CN112094742B - Micro-fluidic chip for synchronously realizing cell electroporation transfection and living cell sorting - Google Patents

Micro-fluidic chip for synchronously realizing cell electroporation transfection and living cell sorting Download PDF

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CN112094742B
CN112094742B CN202010854151.9A CN202010854151A CN112094742B CN 112094742 B CN112094742 B CN 112094742B CN 202010854151 A CN202010854151 A CN 202010854151A CN 112094742 B CN112094742 B CN 112094742B
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flow channel
layer
electroporation
pdms
cell
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CN112094742A (en
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邢晓星
蔡瑶
沈鹏辉
俞度立
柳军旺
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Beijing University of Chemical Technology
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
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    • G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
    • G03F7/16—Coating processes; Apparatus therefor
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03F—PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
    • G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
    • G03F7/26—Processing photosensitive materials; Apparatus therefor
    • G03F7/30—Imagewise removal using liquid means
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03F—PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
    • G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
    • G03F7/26—Processing photosensitive materials; Apparatus therefor
    • G03F7/38—Treatment before imagewise removal, e.g. prebaking
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03F—PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
    • G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
    • G03F7/70—Microphotolithographic exposure; Apparatus therefor
    • G03F7/70216—Mask projection systems

Abstract

A micro-fluidic chip for synchronously realizing cell electroporation transfection and living cell sorting belongs to the field of cell transfection and cell separation in the micro-fluidic chip technology. The side wall electrode with a double-layer inverted ladder-shaped structure is used as the boundary of the micro flow channel, and the electroporation cell transfection and dielectrophoresis screening of living cells are allowed to be carried out synchronously. In the flow channel, living cells can easily generate larger positive dielectric force and can be adsorbed on the upper layer and stimulated by electric pulses to realize cell transfection, and dead cells are limited on the lower layer by the negative dielectric force, so that the separation of the living cells and the dead cells is realized, and the defect of lower cell activity caused by electroporation is overcome. The chip processing adopts a soft photoetching membrane-inverting process, the SU-8 mould is filled with polydimethylsiloxane material, so that the flow channel and the electrode structure are formed in one step, and the microfluidic chip is formed after membrane inversion, so that the cost is low and the processing flow is simple.

Description

Micro-fluidic chip for synchronously realizing cell electroporation transfection and living cell sorting
Technical Field
The invention belongs to the field of cell transfection and cell separation in a microfluidic chip technology, and particularly relates to a microfluidic chip for synchronously realizing cell electroporation transfection and living cell sorting.
Background
Transfection is a technique for introducing foreign biological macromolecules into cells. In the biological research of gene function research, gene expression regulation, mutation analysis, protein production and the like, the application of the protein is more and more extensive. Transfection methods have developed rapidly and exhibit diversity in recent years, which can be specifically subdivided into three categories: namely biological methods, chemical methods, physical methods. Compared with the limitations of biological methods on safety, cell selectivity of chemical methods and the like, the physical methods do not need mediation of carriers and other chemical substances, and the applicable cell types are more abundant, so that the method is widely researched and applied. The patent mainly adopts an electroporation method in a physical method to carry out cell transfection, and the common physical method also comprises the following steps: ultrasonic perforation, microinjection, particle gun method, laser transfection, etc. As early as the twentieth century and the fifties, students such as Weaver and the like discovered the electroporation phenomenon, and the electroporation method instantly improves the permeability of cell membranes under the action of an electric field, so that macromolecules which are difficult to penetrate the cell membranes are transferred into cells, and compared with other transfection technologies, the electroporation method has the characteristics of low toxicity, low cost, high efficiency and the like. With the cross and deep research of micro-electro-mechanical system technology and the subjects of life science, analytical science and the like, the micro-fluidic control electroporation chip gradually becomes a research hotspot of people, and a great deal of previous researches such as a gold plate electrode cell transfection micro-fluidic chip designed by Lin and the like and a coplanar electrode cell transfection micro-fluidic chip designed by He and the like show that the micro-fluidic chip has the advantages of focusing an electric field, reducing required voltage, simplifying a device and the like compared with the traditional electroporation equipment. But the cells still can be affected by electric shock, shearing force and bubbles when passing through the electroporation microfluidic chip and are damaged to different degrees. In order to solve the problem of collection efficiency of successfully transfected living cells and facilitate the analysis and research of transfection results, the introduction of a cell separation technology is a practical and reliable method.
Cell separation as an important purification process has irreplaceable important roles in biological research and analytical diagnosis. The most widely used cell screening method is currently flow cytometry. However, the flow cytometer system is complex and bulky, the detection process depends on expensive biochemical and optical detection equipment, and professional personnel are required to operate. With the gradual maturation of the microfluidic theory and the rapid development of the microfluidic chip processing technology, the cell separation technology is developing towards a more refined operation mode. At present, the cell separation mode based on the microfluidic chip is mainly divided into immunocapture cell separation and label-free cell separation, wherein the immunocapture cell separation refers to the calibration on a cell antibody, and the binding specificity of the antibody and a specific antigen is used as a method for screening cells. The method has the advantages of strong specificity and higher purity of the obtained cells; however, the captured cells have the disadvantage that the captured cells are often provided with antibodies or other labeled fluorescence, which not only changes the physiological state of the cells, but also makes the captured cells difficult to release, and is not beneficial to further observation and study of the culture and state properties of the subsequent cells. The label-free cell separation is realized by applying instantaneous acting force on the surface of cells according to the physical forms of the cells or by using adhesion molecules and the like through the external water force field, the sound field, the electric field and other environments to ensure that different cells are subjected to different external forces in the microfluidic channel, thereby achieving the purpose of cell separation. The hydraulic field cell separation method is very sensitive to flow channel blockage, and the cell concentration of a sample needs to be strictly controlled; the acoustic field device has limited usable material types, complex micro-processing technology and high cost. In contrast, the microfluidic dielectrophoresis cell separation utilizes the difference of different types of cells in dielectric characteristics to control the different types of cells to generate different motion tracks in a non-uniform electric field so as to realize cell separation, has the advantages of simple configuration, easy operation, low cost and the like, is realized on the basis of an electrical method as well as electroporation, and is more suitable for being combined with the electroporation technology.
Adverse effects of electric fields and hydrodynamics in the electroporation chip on cell activity cannot be completely eliminated, and the electroporation technology is combined with the dielectrophoresis technology to remove damaged cells through dielectrophoresis, so that the transfection efficiency and cell activity of collected target cells reach higher levels. One of the biggest characteristics of the microfluidic chip is integrated, so that the possibility is provided for combining the electroporation technology and the dielectrophoresis technology, and basic functions of sample reaction, preparation, separation, detection and the like in the fields of biology, chemistry and the like can be integrated on one chip. The present only relevant technology is parallel metal electrodes designed by Zhihong Li and the like and manufactured by sputtering and etching technology, a polydimethylsiloxane material micro-fluid channel manufactured by a reverse mould technology, and a micro-fluidic chip formed by bonding a glass substrate with the micro-fluid channel, wherein a higher electric field is applied to an electroporation part at the front section of the chip to obtain higher transfection efficiency, and then living cells are separated by a dielectrophoresis part, so that the successfully transfected living cells are finally obtained. However, this design has the following limitations: firstly, the use of the metal thin film electrode can cause the electric field to be uneven, and the electroporation transfection efficiency is influenced; secondly, the chip design of the two functional modules of electroporation and living cell sorting not only increases the length of the chip, but also controls electroporation and dielectrophoresis respectively, so that two electric signals need to be applied to one chip, and the operation complexity is increased. Thirdly, the electrodes and the flow channels of the chip are respectively processed by different materials, so that the process is complex and the cost is high. Different from the prior art, the side wall structure of the three-dimensional ladder-shaped structure constructed by conductive polymer materials such as AgPDMS, C-PDMS and the like can be used as an electrode and a flow channel at the same time, and the structure is prepared by a soft lithography film inversion process, so that the flow channel and the electrode structure are molded at one time, the problem of respective processing is avoided, the process is simplified, and the cost is reduced. The design of three-dimensional ladder-shaped structure lateral wall electrode not only can provide more even electric field for cell electroporation, has realized that electroporation and dielectrophoresis select separately the electrode sharing in addition to make electroporation and dielectrophoresis can go on in step, need not to design electroporation and dielectrophoresis cell separately and select separately the submodule piece, accelerated the whole process of transfection and selection, improved the integrated level of chip, use unified signal of telecommunication, simplified equipment and experimental operation.
Disclosure of Invention
Aiming at the limitation of the prior art, the invention aims to provide a microfluidic chip for synchronously realizing cell electroporation transfection and living cell sorting, which adopts two layers of side wall electrodes with ladder-shaped structures as the boundary of a microchannel and allows electroporation cell transfection and dielectrophoresis screening of living cells to be synchronously carried out. Live cells in the flow channel can easily generate larger positive dielectric force and can be adsorbed on the upper layer and stimulated by electric pulses to realize cell transfection, and dead cells are limited on the lower layer by the negative dielectric force, so that separation of the live cells and the dead cells is realized, and the defect of lower cell activity caused by electroporation is overcome. The chip processing adopts a soft photoetching membrane-inverting process, the SU-8 mould is filled with polydimethylsiloxane material, so that the flow channel and the electrode structure are formed in one step, and the microfluidic chip is formed after membrane inversion, so that the cost is low and the processing flow is simple.
The invention relates to a microfluidic chip for synchronously realizing cell electroporation transfection and living cell sorting, which sequentially comprises the following components in structure from top to bottom: a top layer (1), an electroporation-dielectrophoresis functional layer (2) and a bottom layer (3).
The top layer (1) is used as the top of the electroporation-dielectrophoresis functional layer (2) and is provided with five holes which are respectively a sample inlet (4), a sheath flow inlet (5), a target cell outlet (6) and two waste cell outlets (7);
the electroporation-dielectrophoresis functional layer (2) is composed of a flow channel (11) and a pair of side wall electrodes (12) respectively positioned at two sides of the flow channel (11), the side wall electrodes (12) are integrally of a double-layer flat plate type structure, and the pair of side wall electrodes (12) are symmetrically arranged to form the electroporation-dielectrophoresis functional layer (2) which is parallel to the top layer (1); the flow channel (11) is communicated with the top layer (1) through an inlet and an outlet;
a long straight gap is formed between the two side wall electrodes (12) to form a flow channel (11), and the two side wall electrodes (12) are symmetrical about the flow channel (11); the side wall of any side wall electrode (12) corresponding to the two side edges of the flow channel (11) is of a double-layer structure, the double-layer structure is of an inverted step shape in the longitudinal direction, namely the depth direction, and the upper layer (8) extends out of the flow channel relative to the lower layer (9), so that the flow channel is defined as a structure with a narrow upper layer and a wide lower layer; the electroporation-dielectrophoresis functional layer (2) is sequentially divided into an inflow area (14), a main flow channel (15) and a collection area (16) along the flow direction of fluid in the flow channel (11), and meanwhile, a section is reserved at the front end of the inflow area (14) and the rear end of the collection area (16) to form a pair of isolation channels (13); the electroporation-dielectrophoresis functional layer (2) is provided with a functional layer sample inlet (4 ') at the inlet of an inflow area (14), a first shunt channel (17) extends from the functional layer sample inlet (4') to two side wall electrodes (12) respectively, then the first shunt channel (17) is communicated with a flow channel (11) at the tail of the inflow area (14), and the communication position of the flow channel (11) and the tail end of the first shunt channel (17) is that only a lower layer (9) is communicated but an upper layer (8) is not communicated, so that a bridge-shaped structure (10) is formed; the inflow area (14) is also provided with a functional layer sheath inflow port (5 '), the part of a flow channel (11) of the inflow area (14) connected behind the functional layer sheath inflow port (5') is marked as a second branch flow channel (18), and a section of the flow channel (11) between the inflow area (14) and the collecting area is a main flow channel (15); a second branch flow channel (18) connected with the functional layer sheath inflow port (5') is directly connected with the main flow channel (15); the main flow channel (15) is limited to be located in a narrow strong electric field area at the upper layer and a wide weak electric field area at the lower layer by the double-layer electrodes in the shape of an inverted step in the longitudinal direction, namely the depth direction; the area behind the main flow channel (15) is a collecting area (16), the collecting area (16) comprises the flow channel (11) of the area, the flow channel (11) of the area is a third sub-flow channel (19), namely the third sub-flow channel (19) is directly connected with the main flow channel (15), and the tail end of the third sub-flow channel (19) is connected with a functional layer target cell outlet (6'); a fourth flow channel (20) symmetrically extends from the tail of the main flow channel (15), namely two sides of the starting part of the third flow channel (19), the tail part of the fourth flow channel (20) is connected with a functional layer waste cell outlet (7'), wherein the connecting branch of the main flow channel (15) and the fourth flow channel (20) is only communicated with the lower layer of the main flow channel (15), the upper layer of an electrode penetrating through the whole flow channel (11) blocks the communication between the main flow channel (15) and the fourth flow channels (20) at the two sides at the upper layer, and a bridge-shaped structure (10) is formed at the branch; a pair of isolation trenches (13) are connected to the sample inlet (4 ') and the target cell outlet (6'), respectively, and extend outward, and are filled with an insulating material (21) to insulate the pair of sidewall electrodes (12).
The thickness of the upper layer (8) of the pair of side wall electrodes (12) in the shape of the inverted ladder is preferably 30-40 mu m, the thickness of the lower layer (9) is preferably 50-60 mu m, the width of the upper layer of the flow channel (11) sandwiched between the upper layer and the lower layer is preferably 120-150 mu m, and the width of the lower layer is preferably 200-230 mu m.
The functional layer sample inlet (4 '), the functional layer sheath flow inlet (5'), the functional layer target cell outlet (6 ') and the functional layer waste cell outlet (7') are cylindrical cavities respectively, and the diameters of the cylindrical cavities are all larger than the width of the flow channel (11), the width of the first sub-flow channel (17) and the width of the fourth sub-flow channel (20); and respectively corresponds to the sample inlet (4), the sheath flow inlet (5), the target cell outlet (6) and the waste cell outlet (7) of the top layer (1). The sample inlet (4), the sheath flow inlet (5), the target cell outlet (6) and the two waste cell outlets (7) have the same hole diameter, and the hole diameter is preferably 1.0-1.2 mm.
The structures of the first sub-flow passage (17) and the fourth sub-flow passage (20) in the depth direction and the structures of the flow passages (11) in the depth direction are both narrow at the upper layer and wide at the lower layer.
The bottom layer (3) serves as the bottom of the flow channel (11) and as the carrier part of the device.
The top layer (1) is made of a transparent insulating material, preferably Polydimethylsiloxane (PDMS), glass; the electroporation-dielectrophoresis functional layer (2) is made of a conductive material that can be machined three-dimensionally, such as silver-polydimethylsiloxane (Ag-PDMS), carbon-polydimethylsiloxane (C-PDMS), heavily doped silicon, metal, carbon, and the like. Preferably Ag-PDMS and C-PDMS, and is realized by low-cost reverse mold processing. The bottom layer (3) is made of transparent insulating material, preferably polydimethylsiloxane and glass. The insulating material (21) filling the isolation channel (13) is preferably PDMS or resin.
The preparation method of the microfluidic chip synchronously realizing the cell electroporation transfection and the living cell sorting has the processing flow as shown in figure 4 and is described in detail as follows:
(1) photoetching preparation mold
The photoetching is to use photoresist, mask and ultraviolet light to make microfabrication, and the process is as follows:
cleaning a silicon wafer;
secondly, spin-coating a first layer of photoresist with certain thickness on a silicon wafer by using a spin coater (as shown in the step one in the figure 4);
preparing a required channel pattern on the mask plate; placing a mask plate above a silicon wafer, irradiating the silicon wafer coated with the photoresist in a rotating manner by using ultraviolet light, and carrying out photochemical reaction on the photoresist (as shown in the step two in the figure 4);
fourthly, spin-coating a second layer of photoresist with certain thickness on the silicon wafer by using a spin coater (as shown in the step three in the figure 4);
placing the mask plate above the silicon wafer, irradiating the silicon wafer coated with the photoresist by ultraviolet light, and carrying out photochemical reaction on the photoresist (step four in figure 4);
sixthly, removing the unexposed photoresist by a developing solution through a developing chemical method; accurately copying the pattern on the mask plate onto the photoresist layer, and finishing the SU-8 mold (step five in FIG. 4);
(2) filling of Ag-PDMS conductive polymers
The washed silver powder and PDMS are mixed according to the proportion of 17: 3, uniformly mixing;
secondly, the mixture is transferred to a mortar for full grinding until the mixture is pasty and the whole is moist and glossy;
thirdly, uniformly coating a proper amount of Ag-PDMS on the surface of the SU-8 mould, uniformly pressing to fill the Ag-PDMS into the mould as much as possible, and removing the redundant Ag-PDMS on white paper. (step six as in fig. 4);
heating the filled mold at 70 deg.c for three hr;
(3) reverse mould
Placing the filled mold in an aluminum foil paper box with a proper size, pouring the surface filled with Ag-PDMS upwards into PDMS, and controlling the height to be about 2-3 mm;
heating at 70 deg.c for three hr and curing PDMS after three hr;
thirdly, naturally bonding the poured PDMS and the Ag-PDMS filled in the mould into a whole, and slightly demoulding the whole from the mould (as shown in the seventh step of fig. 4);
(4) substrate bonding
The surface of the device obtained in the last step with a flow channel structure faces upwards, and holes are punched from top to bottom at the positions of an inlet and an outlet, so that the flow channel can be communicated with the outside;
secondly, performing isopropanol ultrasonic cleaning on the punched device, flushing with deionized water, drying with nitrogen, heating to remove water, and keeping the device clean;
thirdly, spin-coating a layer of PDMS on the surface of the glass by using a spin coater, placing the spin-coated glass sheet on a heating plate, heating for 10 minutes at 70 ℃, after the PDMS on the surface of the glass is semi-dried, contacting the surface of the clean device with the flow channel structure with the glass sheet, and lightly pressing to obtain a semi-finished chip (as shown in step eight in FIG. 4). The isolation trenches (13) are filled with a small amount of semi-cured insulating material (21) (see fig. 5);
(5) and (6) assembling. Cutting the PDMS on the electrode part on the side surface of the chip until the electrode is exposed, and connecting the electrode with a lead (23) through silver paste (22) (as shown in figure 6);
when the pair of side wall electrodes (12) are processed by a reverse mould process by conductive polymers, in order to avoid that a large-area shallow groove filled with the side wall electrodes (12) on a mould is difficult to fill completely during filling, increase the filling success rate and optimize the mould pattern, the large-area shallow groove pattern originally used for filling the side wall electrodes on the mould is improved into a separated grid-type filling groove. The sidewall electrodes of the inverse mold after filling are in a grid shape (24) which still has electrical continuity with each other and is easy to fill in processing.
The principle of cell separation of the microfluidic chip synchronously realized by the electroporation transfection and the living cell sorting is shown in fig. 8 and is specifically described as follows:
the sample enters the flow channel from the lower layer (9) through the sample inlet (4), and simultaneously the buffer solution enters the flow channel from the upper layer (8) through the sheath flow inlet (5), and the buffer solution limits the sample at the edge parts of two sides of the flow channel (11); cells in the sample, which are subjected to negative dielectric force or weak positive dielectric force, are limited in the lower layer (9), cells in the sample, which are subjected to strong positive dielectric force, are adsorbed in the upper layer (8), cells, which are subjected to strong positive dielectric force, are subjected to electroporation simultaneously in the upper layer (8), cells, the activity of which is reduced due to the electroporation, fall in the lower layer (9), cells in the lower layer (9) are limited by sheath flow and are limited at the edge parts of two sides of the flow channel (11), and the cells are discharged from the waste cell outlet (7) through the bridge-shaped structure (10). The cells in the upper layer (8) are also restricted by the sheath flow but are discharged from the target cell outlet (6) along the main flow path due to the blocking action of the bridge structure (10).
Transfected cells include, but are not limited to, mammalian cells, bacteria, and the like. The device can also be used as a cell sorting device independently, and is based on positive and negative dielectric force, including but not limited to Circulating Tumor Cell (CTC) sorting, living cell extraction, blood cell extraction, plasma extraction and the like.
The amplitude of the excitation signal voltage for transfection and separation is preferably 600-900V/cm, and the frequency is preferably 100-200 kHz. The flow rate of the sheath flow is preferably 5-10 mul/min, and the flow rate of the sample flow is preferably 10-20 mul/min.
Compared with the prior art, the invention has the following beneficial effects:
the micro-fluidic chip for synchronously realizing the electroporation transfection and the living cell sorting of the cells, which is disclosed by the invention, screens the transfected living cells to improve the cell activity by dielectrophoresis sorting synchronous with the electroporation, and overcomes the problem that the cell activity is generally low in the general electroporation technology; the invention is different from the existing chip design with two functional modules of electroporation and living cell sorting, allows the electroporation and dielectrophoresis cell sorting to share the same module design through the unique inverted step-shaped side wall electrode, realizes the sharing of the transfection and sorting electrodes, enables the transfection and sorting to be carried out synchronously, accelerates the whole process of the transfection and sorting, has the advantages of higher integration level and lighter volume, uses uniform electric signals, and simplifies the equipment. The electroporation and dielectrophoresis are realized on the basis of an electrical method, and markers such as biochemical transfection reagents or antibodies are not needed for assistance, so that the operation is simplified, the cost is reduced, and the possible change to the cell surface is further reduced; the three-dimensional inverted step-shaped side wall structure constructed by the conductive polymer material can be used as an electrode and a flow channel at the same time, and the structure is prepared by a soft lithography film inversion process, so that the flow channel and the electrode structure are formed at one time, the process is simplified, and the cost is reduced.
Drawings
Fig. 1 is a schematic diagram of an overall structure of a microfluidic chip for synchronously realizing cell electroporation transfection and living cell sorting according to the present invention, which sequentially comprises a top layer (1), an electroporation-dielectrophoresis functional layer (2) and a bottom layer (3) from top to bottom.
Fig. 2 is a schematic diagram of the overall structure of the electroporation-dielectrophoresis functional layer (2) of the microfluidic chip, which is realized by synchronously realizing cell electroporation transfection and living cell sorting.
Fig. 3 is a top view of the electroporation-dielectrophoresis functional layer (2) of the microfluidic chip for synchronously realizing cell electroporation transfection and living cell sorting according to the present invention.
FIG. 4 is a flow chart of a process of a microfluidic chip for synchronously performing cell electroporation transfection and living cell sorting according to the present invention.
Fig. 5 is a schematic diagram of filling an isolation channel (13) of a microfluidic chip for synchronously realizing cell electroporation transfection and living cell sorting according to the present invention, wherein the filling is performed before (a) and after (b).
FIG. 6 is a diagram of the operation of connecting the side wall electrode (12) of the microfluidic chip with an external circuit by using silver colloid (21) and a lead (22) for realizing the synchronization of cell electroporation transfection and living cell sorting.
FIG. 7 is a schematic diagram of a grid-shaped (21) electrode of the electroporation-dielectrophoresis functional layer (2) of the microfluidic chip, which is realized by synchronously realizing cell electroporation transfection and living cell sorting.
FIG. 8 is a schematic diagram of a cell separation principle of a microfluidic chip for synchronously realizing cell electroporation transfection and living cell sorting according to the present invention.
The reference numbers are as follows: 1. top layer, 2, electroporation-dielectrophoresis functional layer, 3, bottom layer, 4, sample inlet, 5, sheath flow inlet, 6, target cell outlet, 7, waste cell outlet, 4 ', functional layer sample inlet, 5', functional layer sheath flow inlet, 6 ', functional layer target cell outlet, 7', functional layer waste cell outlet, 8, upper layer, 9, lower layer, 10, bridge structure, 11, flow channel, 12, sidewall electrode, 13, isolation channel, 14, inflow region, 15, main flow channel, 16, collection region, 17, first sub-flow channel, 18, second sub-flow channel, 19, third sub-flow channel, 20, fourth sub-flow channel, 21, insulating material, 22, silver colloid, 23, conducting wire, 24, grid pattern.
Detailed Description
The invention relates to a flow-type electroporation microfluidic chip integrating functions of electroporation cell transfection and perforation survival cell screening, which sequentially comprises the following components in structure from top to bottom: a top layer (1), an electroporation-dielectrophoresis functional layer (2) and a bottom layer (3).
The top layer (1) is used as the top of the electroporation-dielectrophoresis functional layer (2) and is provided with five holes which are respectively a sample inlet (4), a sheath flow inlet (5), a target cell outlet (6) and two waste cell outlets (7);
the electroporation-dielectrophoresis functional layer (2) is composed of a flow channel (11) and a pair of side wall electrodes (12) respectively positioned at two sides of the flow channel (11), the side wall electrodes (12) are integrally of a double-layer flat plate type structure, and the pair of side wall electrodes (12) are symmetrically arranged to form a layer parallel to the top layer (1); the flow channel (11) is communicated with the top layer (1) through an inlet and an outlet;
the electroporation-dielectrophoresis functional layer (2) is composed of a flow channel (11) and a pair of side wall electrodes (12) respectively positioned at two sides of the flow channel (11). A long straight gap clamped between the two side wall electrodes (12) is a flow channel (11), and the two side wall electrodes (12) are symmetrical about the flow channel (11); the side wall of any side wall electrode (12) corresponding to the two side edges of the flow channel (11) is of a double-layer structure, namely, the longitudinal direction, namely the depth direction, is of an inverted step-shaped double-layer structure, and the upper layer (8) extends out of the flow channel relative to the lower layer (9), so that the flow channel is defined as a structure with a narrow upper layer and a wide lower layer; the electroporation-dielectrophoresis functional layer (2) is sequentially divided into an inflow area (14), a main flow channel (15) and a collection area (16) along the flow direction of fluid in the flow channel (11), and meanwhile, a section is reserved at the front end of the inflow area (14) and the rear end of the collection area (16) to form a pair of isolation channels (13); the electroporation-dielectrophoresis functional layer (2) is provided with a functional layer sample inlet (4 ') at the inlet of an inflow area (14), a first shunt channel (17) extends from the functional layer sample inlet (4') to two side wall electrodes (12) respectively, then the first shunt channel (17) is communicated with a flow channel (11) at the tail of the inflow area (14), and the communication position of the flow channel (11) and the tail end of the first shunt channel (17) is that only a lower layer (9) is communicated but an upper layer (8) is not communicated, so that a bridge-shaped structure (10) is formed; the inflow area (14) is also provided with a functional layer sheath inflow port (5 '), the part of a flow channel (11) of the inflow area (14) connected behind the functional layer sheath inflow port (5') is marked as a second sub-flow channel (18), and a section of the flow channel (11) between the inflow area (14) and the collecting area is a main flow channel (15); a second branch flow channel (18) connected with the functional layer sheath inflow port (5') is directly connected with the main flow channel (15); the main flow channel (15) is limited to be located in a narrow strong electric field area at the upper layer and a wide weak electric field area at the lower layer by the double-layer electrodes in the shape of an inverted step in the longitudinal direction, namely the depth direction; the area behind the main flow channel (15) is a collecting area (16), the collecting area (16) comprises the flow channel (11) of the area, the flow channel (11) of the area is a third sub-flow channel (19), namely the third sub-flow channel (19) is directly connected with the main flow channel (15), and the tail end of the third sub-flow channel (19) is connected with a functional layer target cell outlet (6'); a fourth flow channel (20) symmetrically extends from the tail of the main flow channel (15), namely two sides of the starting part of the third flow channel (19), the tail part of the fourth flow channel (20) is connected with a functional layer waste cell outlet (7'), wherein the connecting branch of the main flow channel (15) and the fourth flow channel (20) is only communicated with the lower layer of the main flow channel (15), the upper layer of an electrode penetrating through the whole flow channel (11) blocks the communication between the main flow channel (15) and the fourth flow channels (20) at the two sides at the upper layer, and a bridge-shaped structure (10) is formed at the branch; a pair of isolation trenches (13) are connected to the sample inlet (4 ') and the target cell outlet (6'), respectively, and extend outward, and are filled with an insulating material (21) to insulate the pair of sidewall electrodes (12).
The thickness of the upper layer (8) of the pair of side wall electrodes (12) in the shape of the inverted ladder is preferably 30-40 mu m, the thickness of the lower layer (9) is preferably 50-60 mu m, the width of the upper layer of the flow channel (11) sandwiched between the upper layer and the lower layer is preferably 120-150 mu m, and the width of the lower layer is preferably 200-230 mu m.
The functional layer sample inlet (4 '), the functional layer sheath flow inlet (5'), the functional layer target cell outlet (6 ') and the functional layer waste cell outlet (7') are cylindrical cavities respectively, and the diameters of the cylindrical cavities are all larger than the width of the flow channel (11), the width of the first sub-flow channel (17) and the width of the fourth sub-flow channel (20); and respectively corresponds to a sample inlet (4), a sheath inlet (5), a target cell outlet (6) and a waste cell outlet (7) of the top layer (1). The sample inlet (4), the sheath flow inlet (5), the target cell outlet (6) and the two waste cell outlets (7) have the same hole diameter, and the hole diameter is preferably 1.0-1.2 mm.
The structure of the first sub-runner (17) and the fourth sub-runner (20) in the depth direction and the structure of the runner (11) in the depth direction are both narrow at the upper layer and wide at the lower layer.
The bottom layer (3) serves as the bottom of the flow channel (11) and as the carrier part of the device.
The top layer (1) is made of a transparent insulating material, preferably Polydimethylsiloxane (PDMS), glass; the electroporation-dielectrophoresis functional layer (2) is made of a conductive material which can be machined in three dimensions, such as silver-polydimethylsiloxane (Ag-PDMS), carbon-polydimethylsiloxane (C-PDMS), heavily doped silicon, metal, carbon, etc. Preferably Ag-PDMS and C-PDMS, and is realized by low-cost reverse mold processing. The bottom layer (3) is made of transparent insulating material, preferably polydimethylsiloxane and glass. The insulating material (21) filling the isolation channel (13) is preferably PDMS or resin.
The invention relates to a preparation method of a flow-type electroporation microfluidic chip integrating electroporation cell transfection and perforation survival cell screening functions, the processing flow is shown in figure 4, and the following details are described:
(1) photoetching preparation mold
The photoetching is to use photoresist, mask and ultraviolet light to make microfabrication, and the process is as follows:
firstly, cleaning a silicon wafer.
And secondly, spin-coating a first layer of photoresist with a certain thickness on the silicon wafer by using a spin coater (as shown in the step one in the figure 4).
Preparing a required channel pattern on the mask plate; and (3) placing the mask plate above the silicon wafer, irradiating the silicon wafer coated with the photoresist in a rotating manner by ultraviolet light, and carrying out photochemical reaction on the photoresist (as shown in the step two in the figure 4).
And fourthly, spin-coating a second layer of photoresist with a certain thickness on the silicon wafer by using a spin coater (as shown in the step three in the figure 4).
Placing the mask plate above the silicon wafer, irradiating the silicon wafer coated with the photoresist by ultraviolet light, and enabling the photoresist to generate photochemical reaction (as shown in the step four in the figure 4);
sixthly, removing the unexposed photoresist by a developing solution through a developing chemical method; accurately copying the pattern on the mask plate onto the photoresist layer, and finishing the manufacturing of the SU-8 mold (as shown in the step five in FIG. 4);
(2) filling an Ag-PDMS conductive polymer;
the washed silver powder and PDMS are mixed according to the proportion of 17: 3, and uniformly mixing.
And secondly, transferring the mixture into a mortar for full grinding until the mixture is pasty and the whole is moist and glossy.
And thirdly, selecting a proper amount of paste-shaped Ag-PDMS to be uniformly coated on the surface of the SU-8 mould, contacting the surface coated with the Ag-PDMS with white paper, and uniformly pressing to fill the Ag-PDMS into the SU-8 mould as much as possible. And dragging the mold on the white paper to grind off the redundant Ag-PDMS, so that the blank area of the mold is filled with the Ag-PDMS, and the protruded part of the mold is not provided with the Ag-PDMS and is flush with the surface of the filled Ag-PDMS (step six in FIG. 4).
Fourthly, heating the filled mould for three hours at the temperature of 70 ℃.
(3) Reversing the mold;
putting the filled mold in an aluminum foil paper box with a proper size, pouring the surface filled with Ag-PDMS upwards into PDMS, and controlling the height to be about 2-3 mm.
② heating at 70 ℃ for three hours, curing PDMS after three hours.
Thirdly, naturally bonding the poured PDMS and the Ag-PDMS filled in the mould into a whole, and lightly demoulding the whole from the mould (as shown in the seventh step of FIG. 4).
(4) Bonding a substrate;
the surface of the device obtained in the last step with a flow channel structure faces upwards, and holes are punched from top to bottom at the position of an inlet and an outlet, so that the flow channel can be communicated with the outside.
And secondly, performing isopropanol ultrasonic cleaning on the punched device, flushing with deionized water, drying with nitrogen, heating to remove water, and keeping the device clean.
Thirdly, spin-coating a layer of PDMS on the surface of the glass by using a spin coater, placing the spin-coated glass sheet on a heating plate, heating for 10 minutes at 70 ℃, after the PDMS on the surface of the glass is semi-dried, contacting the surface of the clean device with the flow channel structure with the glass sheet, and lightly pressing to obtain a semi-finished chip (as shown in step eight in FIG. 4). The isolation trenches (13) are filled with a small amount of semi-cured insulating material (21) (see fig. 5).
(5) And (6) assembling. The PDMS of the electrode part on the side of the chip is cut to expose the electrode, and the electrode is connected with a lead (23) through a silver adhesive (22) (see figure 6).
When the pair of side wall electrodes (12) are processed by a reverse mould process by conductive polymers, in order to avoid that a large-area shallow groove filled with the side wall electrodes (12) on a mould is difficult to fill completely during filling, increase the filling success rate and optimize the mould pattern, the large-area shallow groove pattern originally used for filling the side wall electrodes on the mould is improved into a separated grid-type filling groove. The sidewall electrodes of the inverse mold after filling are in a grid shape (24) which still has electrical continuity with each other and is easy to fill in processing.
Examples
In this embodiment, the microfluidic chip for synchronously implementing the cell electroporation transfection and the viable cell sorting obtained in the above embodiment is used for performing a cell transfection and perforation viable cell screening effect test, and the specific steps are as follows:
(1) buffer solutions for experiments were prepared (8mM Na)2HPO4,2mM KH2PO4And 250mM sucrose), and additionally 50 μ l Propidium Iodide (PI) per ml buffer solution as a delivery solution;
(2) and testing the chip.
Firstly, a buffer solution is introduced into the chip, and the tightness of the chip is checked.
And secondly, connecting a circuit, and inspecting the electrical characteristics of the chip by observing the voltage.
(3) Harvesting the cells
Firstly, discarding the old culture solution in a culture vessel;
② washing with 10ml phosphate buffer solution (PBS solution) and discarding;
③ 2ml of trypsin is added, shaken gently and placed in a cell incubator for 6 minutes.
Add 5ml culture liquid, transfer 7ml cell liquid into 15ml test tube, put the test tube into centrifuge, centrifuge 2 minutes at 1500 rpm.
Fifthly, after centrifugation, the supernatant is discarded, 8ml of culture solution is added into the test tube, and the test tube is repeatedly blown and dispersed.
(4) Dyeing; add 2. mu.l Calcein (Calcein) per ml of cell sap and stain for ten minutes.
(5) Cells stained with Calcein were centrifuged twice, the supernatant discarded, placed in the delivery solution, and injected into the chip at a rate of 20. mu.l/min. While a sinusoidal signal with a frequency of 100kHz was applied to the chip, the electric field was maintained at 700V/cm by adjusting the voltage.
(6) The movement track of the cells is observed under a microscope, the cells passing through the chip are collected respectively, 30 mu l of the cells are placed on a glass slide to observe the transfection effect, and the data are recorded by photographing.
In general, cells are dispersedly suspended in the middle of the flow channel when no electric signal is inputted. After the electric signal is input, the cells under the action of strong positive dielectric force are adsorbed to the upper layer, arranged on two sides of the flow channel of the upper layer and discharged from the target cell outlet. The cells under the action of weak positive and negative dielectric forces are adsorbed to the two sides of the lower flow channel and discharged from the waste cell outlet. The cells discharged from the target cell outlet are live cells and have obvious transfection effect, and the cells discharged from the waste cell outlet are dead cells. It is demonstrated that the microfluidic chip obtained by the above embodiment can realize cell transfection and perforation survival cell screening.

Claims (11)

1. A micro-fluidic chip for synchronously realizing cell electroporation transfection and living cell sorting is characterized in that the structure sequentially comprises from top to bottom: a top layer (1), an electroporation-dielectrophoresis functional layer (2) and a bottom layer (3);
the top layer (1) is used as the top of the electroporation-dielectrophoresis functional layer (2) and is provided with five holes which are respectively a sample inlet (4), a sheath flow inlet (5), a target cell outlet (6) and two waste cell outlets (7);
the electroporation-dielectrophoresis functional layer (2) is composed of a flow channel (11) and a pair of side wall electrodes (12) respectively positioned at two sides of the flow channel (11), the side wall electrodes (12) are integrally of a double-layer flat plate type structure, and the pair of side wall electrodes (12) are symmetrically arranged to form the electroporation-dielectrophoresis functional layer (2) which is parallel to the top layer (1); the flow channel (11) is communicated with the top layer (1) through an inlet and an outlet;
a long straight gap is formed between the two side wall electrodes (12) to form a flow channel (11), and the two side wall electrodes (12) are symmetrical about the flow channel (11); the side wall of any side wall electrode (12) corresponding to the two side edges of the flow channel (11) is of a double-layer structure, the double-layer structure is of an inverted step shape in the longitudinal direction, namely the depth direction, and the upper layer (8) extends out of the flow channel relative to the lower layer (9), so that the flow channel is defined as a structure with a narrow upper layer and a wide lower layer; the electroporation-dielectrophoresis functional layer (2) is sequentially divided into an inflow area (14), a main flow channel (15) and a collection area (16) along the flow direction of fluid in the flow channel (11), and meanwhile, a section is reserved at the front end of the inflow area (14) and the rear end of the collection area (16) to form a pair of isolation channels (13); the electroporation-dielectrophoresis functional layer (2) is provided with a functional layer sample inlet (4 ') at the inlet of an inflow area (14), a first shunt channel (17) respectively extends from the functional layer sample inlet (4') to two side wall electrodes (12), then the first shunt channel (17) is communicated with a flow channel (11) at the tail of the inflow area (14), the communication position of the flow channel (11) and the tail end of the first shunt channel (17) is that only a lower layer (9) is communicated but an upper layer (8) is not communicated, thereby forming a bridge structure (10); a functional layer sheath inflow port (5 ') is further arranged in the inflow region (14), the part of a flow channel (11) of the inflow region (14) connected behind the functional layer sheath inflow port (5') is marked as a second sub-flow channel (18), and a section of the flow channel (11) between the inflow region (14) and the collecting region is a main flow channel (15); a second branch flow channel (18) connected with the functional layer sheath inflow port (5') is directly connected with the main flow channel (15); the main flow channel (15) is limited to be located in an upper narrow strong electric field area and a lower wide weak electric field area by the double-layer electrodes in the reverse step shape in the longitudinal direction, namely the depth direction; the area behind the main flow channel (15) is a collecting area (16), the collecting area (16) comprises the flow channel (11) of the area, the flow channel (11) of the area is a third sub-flow channel (19), namely the third sub-flow channel (19) is directly connected with the main flow channel (15), and the tail end of the third sub-flow channel (19) is connected with a functional layer target cell outlet (6'); a fourth flow channel (20) symmetrically extends from the tail of the main flow channel (15), namely two sides of the starting part of the third flow channel (19), the tail part of the fourth flow channel (20) is connected with a functional layer waste cell outlet (7'), wherein the connecting branch of the main flow channel (15) and the fourth flow channel (20) is only communicated with the lower layer of the main flow channel (15), the upper layer of an electrode penetrating through the whole flow channel (11) blocks the communication between the main flow channel (15) and the fourth flow channels (20) at the two sides at the upper layer, and a bridge-shaped structure (10) is formed at the branch; a pair of isolation channels (13) respectively connected with the sample inlet (4 ') and the target cell outlet (6') and extending outwards, and after being filled with an insulating material (21), the pair of side wall electrodes (12) can be insulated;
the functional layer sample inlet (4 '), the functional layer sheath flow inlet (5'), the functional layer target cell outlet (6 '), and the functional layer waste cell outlet (7') correspond to the sample inlet (4), the sheath flow inlet (5), the target cell outlet (6), and the waste cell outlet (7) of the top layer (1), respectively;
the bottom layer (3) serves as the bottom of the flow channel (11) and as the carrier part of the device.
2. The micro-fluidic chip for synchronously realizing cell electroporation transfection and living cell sorting according to claim 1 is characterized in that the upper layer (8) of the pair of inverted step-shaped side wall electrodes (12) is 30-40 mu m thick, the lower layer (9) is 50-60 mu m thick, the upper layer of the middle sandwiched flow channel (11) is 120-150 mu m wide, and the lower layer of the middle sandwiched flow channel is 200-230 mu m wide.
3. The microfluidic chip for realizing the synchronization of the electroporation transfection and the sorting of the living cells according to claim 1, wherein the sample inlet (4), the sheath flow inlet (5), the target cell outlet (6) and the two waste cell outlets (7) have the same diameter of 1.0-1.2 mm.
4. The microfluidic chip for realizing the synchronization of the electroporation transfection and the sorting of the living cells according to claim 1, wherein the structure of the first subchannel (17) and the fourth subchannel (20) in the depth direction and the structure of the channel (11) in the depth direction are both narrow at the upper layer and wide at the lower layer.
5. A microfluidic chip for the simultaneous implementation of electroporation transfection and sorting of living cells according to claim 1, characterized in that the functional layer sample inlet (4 '), the functional layer sheath flow inlet (5'), the functional layer target cell outlet (6 '), and the functional layer waste cell outlet (7') are cylindrical cavities with diameters larger than the width of the flow channel (11) and the width of the first sub-flow channel (17) and the fourth sub-flow channel (20), respectively.
6. The microfluidic chip for the simultaneous implementation of electroporation transfection and sorting of living cells according to claim 1, characterized in that the top layer (1) is made of a transparent insulating material; the electroporation-dielectrophoresis functional layer (2) is made of a conductive material which can be machined in a three-dimensional way and is machined by a low-cost reverse mould process; the bottom layer (3) is made of transparent insulating material; the insulating material (21) filling the isolation channel (13) is selected from PDMS and resin.
7. The microfluidic chip for the synchronous implementation of the electroporation transfection and the sorting of the living cells according to claim 6, wherein the transparent insulating material is selected from Polydimethylsiloxane (PDMS), glass; the conductive material that can be three-dimensionally machined is selected from the group consisting of silver-polydimethylsiloxane (Ag-PDMS), carbon-polydimethylsiloxane (C-PDMS), heavily doped silicon, metal, carbon.
8. The method for preparing the microfluidic chip for synchronously realizing the electroporation transfection and the sorting of the living cells as claimed in claim 1, which comprises the following steps:
(1) photoetching preparation mold
The photoetching is to use photoresist, mask and ultraviolet light to make microfabrication, and the process is as follows:
firstly, cleaning silicon wafer
Spin-coating a first layer of photoresist with a certain thickness on a silicon wafer by using a spin coater;
preparing a required channel pattern on the mask plate; placing a mask plate above a silicon wafer, irradiating the silicon wafer coated with photoresist in a rotating manner by using ultraviolet light, and carrying out photochemical reaction on the photoresist;
spin-coating a second layer of photoresist with a certain thickness on the silicon wafer by using a spin coater;
placing a mask plate above the silicon wafer, irradiating the silicon wafer coated with the photoresist by ultraviolet light, and carrying out photochemical reaction on the photoresist;
sixthly, removing the unexposed photoresist by a developing solution through a developing chemical method; accurately copying the pattern on the mask plate onto the photoresist layer, and finishing the manufacturing of the SU-8 mold;
(2) filling of Ag-PDMS conductive polymers
The washed silver powder and PDMS are mixed according to the proportion of 17: 3, uniformly mixing;
secondly, the mixture is transferred to a mortar for full grinding until the mixture is pasty and the whole is moist and glossy;
uniformly coating a proper amount of Ag-PDMS on the surface of the SU-8 mould, uniformly pressing to fill the Ag-PDMS into the mould as much as possible, and removing the redundant Ag-PDMS on white paper;
heating the filled mold at 70 deg.c for three hr;
(3) reverse mould
Placing the filled mold in an aluminum foil paper box with a proper size, pouring the surface filled with Ag-PDMS upwards into PDMS, and controlling the height to be 2-3 mm;
heating at 70 deg.c for three hr and curing PDMS after three hr;
thirdly, naturally bonding the poured PDMS and the Ag-PDMS filled in the mould into a whole, and lightly demoulding the whole from the mould;
(4) substrate bonding
The surface of the device obtained in the previous step with a flow channel structure faces upwards, and holes are punched from top to bottom at the positions of an inlet and an outlet, so that the flow channel can be communicated with the outside;
secondly, performing isopropanol ultrasonic cleaning on the punched device, flushing with deionized water, drying with nitrogen, heating to remove water, and keeping the device clean;
thirdly, spin-coating a layer of PDMS on the surface of the glass by using a spin coater, placing the spin-coated glass sheet on a heating plate, heating for 10 minutes at 70 ℃, after the PDMS on the surface of the glass is semi-dried, contacting the surface of the clean device with the flow channel structure with the glass sheet, and lightly pressing to obtain a semi-finished chip; the isolation channel (13) is filled with a small amount of semi-cured insulating material (21);
(5) assembly
And cutting the PDMS at the electrode part on the side surface of the chip until the electrode is exposed, wherein the electrode is connected with a lead (23) through silver paste (22).
9. The method of claim 8, wherein the pair of sidewall electrodes (12), when formed by a reverse mold process from a conductive polymer, is characterized in that, in order to avoid that the shallow trench on the mold, which fills the sidewall electrodes (12), with a large area is difficult to fill completely during filling, and increase the filling success rate, the mold pattern can be optimized, and the shallow trench pattern which is originally used for filling the sidewall electrodes with a large area in the mold is modified into a separated grid-type filling trench; the sidewall electrodes of the inverse mold after filling are in the form of a grid (24) which still has electrical continuity with each other and which is easily filled during processing.
10. The method for using the microfluidic chip for realizing the cell electroporation transfection and the living cell sorting synchronously as claimed in claim 1 is characterized by comprising the following steps:
the sample enters the flow channel from the lower layer (9) through the sample inlet (4), and simultaneously the buffer solution enters the flow channel from the upper layer (8) through the sheath flow inlet (5), and the buffer solution limits the sample at the edge parts of two sides of the flow channel (11); cells which are subjected to negative dielectric force or weak positive dielectric force in the sample are limited in the lower layer (9), cells which are subjected to strong positive dielectric force in the sample are adsorbed in the upper layer (8), cells which are subjected to strong positive dielectric force are subjected to electroporation in the upper layer (8), cells with activity reduced due to the electroporation fall in the lower layer (9), cells in the lower layer (9) are limited in the two side edge parts of the flow channel (11) by the limiting effect of sheath flow, and are discharged from a waste cell outlet (7) through the bridge-shaped structure (10); the cells in the upper layer (8) are also restricted by the sheath flow but are discharged from the target cell outlet (6) along the main flow path due to the blocking action of the bridge structure (10).
11. The method of using the chip according to claim 10, wherein the transfected cells are mammalian cells, bacteria; the cell sorting device is independently used as a cell sorting device, is used for sorting Circulating Tumor Cells (CTC), extracting living cells, extracting blood cells and extracting blood plasma based on positive and negative dielectric power;
the amplitude of the voltage of the excitation signal for transfection separation is 600-900V/cm, and the frequency is 100-200 kHz; the sheath flow velocity is 5-10 mul/min, and the sample flow velocity is 10-20 mul/min.
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CN110628568A (en) * 2019-09-30 2019-12-31 北京化工大学 Slide rail type dielectrophoresis electrode structure for high-throughput continuous flow cell separation

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