CN115895876A - Cell electrofusion chip device based on bilateral flow field pairing structure array and preparation method - Google Patents
Cell electrofusion chip device based on bilateral flow field pairing structure array and preparation method Download PDFInfo
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Abstract
The invention relates to the field of cell electrofusion chips, and discloses a cell electrofusion chip device based on a bilateral flow field paired structure array and a preparation method thereof. The invention can realize high-efficiency electrofusion of cells.
Description
Technical Field
The invention relates to the field of cell electrofusion chips, in particular to a cell electrofusion chip device based on a bilateral flow field pairing structure array and a preparation method thereof.
Background
The electrofusion technology is a novel fusion promoting technology established in the last 80 th century. When a cell is placed in a very high electric field, the cell membrane becomes permeable, allowing external molecules to diffuse into the cell, a phenomenon known as electrofusion, also known as electroporation. Using this technique, a number of substances, including DNA, RNA, proteins, drugs, antibodies, and fluorescent probes, can be loaded into cells. Compared with other commonly used methods for introducing foreign substances, electrofusion has many advantages: 1. the electrofusion does not need to use a glass needle like microinjection, does not need technical training and expensive equipment, and can inject millions of cells at one time; 2. electrofusion has few biological or chemical side effects compared to using chemicals; 3. because electrofusion is a physical process and is less dependent on cell type, it is widely used.
Although the cell electrofusion technique has been successfully used in breeding, hybridization studies and cell cloning, several problems remain. The traditional cell synthesis technology depends on random contact of cells to be fused, so that the phenomenon of self-fusion of the same cells is caused, the fusion efficiency is greatly reduced, and the waste of precious cell resources is caused; moreover, if the size difference of the cells to be fused is large, the fusion efficiency is extremely low due to the large difference of transmembrane potentials. The technical scheme mainly aims at improving the pairing efficiency of the heterogenous cells in the cell electrofusion.
Disclosure of Invention
The invention aims to provide a cell electrofusion chip device based on a bilateral flow field pairing structure array and a preparation method thereof, and aims to solve the problem of low pairing efficiency caused by random contact during cell fusion in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme: the cell electrofusion chip device comprises a patterned ITO interdigital electrode layer and a flow path control module, wherein the patterned ITO interdigital electrode layer is connected with an external cell electrofusion instrument, the patterned ITO interdigital electrode layer comprises a substrate layer, an ITO electrode layer and a PDMS bilateral flow field paired structure layer which are sequentially arranged, the PDMS bilateral flow field paired structure layer comprises a bilateral flow field paired structure and a channel, and the bilateral flow field paired structure and the channel are both provided with a plurality of structures and are bilaterally symmetrically arranged.
On the other hand, the technical solution also provides a preparation method of the cell electrofusion chip device based on the bilateral flow field paired structure array, which comprises the following steps:
step one, processing a patterned ITO interdigital electrode layer by adopting a wet etching method;
step two, constructing a PDMS bilateral flow field pairing structure, and constructing by using a PDMS polymer through a soft lithography process and a reverse mold;
step three, preparing a PDMS fusion structure chip, and constructing through a soft lithography process and a reverse mold;
step four, plasma cleaning;
and step five, bonding.
The principle and the advantages of the scheme are as follows: in practical application, aiming at the problem of low fusion efficiency of cell fusion (especially heterologous cell fusion) caused by random contact and membrane potential difference in the prior art, the inventors focused on improving the pairing efficiency of heterologous cells in cell electrofusion and designed the structure (especially pairing structure) of the fusion chip. The micro-fluidic chip technology is adopted, and the micro-scale flow cavity, flow control and electric field control are carried out, so that the aim of accurately controlling cell movement is fulfilled; meanwhile, the method combines the means of microfluid control, dielectrophoresis and the like, and effectively controls the accurate pairing of homologous or heterologous cells under the assistance of microstructures, microelectrodes and the like. According to the chip structure, after cells to be fused enter the capture structure respectively along the channels on the two sides, the patterned ITO interdigital electrode layer is connected with an external cell electrofusion instrument, external electric signals are introduced to the ITO interdigital electrode, an electric field with enough strength is formed between adjacent micro-electrodes, and efficient electrofusion of the cells inside the chip is achieved. The flow path control module can realize sample introduction, capture pairing and sample discharge of cells.
Preferably, as an improvement, an interdigital electrode array is arranged on the ITO electrode layer, the interdigital electrode array is in a comb-tooth structure as a whole, and the interdigital electrode array includes a plurality of interdigital electrodes.
In the technical scheme, the interdigital electrodes are arranged into the array comb-tooth-shaped structure, and after external electric signals are introduced to the ITO interdigital electrodes, an electric field with enough strength is formed between the adjacent micro-electrodes, so that efficient electrofusion of cells in the chip is realized.
Preferably, as a modification, the width of each interdigital electrode is 150-200 μm, and the distance between two adjacent interdigital electrodes is 60-80 μm.
In the technical scheme, the width of the interdigital electrode is optimized so that the interdigital electrode can meet the electric field intensity required by cell perforation under the low voltage condition given by an experiment, and can meet the manufacturing process condition in a laboratory; the width range is a better range verified by experiments, and if the distance between two adjacent interdigital electrodes is too large, the applied voltage for cell perforation is larger, so that the requirement on external electricity is higher; if the width is too narrow, the electrode may not be etched under the existing process conditions, and the cell breakdown may be electrocuted by applying a small voltage, so that it is difficult to explore appropriate experimental parameters.
Preferably, as a modification, the channel has a serpentine structure, and the serpentine channel has a length of 300 to 400 μm, a width of 40 to 50 μm, and a height of 20 to 30 μm.
In the technical scheme, the channel is arranged to be of a snake-shaped structure, so that the space utilization rate can be improved, and a longer length can be extended in a limited space; the serpentine channel facilitates the integration of a plurality of capture units, and improves the flux; the length, width, height and the like of the snake-shaped channel are based on the targeted design of cells selected in experiments, and the requirement of efficient pairing can be met.
Preferably, as an improvement, a plurality of repeated capture units are arranged in the channel, each repeated capture unit comprises a snake-shaped channel section, a capture site structure and a flow resistance adjusting micro-channel, the capture site structure is a circular structure with the diameter of 9-11 μm, and the distance between two adjacent capture sites is 75-150 μm; the flow resistance adjusting micro-channel has the length of 4-6 μm, the width of 4-6 μm and the height of 20-30 μm; the capture site structures are all provided with 7-micrometer openings for realizing the communication of channels on two sides to form a pair of paired structures.
Among this technical scheme, the passageway is whole to be snakelike structure, and each repeated gyration constitutional unit at snakelike structure is the snakelike passageway section promptly, and this scheme all sets up on every snakelike passageway section and catches site and flow resistance regulation passageway, can realize the high flux of chip, through setting up the trompil in catching site department moreover, can realize the intercommunication of both sides passageway, and then realizes pairing and fusing of heterologous cell.
Preferably, as an improvement, flow path control module includes that PDMS fuses structure chip and pipe, and PDMS fuses and is provided with two introduction ports, two play appearance mouths, microchannel and four on the structure chip, and the constitutional part of microchannel all sets up in PDMS fuses the bottom of structure chip with storing up the appearance pond, and introduction port and play appearance mouth set up respectively and correspond to store up directly over the perpendicular in appearance pond, and microchannel constitutional part sets up between four storage appearance ponds.
In the technical scheme, the sample storage pool provides a space for storing the cells to be fused, the cells to be fused are injected into the chip through the micro-channel along the sample inlet, the capture and fusion are realized, and the structural design is reasonable.
Preferably, as an improvement, the diameters of the sample inlet and the sample outlet are both 2-5 mm.
In the technical scheme, the size of the sample inlet and outlet is mainly designed for matching with an external pressure pump pipeline required by an experiment.
Preferably, as an improvement, in the second step and the third step, the soft lithography process and the reverse mold process specifically include the following steps:
(1) Processing a mold with the thickness of 20-35 mu m by using a soft lithography process;
(2) Fixing the mold on an acrylic mold;
(3) Pouring PDMS mixed glue, standing and vacuumizing;
(4) Heating and curing at 60-80 deg.c in a baking oven;
(5) And (4) reversing the mold.
Preferably, as an improvement, in the fourth step, the plasma cleaning condition is that the cleaning time is 10 to 15 seconds; in the fifth step, the bonding process is a hot bonding process, and the bonding temperature is 100-150 ℃. .
In the technical scheme, the plasma cleaning time mainly influences the bonding effect of the chip and the ITO electrode, the too tight bonding can be caused if the cleaning time is too long, cell sap is not easy to enter the structure, and the too short cleaning time can cause the too tight bonding and the easy leakage. In addition, the bonding temperature is verified to be a more appropriate temperature, so that the bonding effect can meet the expected requirement.
Drawings
Fig. 1 is a schematic structural diagram of an entire chip in an embodiment of the present invention.
Fig. 2 is an enlarged schematic view of a pair of mating structures in fig. 1.
Fig. 3 is a schematic plan view of an interdigital electrode in accordance with the present invention.
FIG. 4 is a white light graph of single cell trapping in an embodiment of the present invention.
FIG. 5 is a fluorescence plot of single cell capture in an example of the invention.
FIG. 6 is a white light chart of cell pairing in an embodiment of the present invention.
FIG. 7 is an overlay of paired fluorescence from cells in accordance with an embodiment of the present invention.
FIG. 8 shows white light and fluorescence before and after cell fusion in the example of the present invention.
Detailed Description
The following is a detailed description of the embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents and the like used are commercially available.
Reference numerals in the drawings of the specification include: the device comprises a bilateral flow field pairing structure 1, a channel 2, a capture site structure 3, an inlet sample storage pool I4, an inlet sample storage pool II5, a micro-channel structure part 6, an outlet sample storage pool I7, an outlet sample storage pool II8 and an ITO interdigital electrode 9.
The examples are substantially as shown in figures 1 to 3: the utility model provides a cell electrofusion chip device based on bilateral flow field is to structure array, includes patterning ITO interdigital electrode layer and flow path control module, and patterning ITO interdigital electrode layer forms through conductive adhesive tape and outer cell electrofusion appearance and is connected, and on external signal of telecommunication will be introduced to ITO interdigital electrode 9, will form the electric field of sufficient intensity between the adjacent micro-electrode, realize the high-efficient electrofusion of the inside cell of chip. The flow path control module has the main functions of realizing sample introduction, capture pairing and sample discharge of cell buffer solution in the structure, and comprises a PDMS fusion structure chip and a conduit.
The cell electrofusion chip device based on the bilateral flow field paired structure array sequentially comprises an ITO electrode layer and a PDMS bilateral flow field paired structure layer from bottom to top.
And the ITO electrode layer adopts ITO glass as a substrate, and a patterned interdigital electrode array is etched on the ITO glass by a wet method. The comb tooth distance of the interdigital electrode array is controlled within the range of 60-80 mu m so as to ensure good conductivity and reliability; the width of the comb teeth is in the range of 150-200 μm, and can be determined according to the density of the microelectrode array in actual use.
Referring to fig. 2, the PDMS bilateral flow field paired structure layer includes a bilateral flow field paired structure 1 constructed by PDMS polymer and a channel 2, the bilateral flow field paired structure 1 is arranged symmetrically up and down, and the connection and the contact of paired cells are realized through an opening with a middle length of 7 μm and a middle width of 1 μm. The height of the channel 2 in the PDMS bilateral flow field paired structure layer is 25-30 μm, the channel 2 is arranged in a snake-shaped structure, the overall length of the channel 2 is 300-400 μm, the width is 40-50 μm, and the height is 20-30 μm. A plurality of repeated capture units are arranged in the single-side channel 2, each repeated capture unit comprises a serpentine channel section (namely a rotary unit of the serpentine channel), a capture site structure 3 and a flow resistance adjusting micro-channel (a narrow channel can realize the adjustment of the flow resistance), the capture site structure 3 is a circular structure with the diameter of 9-11 mu m, and the distance between the capture site structures 3 of two adjacent units is 75-150 mu m; the flow resistance adjusting micro-channel has the length of 4-6 μm, the width of 4-6 μm and the height of 20-30 μm; the capture site structures 3 are all provided with openings of 7 μm and 1 μm in width for realizing communication of the channels on both sides to form a pair of paired structures. The symmetrical channels on both sides are connected through a 7 μm opening of the capture site to form a pair of paired structures, i.e., the repeated capture units on both sides are connected to form a paired unit.
The PDMS fusion structure chip is provided with two sample inlets, two sample outlets, a micro-channel structure part and a sample storage pool. The diameter of the sample inlet and the diameter of the sample outlet are both 3mm, and the diameters can be specifically selected according to actual needs in specific application. Two introduction ports are respectively introduction port I and introduction port II, and introduction port I and introduction port II are vertical to run through PDMS and fuse the chip, and the bottom is provided with import respectively and stores up appearance pond I4 and import and store up appearance pond II5. Two play appearance mouths are including going out appearance mouth I and play appearance mouth II, go out appearance mouth I and the equal vertical PDMS that runs through of appearance mouth II and fuse the chip, and the bottom is provided with respectively that the export stores up appearance pond I7 and export and stores up appearance pond II8. Import sample storage pool I4 and import sample storage pool II5, export sample storage pool I7 and export sample storage pool II8 and microchannel structure part 6 all set up in PDMS fuses the bottom of structure chip, and from left to right in proper order for import sample storage pool I4, export sample storage pool II8, microchannel structure part 6, import sample storage pool II5 and export sample storage pool I7. The micro-channels are arranged in a plurality and are respectively communicated between the sample inlet/outlet and the sample storage pool. The PDMS fuses the integrated inlet, outlet and sample storage pool on the structure chip, combines the microchannel that communicates and can realize the kind of introduction of cell suspension, controls the flow of cell in the chip to reach the appearance of cell suspension after fusing.
A preparation method of a cell electrofusion chip device based on a bilateral flow field pairing structure array comprises the following steps:
step one, processing an ITO electrode array: the method is realized by adopting a wet etching process, and specifically comprises the following steps:
s1, selecting ITO glass as a substrate for processing a chip;
s2, spin-coating a layer of SU-8 3005 photoresist with the thickness of 5 microns on the ITO glass;
and S3, etching the interdigital microarray structure on the ITO layer in a photoetching and wet etching mode.
Step two, constructing a PDMS bilateral flow field pairing structure: the method for constructing the bilateral flow field pairing structure and the channel structure by using the PDMS polymer specifically comprises the following steps:
(1) Processing a die with a structure height of 30 mu m by using a soft lithography process, wherein the die structure is a cell suspension liquid storage pool and a micro-channel array structure;
(2) Fixing the mold on an acrylic mold;
(3) Pouring the mixed PDMS mixed glue, standing and vacuumizing;
(4) Curing at 65 deg.c in a baking oven;
(5) And removing the cured PDMS, cutting the PDMS according to the shape adaptation of the ITO interdigital electrode, and punching a sample inlet and a sample outlet which penetrate through the PDMS from the vertical upper part of the sample storage pool.
Step four, plasma cleaning is carried out, and the cleaning time is 10-15 seconds;
and step five, bonding, namely reversely buckling a PDMS chip with a PDMS bilateral flow field matched structure on the ITO electrode, forming a closed cavity with the chip by adopting a thermal bonding process, and performing sample inlet and outlet of the cell suspension only through a sample inlet and a sample outlet, wherein the bonding temperature is 100-150 ℃.
The method comprises the steps of respectively shooting white light and fluorescence of cells which are captured and paired by using a chip by using a fluorescence microscope, powering up by using a cell electrofusion instrument, shooting images of the cells under the white light and the fluorescence again after powering up, and comparing the white light and the fluorescence images of the cells before and after powering up. FIGS. 4-8 are fluorescence images before and after cell pairing and fusion according to the technical scheme, and the chip structure according to the technical scheme can realize efficient pairing and fusion of heterologous cells.
The above description is only an example of the present invention, and the general knowledge of the known specific technical solutions and/or characteristics and the like in the solutions is not described herein too much. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (10)
1. The utility model provides a cell electrofusion chip device based on two side flow field are mated to structure array which characterized in that: the cell electrofusion device comprises a patterned ITO interdigital electrode layer and a flow path control module, wherein the patterned ITO interdigital electrode layer is connected with an extracellular electrofusion instrument, the patterned ITO interdigital electrode layer comprises a substrate layer, an ITO electrode layer and a PDMS bilateral flow field paired structure layer which are sequentially arranged, the PDMS bilateral flow field paired structure layer comprises bilateral flow field paired structures and channels, and the bilateral flow field paired structures and the channels are arranged in a bilaterally symmetrical mode.
2. The cell electrofusion chip device based on the bilateral flow field paired structure array according to claim 1, wherein: the ITO electrode layer is provided with an interdigital electrode array, the interdigital electrode array is integrally of a comb structure, and the interdigital electrode array comprises a plurality of interdigital electrodes.
3. The cell electrofusion chip device based on the bilateral flow field paired structure array according to claim 2, wherein: the width of each interdigital electrode is 150-200 mu m, and the distance between two adjacent interdigital electrodes is 60-80 mu m.
4. The cell electrofusion chip apparatus based on the bilateral flow field paired structure array according to claim 3, wherein: the channel is of a snake-shaped structure, the length of the snake-shaped channel is 300-400 mu m, the width is 40-50 mu m, and the height is 20-30 mu m.
5. The cell electrofusion chip apparatus based on the bilateral flow field paired structure array according to claim 4, wherein: a plurality of repeated capture units are arranged in the channel, each repeated capture unit comprises a serpentine channel section, a capture site structure and a flow resistance adjusting micro-channel, the capture site structure is a circular structure with the diameter of 9-11 mu m, and the distance between two adjacent capture sites is 75-150 mu m; the flow resistance regulating micro-channel has the length of 4-6 μm, the width of 4-6 μm and the height of 20-30 μm; the capture site structures are all provided with 7-micrometer openings for realizing the communication of double-side channels to form a pair of paired structures.
6. The cell electrofusion chip apparatus based on the bilateral flow field paired structure array according to claim 5, wherein: flow path control module includes that PDMS fuses structure chip and pipe, PDMS fuses and is provided with two introduction ports, two play appearance mouth, microchannel and four on the structure chip and stores up the appearance pond, and the constitutional part of microchannel all sets up in PDMS fuses the bottom of structure chip with storing up the appearance pond, and introduction port and play appearance mouth set up respectively and correspond to store up directly over the perpendicular in appearance pond, and microchannel constitutional part sets up and stores up between the appearance pond at four.
7. The cell electrofusion chip apparatus based on the bilateral flow field paired structure array according to claim 6, wherein: the diameters of the sample inlet and the sample outlet are both 2-5 mm.
8. The method for preparing a cell electrofusion chip device based on the double-sided flow field paired structure array according to any one of claims 1 to 7, wherein the method comprises the following steps:
step one, processing a patterned ITO interdigital electrode layer by adopting a wet etching method;
step two, constructing a PDMS bilateral flow field pairing structure, and constructing by using a PDMS polymer through a soft lithography process and a reverse mold;
step three, preparing a PDMS fusion structure chip, and constructing through a soft lithography process and a reverse mold;
step four, plasma cleaning;
and step five, bonding.
9. The method for preparing a cell electrofusion chip device based on the bilateral flow field paired structure array according to claim 8, wherein: in the second step and the third step, the soft lithography process and the reverse mold process specifically comprise the following steps:
(1) Processing a mould with the structure height of 20-35 mu m by using a soft lithography process;
(2) Fixing the mold on an acrylic mold;
(3) Pouring PDMS mixed glue, standing and vacuumizing;
(4) Heating and curing at 60-80 deg.c in a baking oven;
(5) And (4) reversing the mold.
10. The method for preparing a cell electrofusion chip device based on the bilateral flow field paired structure array according to claim 9, wherein: in the fourth step, the plasma cleaning is carried out for 10 to 15 seconds; in the fifth step, the bonding process is a hot bonding process, and the bonding temperature is 100-150 ℃.
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