WO2024114438A1 - Cell electrofusion chip device based on bilateral flow field pairing structure array and preparation method therefor - Google Patents

Abstract

The present invention relates to the field of cell electrofusion chips. Disclosed are a cell electrofusion chip device based on a bilateral flow field pairing structure array and a preparation method therefor. The cell electrofusion chip device comprises a patterned ITO interdigital electrode layer and a flow path control module. The patterned ITO interdigital electrode layer is connected to 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 pairing structure layer which are sequentially arranged. The PDMS bilateral flow field pairing structure layer comprises a bilateral flow field pairing structure and a channel, and the bilateral flow field pairing structure and the channel are symmetrically arranged. According to the present invention, efficient electrofusion of cells can be realized.

Description

A cell electrofusion chip device based on a double-sided flow field pairing structure array and a preparation method thereof Technical Field

The present invention relates to the field of cell electrofusion chips, and in particular to a cell electrofusion chip device based on a double-sided flow field pairing structure array and a preparation method thereof.

Background technique

Electrofusion technology is a new type of fusion-promoting technology established in the 1980s. When cells are placed in a very high electric field, the cell membrane becomes permeable, allowing external molecules to diffuse into the cell. This phenomenon is called electrofusion, also known as electroporation. Using this technology, many substances, including DNA, RNA, proteins, drugs, antibodies and fluorescent probes, can be loaded into cells. Compared with other commonly used methods of introducing exogenous substances, electrofusion has many advantages: First, electrofusion does not require the use of glass needles like microinjection, does not require technical training and expensive equipment, and can be injected into millions of cells at a time; second, compared with the use of chemical substances, electrofusion has almost no biological or chemical side effects; third, because electrofusion is a physical method, it is less dependent on cell types and is therefore widely used.

Although cell electrofusion technology has been successfully applied in breeding, hybridization research and cell cloning, there are still some problems. Traditional cell fusion technology relies on random contact of cells to be fused, which causes the self-fusion of cells of the same species, greatly reducing the efficiency of fusion and causing a waste of precious cell resources; and if the cells to be fused have large differences in scale, the huge difference in transmembrane potential will eventually make the fusion efficiency extremely low. This technical solution mainly focuses on improving the pairing efficiency of heterologous cells in cell electrofusion.

Summary of the invention

The present invention aims to provide a cell electrofusion chip device based on a double-sided flow field pairing structure array and a preparation method thereof, so as to solve the problem of low pairing efficiency caused by random contact during cell fusion in the prior art.

To achieve the above-mentioned purpose, the present invention adopts the following technical scheme: a cell electrofusion chip device based on a double-sided flow field pairing structure array, comprising a patterned ITO interdigitated electrode layer and a flow path control module, the patterned ITO interdigitated electrode layer is connected to an external cell electrofusion instrument, the patterned ITO interdigitated electrode layer comprises a base layer, an ITO electrode layer and a PDMS double-sided flow field pairing structure layer arranged in sequence, the PDMS double-sided flow field pairing structure layer comprises a double-sided flow field pairing structure and a channel, and both the double-sided flow field pairing structure and the channel are provided in plurality and are bilaterally symmetrically arranged.

On the other hand, the present technical solution also provides a method for preparing a cell electrofusion chip device based on a double-sided flow field pairing structure array, comprising the following steps:

Step 1: Processing a patterned ITO interdigital electrode layer using a wet etching method;

Step 2: construct a PDMS double-sided flow field pairing structure using PDMS polymer through soft lithography and reverse molding;

Step 3: Prepare a PDMS fusion structure chip by soft lithography and reverse molding;

Step 4: plasma cleaning;

Step 5: Bonding.

The principle and advantages of this scheme are: in practical application, in view of the problem of low fusion efficiency caused by random contact and membrane potential difference in cell fusion (especially heterologous cell fusion) in the prior art, the inventor focuses on improving the pairing efficiency of heterologous cells in cell electrofusion, and designs the structure of the fusion chip (especially the pairing structure). Microfluidic chip technology is adopted to achieve the purpose of accurately controlling cell movement through microscale flow channels, flow control, and electric field control; at the same time, combined with microfluidic control, dielectrophoresis and other means, with the assistance of microstructures, microelectrodes, etc., the precise pairing of homologous or heterologous cells is effectively controlled. In the chip structure of this scheme, after the cells to be fused enter the capture structure along the channels on both sides, the patterned ITO interdigital electrode layer is connected to the external cell electrofusion instrument, and the external electrical signal will be introduced into the ITO interdigital electrode, and an electric field of sufficient strength will be formed between adjacent microelectrodes to achieve efficient electrofusion of cells inside the chip. The flow control module can realize the sampling, capture pairing and sampling of cells.

Preferably, as an improvement, an interdigital electrode array is provided on the ITO electrode layer, the interdigital electrode array is in a comb-teeth structure as a whole, and the interdigital electrode array includes a plurality of interdigital electrodes.

In this technical solution, by setting the interdigitated electrodes into a comb-tooth structure of an array, after the external electrical signal is introduced into the ITO interdigitated electrodes, an electric field of sufficient strength will be formed between adjacent microelectrodes to achieve efficient electrical fusion of cells inside the chip.

Preferably, as an improvement, the width of the interdigital electrodes is 150-200 μm, and the distance between two adjacent interdigital electrodes is 60-80 μm.

In this technical solution, the optimization of the width of the interdigital electrodes must be able to meet the electric field strength required for cell perforation under the low voltage conditions given in the experiment, and also meet the manufacturing process conditions that can be achieved in the laboratory; the above width range is the preferred range verified by the experiment. If the distance between two adjacent interdigital electrodes is too large, the electric field strength required for cell perforation will be too large. The greater the voltage, the higher the requirements for external electrical equipment; if it is too narrow, the electrode may not be etched out under the existing process conditions, and applying a very small voltage may cause the cells to be electrocuted and killed, making it difficult to explore suitable experimental parameters.

Preferably, as an improvement, the channel has a serpentine structure, and the length of the serpentine channel is 300-400 μm, the width is 40-50 μm, and the height is 20-30 μm.

In the present technical solution, by setting the channel into a serpentine structure, 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 multiple capture units and improves the flux; the length, width, height, etc. of the serpentine channel are targeted designs based on the cells selected for the experiment, which can meet the needs of efficient pairing.

Preferably, as an improvement, a number of repeated capture units are arranged in the channel, and the repeated capture units all include a serpentine channel segment, a capture site structure and a flow resistance regulating microchannel, the capture site structure is a circular structure with a diameter of 9 to 11 μm, and the distance between two adjacent capture sites is 75 to 150 μm; the flow resistance regulating microchannel has a length of 4 to 6 μm, a width of 4 to 6 μm, and a height of 20 to 30 μm; the capture site structures are all provided with 7 μm openings for realizing the connectivity of the channels on both sides, forming a pair of matching structures.

In the present technical solution, the channel as a whole has a serpentine structure, and each repeated rotation structural unit in the serpentine structure is a serpentine channel segment. In the present solution, capture sites and flow resistance adjustment channels are arranged on each serpentine channel segment, which can achieve high throughput of the chip, and by setting openings at the capture sites, the channels on both sides can be connected, thereby realizing the pairing and fusion of heterologous cells.

Preferably, as an improvement, the flow control module includes a PDMS fusion structure chip and a catheter, and the PDMS fusion structure chip is provided with two sample inlets, two sample outlets, a microchannel and four sample storage pools, the structural part of the microchannel and the sample storage pool are both arranged at the bottom of the PDMS fusion structure chip, the sample inlet and the sample outlet are respectively arranged vertically above the corresponding sample storage pool, and the microchannel structural part is arranged between the four sample storage pools.

In this technical solution, the sample storage pool provides space for storing cells to be fused, and the cells to be fused are injected into the chip along the injection port through the microchannel to achieve capture and fusion, 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 this technical solution, the sizes of the sample inlet and outlet are mainly designed to match the external pressure pump pipeline required for the experiment.

Preferably, as an improvement, in step 2 and step 3, the soft lithography process and the reverse molding process specifically include the following steps:

(1) Using soft lithography technology, a mold with a thickness of 20 to 35 μm is processed;

(2) Fixing the mold on an acrylic mold;

(3) Pour in the PDMS mixed glue and let it stand for a while before vacuuming;

(4) Place in an oven and heat at 60-80°C for curing;

(5) Mold making.

Preferably, as an improvement, in step 4, the plasma cleaning condition is 10 to 15 seconds; in step 5, the bonding process is hot bonding and the bonding temperature is 100 to 150°C.

In this technical solution, the plasma cleaning time mainly affects the bonding effect between the chip and the ITO electrode. If the cleaning time is too long, the bonding will be too tight and the cell fluid will not easily enter the structure. If the cleaning time is too short, the bonding will be loose and leak easily. In addition, through verification, the above bonding temperature is a more suitable temperature, which can ensure that the bonding effect meets the expected requirements.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the structure of the entire chip in an embodiment of the present invention.

FIG. 2 is an enlarged schematic diagram of a pair of matching structures in FIG. 1 .

Figure 3 is a schematic plan view of the interdigitated electrodes in an embodiment of the present invention.

FIG. 4 is a white light image of single cell capture in an embodiment of the present invention.

FIG. 5 is a fluorescence image of a single cell captured in an embodiment of the present invention.

FIG. 6 is a white light image of cell pairing in an embodiment of the present invention.

FIG. 7 is a fluorescence overlay image of cell pairs in an embodiment of the present invention.

FIG8 is a white light image and a fluorescence image of cells before and after cell fusion in an embodiment of the present invention.

Detailed ways

The following is further described in detail through specific implementations, but the implementations of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following implementations are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial channels.

The figure marks in the drawings of the specification include: bilateral flow field pairing structure 1, channel 2, capture site structure 3, inlet sample storage pool I4, inlet sample storage pool II5, microchannel structure part 6, outlet sample storage pool I7, outlet sample storage pool II8, ITO interdigital electrode 9.

The embodiment is basically as shown in Figures 1-3: a cell electrofusion chip device based on a double-sided flow field pairing structure array, comprising a patterned ITO interdigital electrode layer and a flow path control module, wherein the patterned ITO interdigital electrode layer is connected to an external cell by a conductive tape. The cell electrofusion instrument forms a connection, and the external electrical signal will be introduced into the ITO interdigital electrode 9, and an electric field of sufficient strength will be formed between adjacent microelectrodes to achieve efficient electrofusion of cells inside the chip. The main function of the flow control module is to realize the injection, capture pairing and sample output of the cell buffer in the above structure. The flow control module includes a PDMS fusion structure chip and a catheter.

A cell electrofusion chip device based on a double-sided flow field pairing structure array comprises, from bottom to top, an ITO electrode layer and a PDMS double-sided flow field pairing structure layer.

The ITO electrode layer uses ITO glass as a substrate, and a patterned interdigital electrode array is etched on the ITO glass by wet etching. The comb tooth distance of the interdigital electrode array is controlled within the range of 60 to 80 μm to ensure good conductivity and reliability; the width of the comb teeth is within the range of 150 to 200 μm, which can be determined according to the density of the microelectrode array in actual use.

As shown in FIG2 , the PDMS double-sided flow field pairing structure layer includes a double-sided flow field pairing structure 1 and a channel 2 constructed using a PDMS polymer. The double-sided flow field pairing structure 1 is symmetrically arranged on the upper and lower sides, and is connected and the contact of paired cells is achieved through an opening with a length of 7 μm and a width of 1 μm in the middle. The height of the channel 2 in the PDMS double-sided flow field pairing structure layer is 25-30 μm, and the channel 2 is arranged in a serpentine structure, and the overall length of the channel 2 is 300-400 μm, the width is 40-50 μm, and the height is 20-30 μm. Several repeated capture units are arranged in the single-sided channel 2, and the repeated capture units all include a serpentine channel section (i.e., a turning unit of the serpentine channel), a capture site structure 3, and a flow resistance adjustment microchannel (a narrow channel that can adjust the flow resistance). The capture site structure 3 is a circular structure with a diameter of 9 to 11 μm, and the distance between the capture site structures 3 of two adjacent units is 75 to 150 μm; the flow resistance adjustment microchannel is 4 to 6 μm long, 4 to 6 μm wide, and 20 to 30 μm high; the capture site structure 3 is provided with a 7 μm opening with a width of 1 μm, which is used to achieve the connection between the two-sided channels, forming a pair of paired structures. The symmetrical channels on both sides are connected through the 7 μm opening of the capture site to form a pair of paired structures, that is, 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 microchannel structure part and a sample storage pool. The diameters of the sample inlet and the sample outlet are both 3mm, and the diameter can be specifically selected according to actual needs during specific applications. The two sample inlets are respectively sample inlet I and sample inlet II, and the sample inlet I and sample inlet II both vertically penetrate the PDMS fusion chip, and the bottom is respectively provided with an import sample storage pool I4 and an import sample storage pool II5. The two sample outlets include a sample outlet I and a sample outlet II, and the sample outlet I and sample outlet II both vertically penetrate the PDMS fusion chip, and the bottom is respectively provided with an export sample storage pool I7 and an export sample storage pool II8. The import sample storage pool I4 and the import sample storage pool II5, the export sample storage pool I7 and the export sample storage pool II8, and the microchannel structure part 6 are all arranged at the bottom of the PDMS fusion structure chip, and from left to right are the import sample storage pool I4, the export sample storage pool II8, the microchannel structure part 6, the import sample storage pool II5 and the export sample storage pool I7. There are multiple microchannels, which are connected to the injection port. The sample inlet/outlet and the sample storage pool. The sample inlet, sample outlet and sample storage pool integrated on the PDMS fusion structure chip, combined with the connected microchannels, can realize the injection of cell suspension, control the flow of cells inside the chip, and discharge the cell suspension after fusion.

A method for preparing a cell electrofusion chip device based on a double-sided flow field pairing structure array comprises the following steps:

Step 1: Processing ITO electrode array: using wet etching process, specifically including:

S1. Select ITO glass as the substrate for processing chips;

S2, spin-coat a 5μm layer of SU-8 3005 photoresist on the ITO glass;

S3. Etching a cross-finger microarray structure on the ITO layer by photolithography and wet etching.

Step 2: Constructing a PDMS double-sided flow field pairing structure: Using PDMS polymer to construct a double-sided flow field pairing structure and a channel structure, specifically including:

(1) Using soft lithography technology, a mold with a structure height and thickness of 30 μm was processed, and the mold structure was a cell suspension sample storage pool and a microchannel array structure;

(2) Fixing the mold on an acrylic mold;

(3) Pour the mixed PDMS glue and let it stand for a while before vacuuming;

(4) Place in an oven at 65°C for curing;

(5) Peel off the cured PDMS, cut it according to the shape of the ITO interdigital electrode, and punch out the inlet and outlet that penetrate the PDMS vertically from above the sample reservoir.

Step 4: plasma cleaning, cleaning time 10 to 15 seconds;

Step 5: Bonding: Place the PDMS double-sided flow field matching structure PDMS chip upside down on the ITO electrode, and use a thermal bonding process to form a closed cavity with the chip. The cell suspension is only sampled in and out through the sample inlet and outlet, and the bonding temperature is 100-150°C.

The cells captured and paired using the chip were photographed under white light and fluorescence using a fluorescence microscope, and then powered on using the cell electrofusion instrument. After powering on, the images of the cells under white light and fluorescence were photographed again, and the white light and fluorescence images of the cells before and after powering on were compared. Figures 4 to 8 are fluorescence images of the cell pairing and fusion before and after the fusion of the technical solution. The chip structure of the technical solution can achieve efficient pairing and fusion of heterologous cells.

The above is only an embodiment of the present invention, and the known specific technical solutions and/or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, Several modifications and improvements may be made, which should also be considered as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The protection scope required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims (10)

1. A cell electrofusion chip device based on a double-sided flow field pairing structure array is characterized in that it includes a patterned ITO interdigital electrode layer and a flow path control module, wherein the patterned ITO interdigital electrode layer is connected to an external cell electrofusion instrument, the patterned ITO interdigital electrode layer includes a substrate layer, an ITO electrode layer and a PDMS double-sided flow field pairing structure layer arranged in sequence, the PDMS double-sided flow field pairing structure layer includes a double-sided flow field pairing structure and a channel, and both the double-sided flow field pairing structure and the channel are provided in plurality and are bilaterally symmetrically arranged.
2. According to the cell electrofusion chip device based on the double-sided flow field pairing structure array described in claim 1, it is characterized in that: an interdigitated electrode array is arranged on the ITO electrode layer, the interdigitated electrode array is a comb-tooth structure as a whole, and the interdigitated electrode array includes a plurality of interdigitated electrodes.
3. According to the cell electrical fusion chip device based on the double-sided flow field pairing structure array as described in claim 2, it is characterized in that the width of the interdigital electrodes is 150-200 μm, and the distance between two adjacent interdigital electrodes is 60-80 μm.
4. According to claim 3, a cell electrofusion chip device based on a double-sided flow field pairing structure array is characterized in that the channel has a serpentine structure, and the length of the serpentine channel is 300-400 μm, the width is 40-50 μm, and the height is 20-30 μm.
5. According to claim 4, a cell electrofusion chip device based on a double-sided flow field pairing structure array is characterized in that: a plurality of repeated capture units are arranged in the channel, and the repeated capture units all include a serpentine channel section, a capture site structure and a flow resistance adjustment microchannel, the capture site structure is a circular structure with a diameter of 9 to 11 μm, and the distance between two adjacent capture sites is 75 to 150 μm; the flow resistance adjustment microchannel has a length of 4 to 6 μm, a width of 4 to 6 μm, and a height of 20 to 30 μm; the capture site structures are all provided with 7 μm openings for realizing the connectivity of the double-sided channels to form a pair of pairing structures.
6. According to claim 5, a cell electrofusion chip device based on a double-sided flow field pairing structure array is characterized in that: the flow path control module includes a PDMS fusion structure chip and a catheter, and the PDMS fusion structure chip is provided with two sample inlets, two sample outlets, a microchannel and four sample storage pools, the structural part of the microchannel and the sample storage pool are both arranged at the bottom of the PDMS fusion structure chip, the sample inlet and the sample outlet are respectively arranged vertically above the corresponding sample storage pool, and the microchannel structural part is arranged between the four sample storage pools.
7. According to claim 6, a cell electrofusion chip device based on a double-sided flow field pairing structure array is characterized in that the diameters of the sample inlet and the sample outlet are both 2 to 5 mm.
8. A cell electrofusion chip device based on a double-sided flow field pairing structure array according to any one of claims 1 to 7 The preparation method of the device is characterized in that it comprises the following steps:

   Step 1: Processing a patterned ITO interdigital electrode layer using a wet etching method;

   Step 2: construct a PDMS double-sided flow field pairing structure using PDMS polymer through soft lithography and reverse molding;

   Step 3: Prepare a PDMS fusion structure chip by soft lithography and reverse molding;

   Step 4: plasma cleaning;

   Step 5: Bonding.
9. The method for preparing a cell electrofusion chip device based on a double-sided flow field pairing structure array according to claim 8 is characterized in that: in step 2 and step 3, the soft lithography process and the reverse molding process specifically include the following steps:

   (1) Using soft lithography technology, molds with a structure height of 20 to 35 μm are processed;

   (2) Fixing the mold on an acrylic mold;

   (3) Pour in the PDMS mixed glue and let it stand for a while before vacuuming;

   (4) Place in an oven and heat at 60-80°C for curing;

   (5) Mold making.
10. According to the method for preparing a cell electrofusion chip device based on a double-sided flow field pairing structure array as described in claim 9, it is characterized in that: in step 4, the plasma cleaning condition is a cleaning time of 10 to 15 seconds; in step 5, the bonding process is hot bonding and the bonding temperature is 100 to 150°C.