patents

Abstract

The application relates to an immune cell electrotransfection micro-fluidic chip based on an electrode array and an electrotransfection method, comprising the following steps: 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 application can realize high-efficiency and controllable cell electrotransfection.

Description

Electrode-array-based immune cell electrotransfection microfluidic chip and electrotransfection method

technical field

The invention relates to the technical field of cell transfection, in particular to an electrode array-based immune cell electrotransfection microfluidic chip and an electrotransfection method.

Background technique

Gene therapy and immune cell therapy are currently hot research directions in the field of biomedicine, the core of which is to conduct gene modification or drug treatment on cells to improve or cure diseases.

Electrotransfection technology, also known as electroporation, is a commonly used transfection technology that instantly breaks down the cell membrane by generating high-intensity electrical pulses, allowing exogenous molecules in the environment to enter the cell. The electroporation method has the advantages of simple operation, high reproducibility, and high transfection rate. In terms of bioengineering, cell perforation can be used to introduce anticancer drugs and anticancer genes into tumor cells to treat tumors. In terms of genetic engineering, this technology can be applied to obtain transgenic new quality animals and plants. Therefore, electrotransfection technology has excellent application prospects in biology and medicine.

Traditional electrotransfection methods for cells mainly include flow electrotransfection technology and single cell electrotransfection technology. Flow electrotransfection technology is to use the appropriate electric field strength to enhance the permeability of the cell membrane under the flowing state of the cell fluid, and introduce foreign molecules into the cell through the pores on the surface of the cell membrane. However, since this method needs to continuously shock the cells, it is easy to cause irreversible damage to the cell membrane, and the cell death rate is high. Single cell electrotransfection technology is a more precise and controllable electroporation technology for single cells. Compared with the electrotransfection of group cells, the single cell electrotransfection technology does not need to continuously shock the cells, and the perforation voltage can be greatly reduced to reduce the damage to the cells, but this also makes the electrotransfection efficiency low and has The possibility of missing the target.

In recent years, microfluidic chip technology has been widely used, and its advantages such as high integration, small volume, high throughput, and low cost make it a powerful tool for cell manipulation and cell research. The microfluidic system is a micro-device and system capable of handling fluids. It integrates traditional operating functional units such as separation, mixing, filtration, and purification of biological samples on a single chip, generates and operates liquids in a closed microchannel network, and can manipulate microliters. An integrated, miniaturized, and automated chemical-biological platform is built with fluids, which have the advantages of small size, easy portability, small reagent usage, fast response, parallel processing, and automation.

Electromotive force is currently the main form of microfluidic distributed force drive. The microfluidic chip uses an alternating electric field to make the polarized dielectric particles generate dielectrophoresis movement under the action of the dipole moment to achieve the capture of cells. The effect forms an alternating current heat flow vortex, and the nucleic acid is trapped in the vortex, and enters the cell through electroporation, thereby realizing the electrotransfection of the cell. However, the single-channel power-on used in traditional microfluidic chips takes up too much volume for the electrodes to achieve multi-channel processing, and is limited by the large channels and low transfection rate. The single-channel utilization rate provided by a single chip and electrode is relatively low. .

However, the microfluidic chips currently on the market still have the following problems in terms of cell capture and electrotransfection: low cell transfection efficiency and uncontrollable cell transfection. Therefore, it is necessary to develop a novel microfluidic chip for electrotransfection of immune cells based on electrode arrays to achieve efficient and controllable cell transfection operations.

Contents of the invention

Therefore, the technical problem to be solved by the invention is to overcome the problems of low cell transfection efficiency and uncontrollable cell transfection in the prior art.

In order to solve the above technical problems, on the one hand, the invention provides a microfluidic chip for electrotransfection of immune cells based on an electrode array, including:

The substrate is provided with an electrode group on the upper surface, and the electrode group includes multiple columns of sub-electrodes, and the sub-electrodes include an upper excitation electrode, a plurality of electric suspension electrodes and a lower excitation electrode; in the connection direction of the upper excitation electrode and the lower excitation electrode, the upper excitation electrode, A plurality of electric levitation electrodes and lower excitation electrodes are arranged at intervals in sequence;

The cover sheet is arranged on the top of the base, the two ends of the cover sheet are respectively provided with an inlet and an outlet through its wall thickness, the lower surface of the cover sheet is provided with a recessed micro channel, and the micro channel is connected to the inlet and the outlet; the micro channel includes a main Channel and channel group, the channel group includes branch channels and multiple columns of sub-channels, the sub-channels include a plurality of microgrooves arranged at intervals, and the plurality of microgrooves are respectively connected to the main channel through branch channels;

Wherein, the lower surface of the cover sheet is in contact with the upper surface of the substrate, and the sub-electrodes and the sub-channels are arranged in one-to-one correspondence; the microgrooves located at one end of the sub-channels are respectively located above the adjacent upper excitation electrode and the electric levitation electrode; The two ends of the microgroove at the other end of the subchannel are respectively located above the adjacent lower excitation electrode and the electric suspension electrode; the two ends of the microgroove located in the middle of the subchannel are respectively located above the two adjacent electric suspension electrodes.

In one embodiment of the invention, there are multiple electrode groups, and multiple sub-electrodes are arranged at intervals in the X direction, and the X direction is parallel to the connection line between the inlet and the outlet;

There are also multiple channel groups, and the channel groups are set in one-to-one correspondence with the electrode groups, and the multiple channel groups are connected through the main channel.

In one embodiment of the invention, a plurality of electrically floating electrodes in the multi-column sub-electrodes are arranged in a rectangular array;

A plurality of microgrooves in the multi-column sub-channels are arranged in a rectangular array.

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

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

In one embodiment of the invention, the present application further includes a microinjection pump, and the microinjection pump communicates with the inlet through a hose.

In one embodiment of the invention, the present application further includes a collecting part, which is in communication with the outlet through a hose.

In one embodiment of the invention, the depths of the main channel, the branch channels and the microgrooves are the same.

In one embodiment of the invention, the depth of the main channel, the branch channels and the microgrooves is 30-60 μm.

In one embodiment of the invention, the widths of the main channel, the branch channels and the microgrooves are the same.

In one embodiment of the invention, the width of the main channel, the branch channels and the microgrooves is 30-100 μm.

In one embodiment of the invention, the cover slip is made of PDMS; and/or,

The base is made of glass.

On the other hand, the invention provides an immune cell electrotransfection method, using the electrode array-based immune cell electrotransfection microfluidic chip in the above embodiment to perform electrotransfection, the steps include:

Capturing and positioning of cells, injecting cell samples into the inlet of the microfluidic chip, connecting high-frequency signal sources to the upper and lower excitation electrodes of the sub-electrodes, capturing the cells to the electric levitation electrodes and positioning the cells into the microgroove ;

Electrotransfection, switching the high-frequency signal to a pulse signal source, setting the voltage of the pulse signal source to a preset value, forming an electric field distribution between the upper excitation electrode of the sub-electrode, multiple electric suspension electrodes and the lower excitation electrode; Under the electric field distribution, the permeability of the cell plasma membrane positioned in the microgroove changes, the surface of the cell plasma membrane perforates, and the nucleic acid in the cell suspension is introduced into the cell to realize the electrotransfection of the cell.

In one embodiment of the invention, microfluidic chip pretreatment is carried out before the capture and localization of cells;

The pretreatment of the microfluidic chip includes: firstly disinfect and clean the microfluidic chip; then connect the microfluidic chip with a high-frequency signal source and a pulse signal source, 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.

In one embodiment of the invention, the cell sample preparation is carried out before the pretreatment of the microfluidic chip, and the cell sample preparation includes: culturing the cell sample to a density of 70%-80%, collecting the cells, centrifuging to remove the supernatant; and then Cells were resuspended in saline to make the cell density 1×10^7cells/mL.

In one embodiment of the invention, cell separation and analysis are performed after electrotransfection, and the cell separation and analysis includes: washing the microfluidic chip with physiological saline, collecting the cells, and performing centrifugation and analysis.

Compared with the prior art, the above-mentioned technical solution of the invention has the following advantages:

The electrode array-based immune cell electrotransfection microfluidic chip and electrotransfection method described in the invention have multiple sub-electrodes arranged on the upper surface of the substrate, and each sub-electrode includes an upper excitation electrode, a plurality of electro-suspension electrodes and Lower the excitation electrode. In this way, the upper excitation electrode, multiple electrically suspended electrodes and the lower excitation electrode are arranged at intervals in sequence, and a more uniform electric field distribution will be formed between the upper excitation electrode and the lower excitation electrode after the pulse signal source is connected to the upper excitation electrode and the lower excitation electrode. , so that in electrotransfection, the survival rate of cells is improved, thereby improving the efficiency of electrotransfection. In this embodiment, multiple columns of sub-channels are provided on the lower surface of the cover sheet, and each column of sub-channels is provided with a plurality of microgrooves arranged at intervals, and the two ends of the microgrooves are located above the adjacent upper excitation electrode and the electric levitation electrode. Above the two adjacent electric suspension electrodes or above the adjacent lower excitation electrode and the electric suspension electrode, after the upper excitation electrode and the lower excitation electrode are connected to the high-frequency signal source, the cell sample flowing in the micro channel will be captured to the into the microgrooves and positioned into the microgrooves. This enables cell samples to be captured and positioned in the microgrooves prior to electrotransfection, thereby enabling controlled electrotransfection of cells.

Description of drawings

In order to make the content of the invention easier to understand clearly, the invention will be described in further detail below according to specific embodiments of the invention in conjunction with the accompanying drawings, wherein

Figure 1 is a schematic diagram of the structure of a microfluidic chip for electrotransfection of immune cells based on electrode arrays;

Figure 2 is an exploded view of the invention of a microfluidic chip for electrotransfection of immune cells based on electrode arrays;

Fig. 3 is a front view of a microfluidic chip for electrotransfection of immune cells based on an electrode array in Fig. 1;

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

Fig. 5 is a schematic diagram of multiple electrode groups in a microfluidic chip for electrotransfection of immune cells based on an electrode array in Fig. 1;

Fig. 6 is a top view of a cover slip in a microfluidic chip for electrotransfection of immune cells based on an electrode array in Fig. 1;

Fig. 7 is a schematic diagram of the projection of multiple electrode groups and microchannels on the cover slip in an electrode array-based immune cell electrotransfection microfluidic chip in Fig. 1;

Fig. 8 is a three-dimensional schematic diagram of a microfluidic chip for electrotransfection of immune cells based on an electrode array in Fig. 7;

Fig. 9 is a flow chart of steps of an immune cell electrotransfection method in Fig. 1 .

Explanation of reference signs in the manual: 100, base; S, electrode group; 110, sub-electrode; 111, upper excitation electrode; 112, electric levitation electrode; 113, lower excitation electrode;

200, cover sheet; 210, entrance; 220, exit; 230, main channel; M, channel group; 240, branch channel; 250, sub-channel; 251, microgroove;

300. Line.

Detailed ways

The invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the invention and implement it, but the examples given are not intended to limit the invention.

Referring to Figures 1 to 8, on the one hand, the invention provides a microfluidic chip for electrotransfection of immune cells based on an electrode array, including:

The substrate 100 is provided with an electrode group S on the upper surface, and the electrode group S includes a multi-column sub-electrode 110, and the sub-electrode 110 includes an upper excitation electrode 111, a plurality of electric suspension electrodes 112 and a lower excitation electrode 113; the upper excitation electrode 111 and the lower excitation electrode 113 connection direction, the upper excitation electrode 111, a plurality of electric suspension electrodes 112 and the lower excitation electrode 113 are successively arranged at intervals; the connection line between the upper excitation electrode 111 and the lower excitation electrode 113 can be a straight line or a curve; the upper excitation electrode 111 and the lower excitation electrode 113 are energized to form an electric field, and a plurality of electric suspension electrodes 112 are located in the electric field;

The cover sheet 200 is arranged on the top of the base 100. The two ends of the cover sheet 200 are respectively provided with an inlet 210 and an outlet 220 through its wall thickness. The lower surface of the cover sheet 200 is provided with a recessed microchannel, and the microchannel communicates with the inlet 210. and outlet 220; the micro channel includes main channel 230 and channel group M, channel group M includes branch channel 240 and multi-column sub-channel 250, and sub-channel 250 includes a plurality of microgrooves 251 arranged at intervals, and a plurality of microgrooves 251 pass branch channels respectively The channel 240 is connected with the main channel 230;

Wherein, the lower surface of the cover sheet 200 is in contact with the upper surface of the substrate 100, and the sub-electrodes 110 and the sub-channels 250 are provided in one-to-one correspondence; the microgrooves 251 located at one end of the sub-channel 250 are respectively located at the adjacent upper excitation electrodes 111 and The top of the electric suspension electrode 112; the microgroove 251 located at the other end of the subchannel 250, the two ends are respectively located above the adjacent lower excitation electrode 113 and the electric suspension electrode 112; the microgroove 251 located in the middle of the subchannel 250, the two ends are respectively It is located above two adjacent electric suspension electrodes 112 .

It should be noted that, during the use of this embodiment, the cell sample (the cell sample includes cells and cell suspension, and there is nucleic acid in the cell suspension) is firstly injected into the inlet 210 through a micro-injection pump. As the micro-injection pump continuously injects the cell sample into the inlet 210 , the cell sample will flow in the micro-channel and flow out from the outlet 220 .

It should be noted that in this embodiment, multiple columns of sub-channels 250 are provided on the lower surface of the cover sheet 200, each column of sub-channels 250 is provided with a plurality of microgrooves 251 arranged at intervals, and multiple columns of sub-electrodes 110 are provided on the upper surface of the substrate 100. , each column of sub-electrodes 110 includes an upper excitation electrode 111 , a plurality of electrically suspended electrodes 112 and a lower excitation electrode 113 . The sub-electrodes 110 and the sub-channels 250 are provided in one-to-one correspondence; the micro-grooves 251 at one end of the sub-channels 250 are respectively located above the adjacent upper excitation electrodes 111 and the electric suspension electrodes 112; the micro-grooves at the other end of the sub-channels 250 251, the two ends are located above the adjacent lower excitation electrode 113 and the electric suspension electrode 112; The cell sample in each microgroove 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 electrode 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 receive high-frequency signals, and the cells in the cell sample are captured by the electric levitation electrode 112 and positioned in the microgroove 251 . Then the high-frequency signal is changed to a pulse signal source. At this time, an electric field distribution will be formed between the upper excitation electrode 111 and the lower excitation electrode 113. A plurality of electric suspension electrodes 112 are located in this electric field distribution, so that the adjacent upper excitation electrodes 111 There is a potential between the electric suspension electrode 112, there is a potential between the adjacent electric suspension electrodes 112, and there is also a potential between the adjacent electric suspension electrodes 112 and the lower excitation electrode 113, so that it can be captured and positioned in each micro The permeability of the cell plasma membrane in the groove 251 is changed, the surface of the cell plasma membrane is perforated, the nucleic acid in the cell suspension is introduced into the cell, and the electrotransfection of the cell is realized.

Specifically, in this embodiment, multiple columns of sub-electrodes 110 are arranged on the upper surface of the substrate 100 , and each column of sub-electrodes 110 includes an upper excitation electrode 111 , a plurality of electric suspension electrodes 112 and a lower excitation electrode 113 . In this way, the upper excitation electrode 111, a plurality of electric suspension electrodes 112 and the lower excitation electrode 113 are arranged at intervals in sequence, and after the pulse signal source is connected to the upper excitation electrode 111 and the lower excitation electrode 113, a pulse signal will be generated between the upper excitation electrode 111 and the lower excitation electrode 113. Form a more uniform electric field distribution, so that in the electrotransfection, the survival rate of the cells is improved, and the efficiency of the electrotransfection is improved. In this embodiment, multiple columns of sub-channels 250 are provided on the lower surface of the cover sheet 200, and each column of sub-channels 250 is provided with a plurality of microgrooves 251 arranged at intervals, and the two ends of the microgrooves 251 are located between the adjacent upper excitation electrodes 111 and Above the electric suspension electrode 112, above two adjacent electric suspension electrodes 112 or above the adjacent lower excitation electrode 113 and the electric suspension electrode 112, after the upper excitation electrode 111 and the lower excitation electrode 113 are connected to the high-frequency signal source, Cell samples flowing in the microchannels are captured and positioned in the microgrooves 251 . In this way, the cell sample can be captured into the microgroove 251 and positioned in the microgroove 251 before electrotransfection, so as to realize controllable cell electrotransfection.

It can be seen that this embodiment can achieve efficient and controllable cell electrotransfection, which is of great significance for the study of biological processes inside cells and the development of gene therapy.

Further, there are multiple electrode groups S, and multiple sub-electrodes 110 are arranged at intervals in the X direction, and the X direction is parallel to the connection line between the inlet 210 and the outlet 220;

There are also multiple channel groups M, and the channel groups M are set in one-to-one correspondence with the electrode groups S, and the multiple channel groups M are connected through the main channel 230 .

Specifically, there are multiple electrode sets S in this embodiment, so that cell capture, positioning, and electrotransfection can be performed on cell samples multiple times; and the efficiency of electrotransfection can be further improved.

Further, a plurality of electric suspension electrodes 112 in the multi-column sub-electrodes 110 are arranged in a rectangular array;

Multiple microgrooves 251 in multiple columns of sub-channels 250 are arranged in a rectangular array.

Specifically, in this embodiment, a plurality of electric levitation electrodes 112 are arranged in a rectangular array, and a plurality of microgrooves 251 are arranged in a rectangular array, which can reduce the inhomogeneity of the electric field, improve the transfection efficiency, and reduce the damage to cells at the same time. and toxicity.

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

Further, the upper excitation electrode 111 and the lower excitation electrode 113 of the sub-electrode 110 are respectively electrically connected to an external signal source through a line 300 .

Specifically, in this embodiment, the upper excitation electrode 111 and the lower excitation electrode 113 are respectively electrically connected to the external signal source through the circuit 300, which facilitates the connection between the upper excitation electrode 111 and the lower excitation electrode 111 by controlling the connection between the circuit 300 and the external signal source. 113 Whether to access the power supply.

Further, the depths of the main channel 230 , the branch channels 240 and the microgrooves 251 are the same.

Specifically, the depths of the main channel 230 , the branch channels 240 and the microgrooves 251 in this embodiment are the same, which facilitates the processing of the main channel 230 , the branch channels 240 and the microgrooves 251 .

Further, the depth of the main channel 230 , the branch channel 240 and the microgroove 251 is 30-60 μm. Optionally, the depth of the main channel 230 , the branch channel 240 and the microgroove 251 is 40 μm.

Further, the main channel 230 , the branch channel 240 and the micro groove 251 have the same width.

Specifically, the widths of the main channel 230 , the branch channels 240 and the microgrooves 251 in this embodiment are the same, which facilitates the processing of the main channel 230 , the branch channels 240 and the microgrooves 251 .

Further, the width of the main channel 230 , the branch channel 240 and the microgroove 251 is 30-100 μm. Optionally, the width of the main channel 230 , the branch channel 240 and the microgroove 251 is 40 μm.

Specifically, the width of the main channel 230 , the branch channel 240 and the microgroove 251 in this embodiment is 30-100 μm, which is convenient for cells to circulate in the microchannel.

The thickness of the cover sheet 200 is 2 mm.

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

Further, the cover sheet 200 is made of PDMS.

Further, the substrate 100 is made of glass.

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

Further, the present application also includes a micro-injection pump, and the micro-injection pump communicates with the inlet 210 through a hose.

Specifically, in this embodiment, a micro-injection pump connected to the inlet 210 is provided, so that a fully automated micro-experiment can be realized. That is, the shell can control the cells by controlling the input speed of the micro-injection pump. During transfection, the cells and nucleic acid can be captured to the same area for reaction, improving the transfection efficiency and nucleic acid utilization rate, and the cells can be observed through a WIFI digital microscope.

Further, the present application also includes a collecting part, and the collecting part communicates with the outlet 220 through a hose.

Specifically, in this embodiment, a collecting component communicated with the outlet 220 is provided, which is convenient for collecting the cell samples after the electrotransfection is completed.

Referring to Fig. 9, on the other hand, the invention provides an immune cell electrotransfection method, using the electrode array-based immune cell electrotransfection microfluidic chip in the above embodiment to perform electrotransfection, the steps include:

For the capture and positioning of cells, the cell sample (cell sample includes cells and cell suspension) is injected into the inlet 210 of the microfluidic chip, and the high-frequency signal source is connected to the upper excitation electrode 111 and the lower excitation electrode 113 of the sub-electrode 110, and the The cells are captured to the electrosuspension electrode 112 and localized into the microgroove 251;

Electrotransfection, switching the high-frequency signal to a pulse signal source, setting the voltage of the pulse signal source to a preset value, and forming a Electric field distribution: Under the electric field distribution, the permeability of the plasma membrane of the cell positioned in the microgroove 251 changes, the surface of the plasma membrane of the cell is perforated, and the nucleic acid in the cell suspension is introduced into the cell to realize electrotransfection of the cell.

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, the cells are captured to the electric levitation electrode 112 and the cells are positioned in the microgroove 251, so as to realize the dynamic capture of cells; The high-frequency signal is switched to a pulse signal source, and an electric field distribution is formed among the upper excitation electrode 111 , the plurality of electric levitation electrodes 112 and the lower excitation electrode 113 of the sub-electrode 110 to realize static electrotransfection. Therefore, this embodiment combines dynamic capture of cells with static electrotransfection to improve the controllability and survival rate of cell transfection; and it uses the principles of dielectrophoresis and alternating current and thermal coupling effects to generate fluid vortices to realize the separation of cells and nucleic acids. Simultaneous capture and transfection. In this embodiment, from the cell capture and positioning step to the electrotransfection step, the electrical signal is gradually converted from a high-frequency signal to a pulse signal, so that the frequency, amplitude and pulse width of the electric field are sequentially reduced to meet the specific needs of the cells; thus Further improve the controllability and survival rate of cell transfection.

The invention realizes the synchronous capture and transfection of cells and nucleic acids, and fills up the gap in the prior art. The invention is applicable to biomedical fields such as basic medical research, immunotherapy, gene modification and biopharmaceuticals.

Further, in the process of capturing and locating the cells, the voltage of the high-frequency signal is changed (for example, increased), or the flow rate of the cell sample is decreased. For example, the cell sample is injected into the inlet 210 by a microsyringe pump, and the flow rate of the cell sample can be controlled by controlling the injection speed of the microsyringe pump. In this example, the cells can be controlled by adjusting the voltage and flow rate. During transfection, the cells and nucleic acid can be captured in the same area for reaction, which improves the transfection efficiency and nucleic acid utilization rate. Cells can be observed through a WIFI digital microscope, transfection After success, adjust the voltage to release the cells, which is highly controllable.

Specifically, this can adjust the conductivity of the cell suspension (the cells in the cell sample are soaked in the cell suspension), thereby improving the cell activity and further improving the cell capture efficiency.

Further, during the process of electrotransfection, the width, amplitude and frequency of the pulse signal can 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 cells and improving the survival rate of cell transfection (because different Cell electrical properties are not the same).

Further, before the capture and positioning of the cells, the microfluidic chip is pretreated;

The pretreatment of the microfluidic chip includes: first disinfecting and cleaning the microfluidic chip; in some embodiments, sterilizing the microfluidic chip with 70% ethanol solution, and washing the microfluidic chip with sterile physiological saline. Surface, then add physiological saline solution containing 2% BSA on the microfluidic chip and incubate for 30 minutes to remove the remaining BSA solution; wash the microfluidic chip with physiological saline; then connect the microfluidic chip to a high-frequency signal source and the pulse signal source, 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 group S and remove oxides on the surface to ensure the effect of electrotransfection.

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

Specifically, the cell sample is prepared to ensure the activity of the cell sample, thereby further improving the cell capture efficiency.

Further, cell separation and analysis are performed after electrotransfection, and the cell separation and analysis includes: washing the microfluidic chip with physiological saline, collecting the cells, and performing centrifugation and analysis. For example, observing changes in cell morphology, detecting gene expression levels, etc.

To sum up, in some comparative examples, due to the inhomogeneity of electric field distribution, cell electrotransfection has problems such as low transfection efficiency and cell damage, which makes its production cost high and utilization rate poor. For this reason, the present invention proposes an electrotransfection microfluidic chip, which utilizes the characteristics that the electrosuspension electrode 112 array can be polarized by an external electric field to generate an induced potential, and increases the number of microgrooves 251 to achieve the above-mentioned flow electroporation. Combining the advantages of transfection with single-cell electrotransfection.

Apparently, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation. For those of ordinary skill in the art, on the basis of the above description, other changes or changes in various forms can also be made. It is not necessary and impossible to exhaustively list all the implementation manners here. The obvious changes or changes derived from this are still within the scope of protection of inventions and creations.

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. An immune cell electrotransfection method, which is characterized in that: comprising the following steps: electrotransfection using the electrode array-based immune cell electrotransfection microfluidic chip of claim 1, the steps comprising:

capturing and positioning cells, namely injecting a cell sample into an inlet of a microfluidic chip, connecting high-frequency signal sources to an upper excitation electrode and a lower excitation electrode of the sub-electrode, capturing the cells to an electric suspension electrode and positioning the cells into a micro-groove;

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

8. The method of claim 7, wherein the method further comprises the step of:

carrying out micro-fluidic chip pretreatment before capturing and positioning the cells;

the microfluidic chip pretreatment comprises: firstly, sterilizing and cleaning a microfluidic chip; 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.

9. The method of claim 8, wherein the immune cell electrotransfection is performed by:

performing cell sample preparation prior to the microfluidic chip pretreatment, the cell sample preparation comprising: 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.

10. The method of claim 9, wherein the immune cell electrotransfection is performed by:

isolation and analysis of cells following electrotransfection, the isolation and analysis of cells comprising: the microfluidic chip was washed with physiological saline, and cells were collected and centrifuged and analyzed.