Micro-fluidic chip device for cell fusion and in-situ sorting

Abstract

The invention discloses a micro-fluidic chip device for cell fusion and in-situ sorting. The micro-fluidic chip device comprises a PDMS (Polydimethylsiloxane) micro-fluidic chip, a PCB (Printed Circuit Board) and a patterned gold electrode micro-array layer which are sequentially arranged from top to bottom, the PDMS micro-fluidic chip is provided with a sample inlet, a sample outlet and a collection cavity, and the sample inlet, the sample outlet and the collection cavity are communicated through the experiment cavity; a plurality of independent channels and a capturing cavity connected with the independent channels are arranged in the experiment cavity, and a double-cavity capturing structure is arranged at an inlet of the capturing cavity; and an independent microelectrode matched with the double-cavity capture structure is arranged on the patterned gold electrode microarray. The independent microelectrodes on the patterned gold electrode microarray layer are matched with the double-cavity capture structure, so that cell capture, cell electrofusion and cell in-situ release can be realized in the same chip, and cells in the double-cavity capture structure can be controlled more accurately.

Description

Technical Field

[0001] The invention relates to the technical field of cell electrofusion, and in particular to a microfluidic chip device for cell fusion and in-situ sorting. Background Art

[0002] Cell electrofusion is an emerging cell engineering technology developed in the 1980s. The principle is that when cells are exposed to a sufficiently high electric field, a potential difference will be generated on both sides of the cell membrane. When the potential difference reaches a certain threshold, the phospholipid molecules on the cell membrane will be disturbed to a certain extent. Under the action of the electric field force, the phospholipid molecules will move and rearrange, causing the cell membrane to be perforated at a certain position; when a sufficient number and size of holes appear in the contact area of ​​two cells close to each other, these holes continue to expand, causing the cell membrane in the cell contact area to rupture and disappear, thereby promoting cell membrane fusion. Cell electroporation is an important step in cell fusion, and this technology has been widely used in cell transfection, that is, loading DNA, RNA, protein, drugs, etc. into cells. Compared with other fusion methods, electrofusion has many advantages: First, compared with biological methods (virus methods) and chemical methods (PEG, etc.), electrofusion causes less damage to cells; second, it is highly controllable. Because the electrofusion method is controlled by an electric field, cells can be paired in a certain direction according to the electric field design and fuse. Currently, there are a variety of commercial cell electrofusion instruments, and cell electrofusion is widely used in cell electrofusion.

[0003] At present, after cell fusion, selective culture medium and subcloning operations are still often used to separate target cells, which is time-consuming and laborious. Single-cell release technology based on microfluidic chips has been widely used, but there is no chip that can simultaneously achieve cell fusion and cell release. Therefore, the separation of fused cells is still a big challenge, which makes it difficult to further apply fused cells. Therefore, it is necessary to provide a chip device that can achieve efficient cell electrical fusion and cell release through innovations in structure, microelectrodes and methods. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a microfluidic chip device for cell fusion and in-situ sorting.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] Provided is a microfluidic chip device for cell fusion and in-situ sorting, comprising a PDMS microfluidic chip, a PCB circuit board and a patterned gold electrode microarray layer arranged in sequence from top to bottom; the PDMS microfluidic chip is provided with an inlet, an outlet and a collection cavity, and the inlet, outlet and collection cavity are all connected through an experimental cavity; a plurality of independent channels and capture cavities connected to the independent channels are provided in the experimental cavity, the independent channels are connected to the capture cavity to form an S-shaped structure, and a double-cavity capture structure is provided at the entrance of the capture cavity; an independent microelectrode matching the double-cavity capture structure is provided on the patterned gold electrode microarray, and the double-cavity capture structure cooperates with the independent microelectrode to realize cell capture, cell electrical fusion and cell in-situ release.

[0007] Furthermore, one end of several independent microelectrodes is connected to form a common electrode, which is connected to a host computer, and the other end forms a capture electrode matching several capture chambers, which is connected to a host computer; one end of the capture electrode spans the entire capture chamber, and the other end is located at the lower right side of the capture chamber.

[0008] Furthermore, the dual-cavity capture structure includes a first microcolumn, a second microcolumn and a mountain-shaped capture seat. The first microcolumn and the second microcolumn are arranged in a straight line at the entrance of the capture cavity, and a gap is left between the second microcolumn and the first microcolumn and the capture cavity. The mountain-shaped capture seat is arranged between the first microcolumn and the second microcolumn, and capture ports are formed between the first microcolumn and the mountain-shaped capture seat, and between the second microcolumn and the mountain-shaped capture seat.

[0009] Furthermore, the first micro-column, the second micro-column and the U-shaped capture seat are all provided with rounded corners at the capture opening.

[0010] Furthermore, the first microcolumn and the second microcolumn are both provided with arc surfaces on the sides facing each other; and the capture side of the mountain-shaped capture seat is provided with a pointed tip.

[0011] Furthermore, the width of the independent channel is 30 μm, and the width of the capture port is 4 μm.

[0012] Furthermore, the thickness of the PDMS microfluidic chip is 23 μm, and the thickness of the independent microelectrode is 100 nm.

[0013] Furthermore, the inner corners of the capture cavity are all provided with chamfers.

[0014] Furthermore, the collection chamber comprises a first collection chamber and a second collection chamber, the first collection chamber and the second collection chamber are respectively arranged on both sides of the sample outlet, and the first collection chamber and the second collection chamber are both provided with collection ports.

[0015] Further, the inlet section of the independent channel is parallel to the outlet section of the capture chamber.

[0016] The beneficial effects of the present invention are:

[0017] The present invention includes a PDMS microfluidic chip and a microelectrode array with a specific arrangement and structure matched therewith. The PDMS chip contains 25 independent microchannels and capture cavities, and the double-cavity capture structure is at the entrance of the capture cavity. The microelectrode array matches the capture cavity structure of the PDMS chip, one end is a common end, and the other end is composed of 25 pairs of independent electrodes, which can realize independent and precise control of cells in 25 capture cavities. After cell electrofusion, the cell release is realized by buffer replacement and negative dielectrophoresis force; the present invention can realize cell electrofusion and cell in situ release in the same chip.

[0018] The PDMS microfluidic chip of the present invention is assembled after being processed with a gold electrode microarray layer, and high-efficiency cell capture is achieved through a double-cavity capture structure in the chip through fluid dynamics; an electric signal is applied to complete electrical fusion of the cells; a buffer is replaced, and an electric signal is applied to release the target cells in situ after cell fusion, and accurate and independent control of the target cells is achieved through the independent design of a PCB circuit board and electrodes.

[0019] The gold electrode microarray layer of the present invention has a unique electrode layout and is matched one-to-one with the capture cavity. By optimizing the electrode structure and electrode arrangement, cell fusion and release functions can be simultaneously realized; independent channels and capture cavities are arranged in the PDMS microfluidic chip, and the independent channels are connected with the capture cavity to form an S structure; in-situ integrated operations of cell pairing, fusion and precise release are simultaneously realized on the same chip. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the structure of a microfluidic chip device for cell fusion and in-situ sorting according to the present invention;

[0021] Figure 2 A top view of the PDMS microfluidic chip layer of the present invention;

[0022] Figure 3 A top view of the gold electrode microarray layer of the present invention;

[0023] Figure 4 It is a schematic diagram of the structure of the channel and the double-cavity capture structure in the present invention;

[0024] Figure 5 It is a schematic diagram of the structure inside the capture chamber of the present invention;

[0025] Figure 6 It is a schematic diagram of the workflow of the present invention;

[0026] Figure 7 Comparison diagram of electric field intensity gradient distribution under different electrode arrangement modes;

[0027] The main components in the figure are described as follows:

[0028] 1. Gold electrode microarray layer; 2. PDMS microfluidic chip; 3. PCB circuit board; 4. Capture chamber; 5. Sample inlet; 6. Sample outlet; 7. First collection chamber; 8. Second collection chamber; 9. Independent channel; 10. First microcolumn; 11. Second microcolumn; 12. Mountain-shaped capture seat; 13. Capture port. DETAILED DESCRIPTION

[0029] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0030] like Figure 1 As shown, the microfluidic chip device for cell fusion and in-situ sorting includes a PDMS microfluidic chip 2, a PCB circuit board 3 and a patterned gold electrode microarray layer 1 which are arranged in sequence from top to bottom.

[0031] like Figure 2 As shown, the PDMS microfluidic chip 2 is provided with an inlet 5, an outlet 6 and a collection chamber, and the inlet 5, the outlet 6 and the collection chamber are all connected through the experimental chamber. A plurality of independent channels 9 and a capture chamber 4 connected to the independent channels 9 are provided in the experimental chamber, and the independent channels 9 are connected to the capture chamber 4 to form an S-shaped structure, the inlet section of the independent channel 9 is parallel to the outlet section of the capture chamber 4, and a double-cavity capture structure is provided at the inlet of the capture chamber 4. In this embodiment, the independent channels 9 and the capture chamber 4 are preferably provided at 25 locations.

[0032] like Figure 3 As shown, the patterned gold electrode microarray 1 is provided with an independent microelectrode matching the dual-cavity capture structure, and the dual-cavity capture structure cooperates with the independent microelectrode to realize cell capture, cell electrofusion and cell in situ release. One end of several independent microelectrodes is connected to form a common electrode, and the other end forms a capture electrode matching several capture cavities 4, and the common electrode is connected to a host computer. One end of the capture electrode spans the entire capture cavity 4, and the other end is located at the lower right side of the capture cavity 4. The gold electrode pattern is an optimized pattern, so as to meet the electric field and electric field intensity gradient requirements for cell fusion and cell release. In this embodiment, 25 independent microelectrodes are provided, and each independent microelectrode matches the capture cavity 4. The host computer provides an electrical signal to the patterned gold electrode microarray layer 2 to realize independent control of the 25 independent microelectrodes.

[0033] like Figure 4 and 5As shown, the dual-cavity capture structure includes a first microcolumn 10, a second microcolumn 11 and a mountain-shaped capture seat 12. The first microcolumn 10 and the second microcolumn 11 are both in a straight line shape, and the mountain-shaped capture seat 12 has a "mountain"-shaped outer contour. The first microcolumn 10 and the second microcolumn 11 are arranged in a straight line at the entrance of the capture chamber 4. There is a gap between the second microcolumn 11 and the first microcolumn 10 and the capture chamber 4. The mountain-shaped capture seat 12 is arranged between the first microcolumn 10 and the second microcolumn 11. A capture port 13 is formed between the first microcolumn 10 and the mountain-shaped capture seat 12, and between the second microcolumn 11 and the mountain-shaped capture seat 12. The first microcolumn 10, the second microcolumn 11 and the mountain-shaped capture seat 12 are all provided with rounded corners at the capture port 13. The first microcolumn 10 and the second microcolumn 11 are both provided with arc surfaces on the opposite sides. The rounded corners and the arc surfaces reduce the electric field intensity gradient of the capture chamber, which can ensure the smooth progress of the cell release process. The first microcolumn 10 and the second microcolumn 11 are changed to arc shapes, which can better optimize the electric field and meet the cell electric sorting operation. The capture side of the mountain-shaped capture seat 12 is provided with a tip, which can separate and limit the two cells to be fused.

[0034] In this embodiment, the width of the independent channel 9 is preferably 30 μm; the width of the capture port 13 is 4 μm to prevent cells from entering the opposite side.

[0035] In this embodiment, the thickness of the PDMS microfluidic chip 2 is 23 μm, and the thickness of the independent microelectrode is 100 nm. The patterned gold electrode microarray 1 is prepared by soft lithography on quartz glass with 100 nm gold and 5 nm chromium metal layers on the surface, and then by wet etching process to prepare independent microelectrode patterns.

[0036] In this embodiment, the inner corners of the capture chamber 4 are all provided with chamfers, which can optimize the electric field required for cell release.

[0037] In this embodiment, the collection chamber includes a first collection chamber 7 and a second collection chamber 8. The first collection chamber 7 and the second collection chamber 8 are respectively arranged on both sides of the sample outlet 6. The first collection chamber 7 and the second collection chamber 8 are both provided with collection ports. The first collection chamber 7 and the second collection chamber 8 can collect different types of target cells according to experimental requirements.

[0038] In the present invention, the electric field strength gradient is crucial to the cell release function. Cells will move towards the place with weak electric field strength gradient under the culture medium. When the electric field strength gradient of the capture chamber is strong, the cells will move away from the capture chamber, thus realizing the cell release function. In the electrode, the most important thing is the configuration of the electrode and the structure. The shape of the electrode and the position of the relative structure have a great influence on the electric field distribution. The electric field generated by the configuration proposed in the present invention needs to satisfy both the electric field of cell electrofusion and the electric field of cell electrorelease, so the designed electrode structure is optimized according to the structure.

[0039] like Figure 7 The independent microelectrode in the present invention has a long electrode on the top and a short electrode on the bottom right. Compared with other arrangement modes (long electrodes on the top and bottom; short electrodes on the top and bottom; short electrodes on the top and long electrodes on the bottom), only the electrode arrangement in this mode can achieve better cell release operation ( Figure 7 Comparison between B and C in Figure 1).

[0040] The method of the microfluidic chip device for cell fusion and in situ sorting specifically comprises the following steps: Figure 6 As shown,

[0041] S1, preparing chip gold electrode microarray layer 1 and PMDS microfluidic chip layer 2;

[0042] S2, pre-treating the PDMS microfluidic chip 2: placing the PDMS microfluidic chip 2 in plasma for 15 seconds, and then aligning the PDMS microfluidic chip 2 and the patterned gold electrode microarray 1 under a microscope;

[0043] S3, adding a cell suspension at a flow rate of 0.5 μL / min through the injection port 5, wherein the cell suspension is RPMI 1640 cell culture medium; after 5 minutes, the cells complete pairing in the capture chamber 4, and the injection is stopped;

[0044] S4, adding a low osmotic pressure cell fusion buffer with an osmotic pressure of 110 mOsm / kg through the injection port 5 at a flow rate of 0.5 μL / min, and stopping after 10 minutes. At this time, the cells absorb water and swell to achieve tight squeezing;

[0045] S5, applying an electric pulse signal to the capture chamber 4 through the host computer and the independent microelectrode to complete the electric fusion of the cells; the field strength of the electric pulse signal is 1.5 kV / cm, the pulse width is 10 μs, the number of pulses is 5, and the pulse interval time is 1 s;

[0046] S6, adding RPMI 1640 cell culture medium through injection port 5 at a flow rate of 0.5 μL / min, stopping after 10 minutes, and adjusting the flow rate to 0.2 μL / min after the cells show obvious dehydration and shrinkage, in preparation for cell release;

[0047] S7. A sinusoidal signal of 0.5 kV / cm and a frequency of 1 MHz is continuously applied to the target fusion cells through the host computer and the independent microelectrode. The in situ target cells are released. The sinusoidal signal is applied for 10 seconds and then stopped. The collection port on the collection chamber 7 is opened and the target cells are collected at the collection port of the collection chamber 7.

Claims

1. A microfluidic chip device for cell fusion and in situ sorting, characterized in that: It comprises a PDMS microfluidic chip (2), a PCB circuit board (3) and a patterned gold electrode microarray layer (1) which are arranged in sequence from top to bottom; The PDMS microfluidic chip (2) is provided with a sample inlet (5), a sample outlet (6) and a collection chamber, and the sample inlet (5), the sample outlet (6) and the collection chamber are all connected through the experimental chamber; The experimental chamber is provided with a plurality of independent channels (9) and a capture chamber (4) connected to the independent channels (9); the independent channels (9) and the capture chamber (4) are connected to form an S-shaped structure; a double-chamber capture structure is provided at the entrance of the capture chamber (4); The patterned gold electrode microarray (1) is provided with independent microelectrodes matching the dual-cavity capture structure, and the dual-cavity capture structure cooperates with the independent microelectrode to achieve cell capture, cell electrical fusion and cell in situ release.

2. The microfluidic chip device for cell fusion and in situ sorting according to claim 1, characterized in that: One end of the plurality of independent microelectrodes is connected to form a common electrode, and the other end forms a capture electrode matching the plurality of capture chambers (4), wherein the common electrode is connected to a host computer; one end of the capture electrode spans the entire capture chamber (4), and the other end is located at the lower right side of the capture chamber (4).

3. The microfluidic chip device for cell fusion and in situ sorting according to claim 1, characterized in that: The double-chamber capture structure comprises a first microcolumn (10), a second microcolumn (11) and a mountain-shaped capture seat (12); the first microcolumn (10) and the second microcolumn (11) are arranged in a straight line at the entrance of the capture chamber (4) facing each other; a gap is left between the second microcolumn (11) and the first microcolumn (10) and the capture chamber (4); the mountain-shaped capture seat (12) is arranged between the first microcolumn (10) and the second microcolumn (11); and a capture port (13) is formed between the first microcolumn (10) and the mountain-shaped capture seat (12), and between the second microcolumn (11) and the mountain-shaped capture seat (12).

4. The microfluidic chip device for cell fusion and in situ sorting according to claim 3, characterized in that: The first micro-column (10), the second micro-column (11) and the mountain-shaped capture seat (12) are all provided with rounded corners at the capture opening (13).

5. The microfluidic chip device for cell fusion and in situ sorting according to claim 3, characterized in that: The first microcolumn (10) and the second microcolumn (11) are both provided with arc surfaces on the sides facing each other; and the capture side of the mountain-shaped capture seat (12) is provided with a pointed tip.

6. The microfluidic chip device for cell fusion and in situ sorting according to claim 3, characterized in that: The width of the independent channel (9) is 30 μm, and the width of the capture port (13) is 4 μm.

7. The microfluidic chip device for cell fusion and in situ sorting according to claim 1, characterized in that: The thickness of the PDMS microfluidic chip (2) is 23 μm, and the thickness of the independent microelectrode is 100 nm.

8. The microfluidic chip device for cell fusion and in situ sorting according to claim 1, characterized in that: The inner corners of the capture chamber (4) are all provided with chamfers.

9. The microfluidic chip device for cell fusion and in situ sorting according to claim 1, characterized in that: The collection chamber comprises a first collection chamber (7) and a second collection chamber (8); the first collection chamber (7) and the second collection chamber (8) are respectively arranged on both sides of the sample outlet (6); and the first collection chamber (7) and the second collection chamber (8) are both provided with collection ports.

10. The microfluidic chip device for cell fusion and in situ sorting according to claim 1, characterized in that: The inlet section of the independent channel (9) is parallel to the outlet section of the capture chamber (4).

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