A high-throughput cell electrofusion chip device based on double-sided injection and double-concave capture pairing structure array and a preparation method thereof

Abstract

The application relates to the field of cell electrofusion chips, and discloses a cell electrofusion chip device based on a high-throughput double-concave capture pairing structure array and a preparation method thereof. The cell electrofusion chip device based on the double-concave capture pairing structure array comprises, from bottom to top, an electrode layer and a PDMS double-side flow field capture structure layer, the electrode layer is connected with an external cell electrofusion instrument, the PDMS double-side flow field capture structure layer comprises a plurality of array-arranged capture pairing structures and channels, each capture pairing structure comprises a pairing structure located in the middle and two capture structures located on the two sides of the pairing structure, and the two capture structures are symmetrically arranged. The application can realize rapid and efficient fusion of cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell electrofusion chip, in particular to a high-throughput double concave capture pairing structure array cell electrofusion chip device based on double-side injection and a preparation method thereof. BACKGROUND

[0002] Electrofusion technology is a new type of fusion promotion 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, which is called electrofusion or 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 foreign 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 inject millions of cells at a time; second, compared with chemical substances, electrofusion has almost no biological or chemical side effects; third, because electrofusion is a physical method, it is less dependent on cell type and is widely applicable.

[0003] 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 self-fusion of the same cells, greatly reduces the fusion efficiency, and wastes valuable cell resources; and if the size difference between the cells to be fused is large, the large difference in transmembrane potential will eventually result in a very low fusion efficiency. SUMMARY

[0004] The present application aims to provide a high-throughput double concave capture pairing structure array cell electrofusion chip device based on double-side injection and a preparation method thereof, to solve the problem of low fusion efficiency in the prior art cell fusion technology.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: a high-throughput double concave capture pairing structure array cell electrofusion chip device based on double-side injection, comprising an electrode layer and a PDMS double-side flow field capture structure layer arranged in sequence from bottom to top, the electrode layer being connected with an external cell electrofusion instrument, the PDMS double-side flow field capture structure layer comprising a plurality of array-arranged capture pairing structures and channels, each capture pairing structure comprising a pairing structure in the middle and two capture structures on both sides of the pairing structure, the two capture structures being symmetrically arranged.

[0006] On the other hand, the technical scheme provides a preparation method of a high-throughput double concave capture pairing structure array cell electrofusion chip device based on double-side injection, comprising the following steps:

[0007] Step one, using wet etching method to process electrode layer;

[0008] Step two, constructing PDMS double-sided flow field capture structure layer, using PDMS polymer to construct through soft lithography process and reverse mold;

[0009] Step three, plasma cleaning;

[0010] Step four, bonding.

[0011] The principle and advantages of the present scheme are: in practical application, aiming at the low fusion efficiency caused by random contact and membrane potential difference in the cell fusion (especially the fusion of heterologous cells with large size difference) in the prior art, the inventors focus on improving the pairing efficiency of heterologous cells in cell electrofusion, and plan to innovate the structure of the cell fusion chip and develop a high-throughput microfluidic capture pairing fusion chip. The chip structure is designed through the structure of the microfluidic chip, so that the heterologous cells enter the structure from different directions and are captured, and the heterologous cells are controlled to pair efficiently. In the technical scheme, in order to realize efficient cell fusion, the structure of the electrofusion chip is optimized and designed, and through the upgrading of the channel structure and the capture structure, when the cells to be fused enter the capture pairing structure along the channel, the electrode layer is connected with the external cell electrofusion instrument, the external electric signal is introduced to the electrode, and a strong electric field is formed between the adjacent microelectrodes, realizing efficient electrofusion of the cells in the chip. The flow path control module can realize cell sampling, capture pairing and sample discharge. In addition, in order to realize high throughput, the technical scheme adopts a double-sided flow field capture structure, which improves the pairing efficiency of heterologous cells under the premise of ensuring high throughput.

[0012] Preferably, as an improvement, the capture structure is surrounded by the channel side wall and the micro column, one end of the capture structure is communicated with the pairing structure, and the other end of the capture structure is provided with an inner concave arc segment.

[0013] In the technical scheme, the arc segment cooperates with the micro column to realize rapid capture of cells, and then realizes efficient pairing.

[0014] Preferably, as an improvement, the radius of the arc segment is 12-16 μm.

[0015] In the technical scheme, the radius of the arc segment is determined based on the diameter of the selected experimental cells. If the radius is too large, the cells will directly enter the pairing chamber without being first stuck in the capture site during the capture process, causing multi-cell capture; if the radius is too small, the cells will be difficult to be captured in the capture site, affecting the capture efficiency.

[0016] Preferably, as an improvement, the matching structure comprises a capture chamber and two concave micro-pillars arranged in the capture chamber, the two concave micro-pillars enclose two symmetrical capture sites, and the middle part of the capture site is provided with an opening for realizing communication and contact of the cells to be matched.

[0017] In the technical solution, when the cells are captured, the cells are inhaled along the channel into the matching chamber by negative pressure suction operation, and the two cells to be matched are contacted at the opening of the matching site.

[0018] Preferably, as an improvement, the width of the opening is 8-14 microns.

[0019] In the technical solution, the width of the opening is determined based on the diameter of the selected experimental cells and the effective contact area required for cell fusion. If the width is too large, the cells will pass through the opening to the other side when inhaled into the matching chamber; if the width is too small, the cells will be difficult to fuse due to insufficient contact during the fusion process.

[0020] Preferably, as an improvement, it further comprises a PDMS fusion structure chip matched with the channel, the PDMS fusion structure chip is provided with two sample inlets, two sample outlets, structure micro-pillars, a micro-channel and a sample storage pool, the structure micro-pillars, the micro-channel and the sample storage pool are arranged at the bottom of the PDMS fusion structure chip, and the two sample inlets and the sample outlets are arranged vertically through the PDMS fusion structure chip and above the sample storage pool.

[0021] In the technical solution, the PDMS fusion structure chip is mainly used to realize the sample inlet, capture matching and sample outlet of the cell buffer in the above structure.

[0022] Preferably, as an improvement, the electrode layer is provided with an interdigital electrode array, the distance between adjacent two interdigital electrodes is in the range of 60-80 microns, and the width of the interdigital electrode is 150-200 microns.

[0023] In the technical solution, the width of the interdigital electrode not only meets the required electric field intensity for cell perforation under the given low voltage condition, but also facilitates preparation and processing. The above-mentioned width of the interdigital electrode is a suitable width range verified by practice. In addition, the distance between adjacent two interdigital electrodes has a great influence on the difficulty of cell perforation. If the distance is too large, the voltage required to achieve cell perforation will be higher, and the requirement for external electrical appliances will be higher. If the distance is too narrow, the electrode may not be etched out under the existing process conditions, and the cell may be electrocuted by a small voltage.

[0024] Preferably, as an improvement, in step three, the plasma cleaning condition is a cleaning time of 10-15 seconds.

[0025] In the technical solution, the time of plasma cleaning mainly affects the bonding effect of the chip and the ITO electrode, and too long cleaning time will cause too tight bonding, and the cell fluid is not easy to enter the structure, and too short cleaning time will cause loose bonding and easy leakage.

[0026] Preferably, as an improvement, in step four, the bonding process is thermal bonding, and the bonding temperature is 100-150 DEG C.

[0027] In the technical solution, through practice verification, the bonding temperature is a more appropriate temperature, which can ensure that the bonding effect meets the expected requirements. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The figure is a schematic diagram of an ITO interdigital electrode structure of the application.

[0029] Figure 2 The figure is a schematic diagram of a PDMS double-sided flow field capture structure layer in an embodiment of the application.

[0030] Figure 3 The figure is a schematic diagram of a capture pairing structure in an embodiment of the application.

[0031] Figure 4 The figure is a schematic diagram of a PDMS fusion chip. DETAILED DESCRIPTION

[0032] The following will be further described in detail through specific embodiments, but the embodiments of the application are not limited thereto. If not specifically indicated, the technical means used in the following embodiments are conventional means known to those skilled in the art; the experimental methods used are conventional methods; the materials, reagents, etc. used can be obtained from commercial channels.

[0033] The reference signs in the drawings of the specification include: interdigital electrode array 1, double-sided flow field capture structure 2, channel 3, capture structure 4, pairing structure 5, circular arc segment 6, sample inlet I 7, sample inlet II 8, inlet sample storage pool I 9, inlet sample storage pool II 10, sample outlet I 11, sample outlet II 12, outlet sample storage pool I 13, outlet sample storage pool II 14.

[0034] The embodiment is basically as shown in the drawings: a high-throughput double-concave capture pairing structure array-based cell electrofusion chip device based on double-sided sampling, comprising an electrode layer and a PDMS double-sided flow field capture structure layer arranged in order from bottom to top. Figures 1-2

[0035] ​The electrode layer is an ITO electrode layer, a patterned interdigital electrode array 1 is etched on the ITO glass by a wet etching method, and the ITO electrode layer is connected to an external cell fusion instrument through a conductive adhesive tape. External electrical signals will be introduced to the ITO interdigital electrodes, and a strong electric field will be formed between adjacent microelectrodes to achieve efficient electrical fusion of cells inside the chip. The comb tooth distance of the interdigital electrode array 1 is controlled within the range of 60-80 μm to ensure good conductivity and reliability. The width of the comb tooth is determined according to the density of the microelectrode array, and the specific width range is 150-200 μm.

[0036] The PDMS double-sided flow field capture structure layer uses PDMS polymer to construct a double-sided flow field capture structure 2 and a channel 3 connected thereto, which is mainly used for realizing sample injection, capture pairing and sample discharge of cell buffer. In this embodiment, the height of the double-sided flow field capture structure 2 and the channel 3 is 30 μm, and the double-sided flow field capture structure 2 is composed of 12×18 capture pairing structures. In actual implementation, different numbers of capture pairing structures can be set according to actual needs. A single capture pairing structure includes a capture structure 4 arranged on both sides and a pairing structure 5 located in the middle. The capture structure 4 is surrounded by a channel side wall and a microcolumn, and two capture structures 4 are respectively located on the upper and lower sides of the pairing structure and are symmetrically arranged to capture two different cells respectively. The pairing structure 5 is composed of a capture chamber and a double-concave microcolumn. One side of the capture site of the capture structure is a channel wall, and the other side is provided with a circular arc segment 6 with a radius of 14 μm. The capture structure 4 and the capture chamber of the pairing structure 5 are connected through a channel with a length of 12 μm and a width of 14 μm. The pairing structure 5 is a double-concave pairing structure, which is surrounded by two independent concave microcolumns to form two symmetrical 12 μm circular arc capture sites. There is a 10 μm opening in the middle to realize communication and contact between paired cells.

[0037] The channel includes a main channel and a bypass channel; the main channel has a width of 57 μm and a total length of 5806 μm; the bypass channel has a width of 35 μm and a length of 180 μm. The scheme also includes a PDMS fusion chip, which is combined with Figure 3As shown, the PDMS fusion chip is provided with two sample inlets, two sample outlets, sample storage pools, microchannels and capture pairing structures (capture microcolumns). The two sample inlets are sample inlet I 7 and sample inlet II 8, and the sample inlet I 7 and the sample inlet II 8 are located at two ends of the PDMS fusion chip. The sample inlet I 7 and the sample inlet II 8 both vertically penetrate the PDMS fusion chip, and the bottoms are respectively provided with inlet sample storage pool I 9 and inlet sample storage pool II 10. The two sample outlets include sample outlet I 11 and sample outlet II 12, and the sample outlet I 11 and the sample outlet II 12 are also located at two ends of the PDMS fusion chip. The sample outlet I 11 and the sample outlet II 12 both vertically penetrate the PDMS fusion chip, and the bottoms are respectively provided with outlet sample storage pool I 13 and outlet sample storage pool II 14. The inlet sample storage pool I 9 and the inlet sample storage pool II 10, the outlet sample storage pool I 13 and the outlet sample storage pool II 14 are all located in the same plane with the microchannels and the capture pairing structures.

[0038] The connecting line of the inlet sample storage pool I 9 and the outlet sample storage pool I 13 and the connecting line of the inlet sample storage pool II 10 and the outlet sample storage pool II 14 intersect with each other. Such intersection is firstly to meet the requirement that two kinds of cells are respectively introduced from two sides to be captured and inhaled into the pairing structure in the capture pairing operation, so as to avoid pollution and affect pairing caused by introducing two kinds of cells from the same inlet; secondly, the inlet and outlet sample ports are arranged in a diagonal line, so that the cells introduced into the structure can flow into each channel, avoiding the consequence of not capturing pairing caused by the cells flowing into only part of the channels under the influence of pressure. In the embodiment, the diameters of the sample inlet and the sample outlet are both 3 mm, which can be flexibly adjusted according to actual conditions during specific use.

[0039] A preparation method of a cell electrofusion chip device based on a double concave capture pairing structure array of double-sided sample introduction, comprising the following steps:

[0040] Step one, processing ITO electrode array, using wet etching process, specifically including:

[0041] S1, selecting ITO glass as the substrate for processing the chip;

[0042] S2, spin coating a layer of 5 μm SU-8 3005 photoresist on the ITO glass;

[0043] S3, etching the interdigital microarray structure on the ITO layer by means of photoetching and wet etching.

[0044] Step two, constructing PDMS double-sided flow field capture pairing structure: using PDMS polymer to construct double-sided flow field capture pairing structure and channel structure, specifically including:

[0045] (1) Using soft lithography process, processing the mold with a structure height of 30 μm, the mold structure is a cell suspension sample pool, a sample inlet, a sample outlet and a microchannel array structure;

[0046] (2) Fix the mold on an acrylic mold;

[0047] (3) Pour the mixed PDMS glue, and after static, vacuumize;

[0048] (4) Place in the oven at 65°C for curing;

[0049] (5) Remove the cured PDMS, according to the shape of the ITO interdigital electrode, trim and remove the sample inlet and outlet glue.

[0050] Step three, plasma cleaning, plasma cleaning time is 10-15 s;

[0051] Step four, bonding, place the PDMS double-sided flow field capture pairing structure PDMS chip upside down on the ITO electrode, use thermal bonding process, and form a closed cavity with the chip, only through the sample inlet and outlet to sample the cell suspension.

[0052] The operation steps when using are as follows:

[0053] Step I: First seal the sample inlet II8 and the sample outlet II12, add cell I suspension to the sample inlet I7, and apply negative pressure to the sample outlet I11, so that cell I enters the channel and is efficiently captured in the capture structure at a high flow rate.

[0054] Step II: Replace the cell I suspension with PM buffer to flush the channel for 1 min.

[0055] Step III: Apply greater pressure to the sample outlet I11, and in a very short time, cell I located in the capture structure is sucked into the pairing structure and stuck in the groove.

[0056] Step IV: Seal the sample inlet I7 and the sample outlet I11, open the sample inlet II8 and the sample outlet II12, and capture cell II in the capture structure and then suck it into the groove of the pairing structure. Due to the design of the structure and the high negative pressure for a short time, cell I will not be sucked out of the pairing chamber.

[0057] Step V: Use the ITO electrode to apply an alternating current signal first, so that the two cells (cell I and cell II) in the pairing chamber are clamped into the double-groove pairing structure due to the dielectrophoresis force and tightly contact, and the pairing is completed.

[0058] Step VI: Apply a direct current pulse signal and a fusion signal to fuse the heterologous cells.

[0059] The above-mentioned are only embodiments of the present application, and common technical solutions and / or common knowledge of the scheme are not described in detail. It should be pointed out that, for those skilled in the art, without departing from the technical solutions of the present application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the present application, and these will not affect the effect and practicality of the patent. The protection scope claimed in the present application should be subject to the content of its claims, and the specific implementation mode and the like recorded in the specification can be used to explain the content of the claims.

Claims

1. A cell electrofusion chip device based on a high-throughput biconcave capture pairing structure array with dual-sided sampling, characterized in that: The device includes an electrode layer and a PDMS dual-sided flow field capture structure layer arranged sequentially from bottom to top. The electrode layer is connected to an external cell electrofusion instrument. The PDMS dual-sided flow field capture structure layer includes several arrayed capture pairing structures and channels. Each capture pairing structure includes a pairing structure located in the middle and capture structures located on both sides of the pairing structure, with the two capture structures arranged symmetrically. The device also includes a PDMS fusion structure chip that cooperates with the channels. The PDMS fusion structure chip is provided with two inlet ports, two outlet ports, structural micropillars, microchannels, and a sample reservoir. The structural micropillars, microchannels, and sample reservoir are all located at the bottom of the PDMS fusion structure chip. The two inlet ports and two outlet ports are both vertically penetrating the PDMS fusion structure chip and located above the sample reservoir. The two inlet ports are located at both ends of the PDMS fusion chip, and the two outlet ports are also located at both ends of the PDMS fusion chip.

2. The cell electrofusion chip device based on a high-throughput biconcave capture pairing structure array with dual-sided sampling according to claim 1, characterized in that: The capture structure is formed by the channel sidewall and micropillars. One end of the capture structure is connected to the pairing structure, and the other end of the capture structure is provided with a concave arc segment.

3. The cell electrofusion chip device based on a high-throughput biconcave capture pairing structure array with dual-sided sampling according to claim 2, characterized in that: The radius of the arc segment is 12~16μm.

4. The cell electrofusion chip device based on a high-throughput biconcave capture pairing structure array with dual-sided sampling according to claim 3, characterized in that: The pairing structure includes a capture chamber and two concave micropillars disposed within the capture chamber. The two concave micropillars form two symmetrical capture sites, and an opening is provided in the middle of the capture site to enable communication and contact between the cells to be paired.

5. The cell electrofusion chip device based on a high-throughput biconcave capture pairing structure array with dual-sided sampling according to claim 4, characterized in that: The width of the opening is 8~14μm.

6. A cell electrofusion chip device based on a high-throughput biconcave capture pairing structure array with dual-sided sampling according to claim 5, characterized in that: The electrode layer is an ITO electrode layer, and an interdigitated electrode array is disposed on the ITO electrode layer. The distance between two adjacent comb teeth of the interdigitated electrode array is in the range of 60~80 μm, and the width of the interdigitated electrode is 150~200 μm.

7. A method for fabricating a cell electrofusion chip device based on a high-throughput biconcave capture pairing structure array with dual-sided sampling, as described in any one of claims 1-6, characterized in that, Includes the following steps: Step 1: The electrode layer is fabricated using a wet etching method; Step 2: Construct a PDMS double-sided flow field trapping structure layer, which is built using PDMS polymer through soft lithography and casting. Step 3: Plasma cleaning; Step 4: Bonding.

8. The method for fabricating a cell electrofusion chip device based on a high-throughput biconcave capture pairing structure array with dual-sided sampling according to claim 7, characterized in that: In step three, the plasma cleaning condition is a cleaning time of 10-15 seconds.

9. The method for fabricating a cell electrofusion chip device based on a high-throughput biconcave capture pairing structure array with dual-sided sampling according to claim 8, characterized in that: In step four, the bonding process is thermal bonding, and the bonding temperature is 100~150℃.

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