Cell three-dimensional rotation and electroporation micro-fluidic chip based on alternating current electric field regulation and control

Abstract

The invention relates to a cell three-dimensional rotation and electroporation micro-fluidic chip based on alternating current electric field regulation and control, which comprises a first PDMS layer, a second PDMS layer and a third PDMS layer which are sequentially stacked, a first PDMS channel and a second PDMS channel are arranged in the first PDMS layer, a third PDMS channel is arranged in the second PDMS layer, and the third PDMS channel is arranged in the third PDMS layer. A fourth PDMS channel, a fifth PDMS channel, a fluid inlet and a fluid outlet are formed in the third PDMS layer. According to the embodiment of the invention, the electric field is applied to the four exciting electrodes to form the rotating electric field, the negative dielectrophoresis force generated by the rotating electric field generates the rotating torque on the cells, the cells are controlled to rotate three-dimensionally, and the cells are driven to rotate three-dimensionally in different directions and at different speeds by changing the parameters of the applied electric field, so that the three-dimensional rotation of the cells is realized. Therefore, many genetic information and structural details of observed cells and particles can be observed, the applied voltage is small, additional modification on the cells is not needed, and the control is accurate.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell analysis, and particularly relates to a microfluidic chip for three-dimensional rotation and electroporation of cells based on alternating current (AC) electric field regulation. Background Art

[0002] The manipulation of particles, cells, and multicellular organisms is a very important part of the field of cell analysis technology. Among them, precise rotational manipulation can perform cell observation and analysis more accurately and comprehensively, and has important impacts in the fields of single-cell analysis, drug discovery, and biological research.

[0003] If the selection manipulation can only be achieved on a plane, then the analysis process will lack an observation dimension, and a lot of genetic information and structural details of the observed cells and particles cannot be observed. Therefore, the three-dimensional rotational manipulation of particles, cells, and multicellular organisms has very important significance in the field of cell analysis, and can also be applied to the selective electroporation of cells. However, the existing three-dimensional rotational manipulation technologies have problems such as complex operation, the need for an external robotic manipulation system, high cost, and limited throughput. Summary of the Invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a microfluidic chip for three-dimensional rotation and electroporation of cells based on AC electric field regulation. The technical problems to be solved by the present invention are realized through the following technical solutions:

[0005] An embodiment of the present invention provides a microfluidic chip for three-dimensional rotation and electroporation of cells based on AC electric field regulation, including: a first polydimethylsiloxane (PDMS) layer, a second PDMS layer, and a third PDMS layer stacked in sequence, wherein,

[0006] a first PDMS channel and a second PDMS channel are provided in the first PDMS layer, a third PDMS channel is opened in the second PDMS layer, and a fourth PDMS channel, a fifth PDMS channel, a fluid inlet, and a fluid outlet are provided in the third PDMS layer;

[0007] the first PDMS channel and the second PDMS channel are arranged opposite to each other, and both the first PDMS channel and the second PDMS channel have side edges parallel to the third PDMS channel and located below the third PDMS channel; the first PDMS channel and the second PDMS channel are both filled with liquid metal; one end of the first PDMS channel forms a first excitation electrode, and the other end is used for connecting an electrode lead; one end of the second PDMS channel forms a second excitation electrode, and the other end is used for connecting an electrode lead;

[0008] One end of the third PDMS channel forms a channel inlet for injecting a cell-containing solution, and the other end forms a channel outlet for discharging the cell-containing solution. The channel inlet is in communication with the fluid inlet, and the channel outlet is in communication with the fluid outlet.

[0009] The fourth PDMS channel and the fifth PDMS channel are arranged opposite to each other. Both the fourth PDMS channel and the fifth PDMS channel have a side parallel to the third PDMS channel and located below the third PDMS channel. The fourth PDMS channel and the fifth PDMS channel are both filled with liquid metal. One end of the fourth PDMS channel forms a third excitation electrode, and the other end is used to connect to an electrode lead. One end of the fifth PDMS channel forms a fourth excitation electrode, and the other end is used to connect to an electrode lead.

[0010] The inner ends of the first PDMS channel, the second PDMS channel, the fourth PDMS channel, and the fifth PDMS channel are all located within the third PDMS channel.

[0011] In one embodiment of the present invention, the first PDMS channel, the second PDMS channel, the fourth PDMS channel, and the fifth PDMS channel each include a first vertical segment, a parallel segment, and a second vertical segment, where the parallel segment is connected between the first vertical segment and the second vertical segment.

[0012] In one embodiment of the present invention, the electric field phases applied to the first excitation electrode, the second excitation electrode, the third excitation electrode, and the fourth excitation electrode increase by 90° in sequence.

[0013] In one embodiment of the present invention, the electric field phases applied to the first excitation electrode, the second excitation electrode, the third excitation electrode, and the fourth excitation electrode are 0°, 90°, 180°, and 270° in sequence.

[0014] In one embodiment of the present invention, when an electric field is applied, the time-averaged dielectrophoretic force acting on the cell is:

[0015]

[0016] The time-averaged electro-rotation torque is:

[0017]

[0018] Where K(ω) is the CM factor, Im[K(ω)] is the imaginary part of the CM factor, Re[K(ω)] is the real part of the CM factor, r is the cell radius, E is the electric field strength, * is the conjugate complex number, and ~ is the complex amplitude. is a gradient operator;

[0019]

[0020] wherein, is the complex dielectric constant of the cell, is the complex dielectric constant of the solution;

[0021] ε * = ε - j(σ / ω)

[0022] wherein, ε is the dielectric constant, σ is the conductivity, ω is the angular frequency, and j is the imaginary unit.

[0023] In one embodiment of the present invention, the liquid metal includes bismuth-based liquid metal.

[0024] In one embodiment of the present invention, metal connectors are provided at both the fluid inlet and the fluid outlet.

[0025] In one embodiment of the present invention, the first PDMS channel has a thickness of 50 μm and a width of 150 μm;

[0026] The second PDMS channel has a thickness of 50 μm and a width of 150 μm;

[0027] The third PDMS channel has a thickness of 75 μm and a width of 75 μm;

[0028] The fourth PDMS channel has a thickness of 50 μm and a width of 150 μm;

[0029] The fifth PDMS channel has a thickness of 50 μm and a width of 150 μm.

[0030] Another embodiment of the present invention provides a preparation method of a microfluidic chip for three-dimensional cell rotation and electroporation regulated by an alternating electric field, for preparing the microfluidic chip for three-dimensional cell rotation and electroporation regulated by an alternating electric field as described in the above embodiment, including steps: processing of the first PDMS layer and the third PDMS layer; processing of the PDMS stamp; processing of the second PDMS layer; bonding of the chip, wherein,

[0031] The processing of the first PDMS layer and the third PDMS layer includes steps:

[0032] S1. Cover a photoresist on a glass substrate, and perform exposure and development using a channel mask to form channel molds of the first PDMS layer and the third PDMS layer;

[0033] S2. Pour the prepared PDMS solution onto the channel mold after silane treatment and cure it to form a PDMS channel, and drill holes in the cured PDMS to form ports of the PDMS channel;

[0034] S3. Bond the cured PDMS to the PC film so that the PDMS channel faces the PC film, and inject liquid metal from the port of the PDMS channel to form a metal electrode;

[0035] S4. Separate the PDMS from the PC film to form the first PDMS layer or the third PDMS layer;

[0036] The processing of the PDMS stamp includes the steps:

[0037] S1. Pour the prepared PDMS solution onto the glass substrate after silane treatment and cure it to form a PDMS stamp, and modify the PDMS stamp to obtain a modified PDMS stamp;

[0038] The processing of the second PDMS layer includes the steps:

[0039] S1. Cover a photoresist on the glass substrate and use a channel mask for exposure and development to form a mold with a channel pattern;

[0040] S2. Pour the prepared PDMS solution into the mold with the channel pattern and press the modified PDMS stamp on the channel pattern to form a second PDMS layer with the PDMS stamp;

[0041] The bonding of the chip includes the steps:

[0042] S1. Align and bond the side with liquid metal on the third PDMS layer to the side with the third PDMS channel on the second PDMS layer, and then remove the PDMS stamp;

[0043] S2. Drill holes at both ends of the third PDMS channel on the second PDMS layer so that the holes penetrate the second PDMS layer and the third PDMS layer, form a channel inlet and a channel outlet on the second PDMS layer, and form a fluid inlet and a fluid outlet on the third PDMS layer;

[0044] S3. Align and bond the side with liquid metal on the first PDMS layer to the second PDMS layer.

[0045] Compared with the prior art, the beneficial effects of the present invention:

[0046] The three-dimensional rotation and electroporation microfluidic chip based on alternating current electric field regulation of the present invention applies an electric field on four excitation electrodes to form a rotating electric field. The negative dielectrophoretic force generated by the rotating electric field generates a rotating torque on the cells, manipulates the cells to perform three-dimensional rotation, and drives the cells to complete three-dimensional rotation in different directions and at different speeds by changing the parameters of the applied electric field. Thus, a lot of genetic information and structural details of the observed cells and particles can be observed. The applied voltage is small, there is no need for external modification of the cells, and the manipulation is precise. The rotation manipulation function is combined with the electroporation technology, which can rotate the cells to any side facing the electric field direction to achieve selective electroporation. The chip designed by the present invention makes the three-dimensional rotation manipulation technology have the advantages of simple operation, no need for an external robot manipulation system, low cost, and high throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 FIG. is a schematic structural diagram of a three-dimensional rotation and electroporation microfluidic chip based on alternating current electric field regulation provided by an embodiment of the present invention;

[0048] Figure 2 FIG. is a schematic structural diagram of a first PDMS layer, a first excitation electrode, and a second excitation electrode provided by an embodiment of the present invention;

[0049] Figure 3 FIG. is a schematic structural diagram of a second PDMS layer provided by an embodiment of the present invention;

[0050] Figure 4 FIG. is a schematic structural diagram of a third PDMS layer, a third excitation electrode, and a fourth excitation electrode provided by an embodiment of the present invention;

[0051] Figure 5 FIG. is a schematic diagram of the principle of three-dimensional rotation of cells provided by an embodiment of the present invention;

[0052] Figure 6 FIG. is a diagram of the experimental results of three-dimensional rotation manipulation of a yeast provided by an embodiment of the present invention;

[0053] Figure 7 FIG. is a diagram of the experimental results of electroporation of a yeast provided by an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0054] The following further describes the present invention in detail with reference to specific embodiments, but the embodiments of the present invention are not limited thereto.

[0055] Embodiment 1

[0056] Please refer to FIGS. 1, Figure 2 , Figure 3 , Figure 4 and Figure 5 , Figure 1Schematic diagram of the structure of a three-dimensional cell rotation and electroporation microfluidic chip based on AC electric field regulation provided by an embodiment of the present invention. Figure 2 Schematic diagram of the structure of the first PDMS layer, the first excitation electrode, and the second excitation electrode provided by an embodiment of the present invention. Figure 3 Schematic diagram of the structure of the second PDMS layer provided by an embodiment of the present invention. Figure 4 Schematic diagram of the structure of the third PDMS layer, the third excitation electrode, and the fourth excitation electrode provided by an embodiment of the present invention. Figure 5 Schematic diagram of the principle of three-dimensional cell rotation provided by an embodiment of the present invention.

[0057] The three-dimensional cell rotation and electroporation microfluidic chip based on AC electric field regulation in this embodiment includes: a first PDMS layer 1, a second PDMS layer 2, and a third PDMS layer 3 stacked in sequence. It can be understood that the second PDMS layer 2 covers the first PDMS layer 1, and the third PDMS layer 3 covers the second PDMS layer 2, that is, the first PDMS layer 1 is the bottom layer, the second PDMS layer 2 is the middle layer, and the third PDMS layer 3 is the top layer.

[0058] Specifically, a first PDMS channel 4 and a second PDMS channel 5 are provided in the first PDMS layer 1. The first PDMS channel 4 and the second PDMS channel 5 are arranged opposite to each other. Both the first PDMS channel 4 and the second PDMS channel 5 are filled with liquid metal; one end of the first PDMS channel 4 serves as the channel inlet, where liquid metal is injected to form the first excitation electrode 401, and the other end serves as the outlet for connecting the electrode lead; one end of the second PDMS channel 5 serves as the channel inlet, where liquid metal is injected to form the second excitation electrode 501, and the other end serves as the outlet for connecting the electrode lead.

[0059] Specifically, a third PDMS channel 6 is opened in the second PDMS layer 2. One end of the third PDMS channel 6 forms a channel inlet 601, and the other end forms a channel outlet 602. Among them, the channel inlet 601 is used to inject a solution containing cells, and the channel outlet 602 is used to discharge the solution containing cells injected through the channel inlet 601.

[0060] Specifically, a fourth PDMS channel 7, a fifth PDMS channel 8, a fluid inlet 9, and a fluid outlet 10 are provided on the third PDMS layer 3. The fourth PDMS channel 7 and the fifth PDMS channel 8 are oppositely arranged. Both the fourth PDMS channel 7 and the fifth PDMS channel 8 are filled with liquid metal; one end of the fourth PDMS channel 7 serves as a channel inlet, where liquid metal is injected to form a third excitation electrode 501, and the other end serves as a channel outlet for connecting an electrode lead; one end of the fifth PDMS channel 8 serves as a channel inlet, where liquid metal is injected to form a fourth excitation electrode 801, and the other end serves as a channel outlet for connecting an electrode lead. Further, the channel inlet 601 and the channel outlet 602 of the third PDMS channel 6 extend to the third PDMS layer 3 and are connected to the fluid inlet 9 and the fluid outlet 10, so as to connect the fluid inlet 9 with the channel inlet 601 for injecting a solution containing cells; the fluid outlet 10 is connected to the channel outlet 602 for discharging the injected solution containing cells.

[0061] Further, both the first PDMS channel 4 and the second PDMS channel 5 have parallel segments parallel to the third PDMS channel 6 and located below the third PDMS channel 6. As Figure 5 shown, the parallel segment in the first PDMS channel 4 close to the side of the second PDMS channel 5 is the inner end portion, and the inner end portion of the first PDMS channel 4 is located within the third PDMS channel 6. The parallel segment in the second PDMS channel 5 close to the side of the first PDMS channel 4 is the inner end portion, and the inner end portion of the second PDMS channel 5 is located within the third PDMS channel 6.

[0062] Further, both the fourth PDMS channel 7 and the fifth PDMS channel 8 have parallel segments parallel to the third PDMS channel 6 and located below the third PDMS channel 6. The parallel segment in the fourth PDMS channel 7 close to the side of the fifth PDMS channel 8 is the inner end portion, and the inner end portion of the fourth PDMS channel 7 is located within the third PDMS channel 6. The parallel segment in the fifth PDMS channel 8 close to the side of the fourth PDMS channel 7 is the inner end portion, and the inner end portion of the fifth PDMS channel 8 is located within the third PDMS channel 6.

[0063] In a specific embodiment, the first PDMS channel 4, the second PDMS channel 5, the fourth PDMS channel 7, and the fifth PDMS channel 8 all include a first vertical segment 110, a parallel segment 120, and a second vertical segment 130, wherein the parallel segment 120 is connected between the first vertical segment 110 and the second vertical segment 130. Further, the inner end portions of the parallel segments 120 in the first PDMS channel 4, the second PDMS channel 5, the fourth PDMS channel 7, and the fifth PDMS channel 8 are located within the third PDMS channel 6.

[0064] In a specific embodiment, the thickness of the first PDMS channel 4 is 50 μm, and the width is 150 μm; the thickness of the second PDMS channel 5 is 50 μm, and the width is 150 μm; the thickness of the third PDMS channel 6 is 50 μm, and the width is 150 μm; the thickness of the fourth PDMS channel 7 is 75 μm, and the width is 75 μm; the thickness of the fifth PDMS channel 8 is 75 μm, and the width is 75 μm.

[0065] It can be understood that the first PDMS channel 4, the second PDMS channel 5, the fourth PDMS channel 7, and the fifth PDMS channel 8 are all recessed areas formed in the PDMS layer, which do not penetrate the PDMS layer, and the side filled with liquid metal faces the second PDMS layer 2, while the third PDMS channel 6 is a channel that penetrates the second PDMS layer 2, so that the first PDMS layer 1, the second PDMS layer 2, and the third PDMS layer 3 form a sealed structure.

[0066] In a specific embodiment, metal connectors are provided at both the fluid inlet 9 and the fluid outlet 10.

[0067] Specifically, the fluid inlet 9 is connected to the channel inlet 601 and is provided with a metal connector for connecting to an injection cell solution injection pump to inject a solution containing cells; the fluid outlet 10 is connected to the channel outlet 602 and is provided with a metal connector for discharging the injected solution containing cells.

[0068] In a specific embodiment, the liquid metal filled in the first PDMS channel 4, the second PDMS channel 5, the fourth PDMS channel 7, and the fifth PDMS channel 8 includes bismuth-based liquid metal. Further, the first excitation electrode 401, the second excitation electrode 501, the third excitation electrode 701, and the fourth excitation electrode 801 are all bismuth-based liquid metal electrodes.

[0069] In a specific embodiment, the electric field phases applied to the first excitation electrode 401, the second excitation electrode 501, the third excitation electrode 501, and the fourth excitation electrode 801 increase by 90° in sequence to form a rotating electric field for manipulating the cells to perform three-dimensional rotation.

[0070] In this embodiment, the phases of the electric fields applied to the first excitation electrode 401, the second excitation electrode 501, the third excitation electrode 701, and the fourth excitation electrode 801 are 0°, 90°, 180°, and 270° respectively.

[0071] When an electric field is applied, the time-averaged dielectrophoretic force acting on the cells is:

[0072]

[0073] The time-averaged electro-rotation torque is:

[0074]

[0075] Among them, K(ω) is the CM factor, Im[K(ω)] is the imaginary part of the CM factor, Re[K(ω)] is the real part of the CM factor, r is the cell radius, E is the electric field strength, * is the conjugate complex number, and ~ is the complex amplitude. is the gradient operator;

[0076]

[0077] Among them, is the complex dielectric constant of the cell, is the complex dielectric constant of the solution;

[0078] ε * = ε - j(σ / ω) (4)

[0079] Among them, ε is the dielectric constant, σ is the conductivity, ω is the angular frequency, and j is the imaginary unit.

[0080] It can be seen from formula (1) that the dielectrophoretic force mainly depends on the non-uniformity of the electric field and the CM (Clausius-Mossotti) factor. That is, by changing the CM factor, the time-averaged dielectrophoretic force acting on the cell can be changed, thereby changing the rotation direction of the cell. When Re[K(ω)] is negative, the cell is subjected to a negative dielectrophoretic force, causing the cell to move away from the strong electric field region. When Im[K(ω)] is positive, the cell moves along the direction in which the electric field phase increases; when Im[K(ω)] is negative, the cell moves along the direction in which the electric field phase decreases.

[0081] It can be seen from formula (2) that the direction of electro-rotation is determined by Im[K(ω)]. When the electro-rotation torque is positive, the cell moves along the same direction as the electric field propagation direction. When the electro-rotation torque is negative, the cell moves along the direction opposite to the electric field propagation direction.

[0082] It should be noted that the CM factor can be changed by changing the frequency of the electric field applied to the first excitation electrode 401, the second excitation electrode 501, the third excitation electrode 701, and the fourth excitation electrode 801, or the conductivity of the conductive solution contained in the third PDMS channel 6, so as to manipulate the cell to perform three-dimensional rotation with different rotation directions and speeds.

[0083] The three-dimensional rotation and electroporation microfluidic chip based on AC electric field regulation in this embodiment applies electric fields on four excitation electrodes to form a rotating electric field. The negative dielectrophoretic force generated by the rotating electric field generates a rotating torque on the cells, controlling the cells to perform three-dimensional rotation. By changing the parameters of the applied electric field, the cells are driven to complete three-dimensional rotation in different directions and at different speeds, so that a lot of genetic information and structural details of the observed cells and particles can be observed. The applied voltage is small, there is no need for external modification of the cells, and the manipulation is precise. Combining this rotation control function with the electroporation technology, the cells can be rotated to any side facing the electric field direction to achieve selective electroporation. The chip designed in the present invention makes the three-dimensional rotation control technology have the advantages of simple operation, no need for an external robot control system, low cost, and high throughput.

[0084] Embodiment 2

[0085] Based on Embodiment 1, this embodiment provides a preparation method for a three-dimensional rotation and electroporation microfluidic chip based on AC electric field regulation, which is used to prepare the chip as described in Embodiment 1. The preparation method includes the following steps: Step 1, processing of the first PDMS layer 1 and the third PDMS layer 3; Step 2, processing of the PDMS stamp; Step 3, processing of the second PDMS layer 2; Step 4, bonding of the chip.

[0086] Step 1, processing of the liquid metal electrode layer, i.e., the first PDMS layer 1 and the third PDMS layer 3.

[0087] Taking the depth (thickness) of the channels on the first PDMS layer 1 and the third PDMS layer 3 as 50 μm as an example, the processing of the first PDMS layer 1 and the third PDMS layer 3 specifically includes the following steps:

[0088] S1. Cover the photoresist on the glass substrate, and use the channel mask for exposure and development to form the channel molds of the first PDMS layer 1 and the third PDMS layer 3. Specifically, it includes the following steps:

[0089] 1) Pretreatment of the glass substrate: First, wash the glass substrate by hand with a cleaning agent, then place the glass substrate in acetone and isopropanol for ultrasonic cleaning for 10 min in sequence, then rinse with ionized water and dry with nitrogen; finally, place the dried glass substrate in an oven and heat it at 80 °C for 15 min.

[0090] 2) Covering of the photoresist: In order to obtain channels with a depth of 50 μm, the photoresist of the model DuPont photosensitive dry film ST925 is used. Since the thickness of a single layer of photoresist is 25 μm, two layers of dry film are selected for covering here.

[0091] First, select a washed glass substrate, drop a small amount of water droplets on the surface to facilitate the removal of air bubbles between the photoresist and the glass substrate; then, cover the photoresist on the glass substrate through a thermal laminator. After completion, perform pre-baking first, that is, place the glass substrate with the photoresist attached on a hot plate at 60 °C, and the pre-baking duration is 30 min.

[0092] 3) Exposure: Under a UV lamp, place the channel mask (MASK) on the photoresist, and make the side of the MASK with ink closely adhere to the photoresist. Then, press it tightly with a transparent plate and place it under the UV lamp for exposure.

[0093] 4) Development: Before development, post-baking is required, that is, heat on a hot plate, increase the temperature from 60 °C to 95 °C, and then maintain it at 95 °C for 35 min. Next, place the cooled mold in a 1 wt% concentration of Na2CO3 developer solution for development. After developing for 10 min, take it out, wash it with plasma water, dry it with nitrogen, and then bake it in an oven at 80 °C for 10 - 20 min.

[0094] Through the above steps, the channel molds of the first PDMS layer 1 and the third PDMS layer 3 are formed, and the patterns of the channels on the first PDMS layer 1 and the third PDMS layer 3 are on the channel molds.

[0095] S2. Pour the prepared PDMS solution on the channel mold after silane treatment and cure it to form a PDMS channel, and punch holes in the cured PDMS to form the ports of the PDMS channel. Specifically, it includes the steps:

[0096] 1) Pouring PDMS: Mix PDMS and a curing agent according to a mass ratio of 10:1, stir with a clean glass rod for 15 - 20 min, and evacuate for 30 min to ensure that the bubbles in the evenly stirred mixture completely disappear. Then, perform silanization treatment on the channel mold to deposit a layer of silane on the surface of the channel mold, which helps the PDMS not to adhere to the channel mold and is easy to detach the PDMS channel from the mold. Finally, pour the prepared PDMS solution on the channel mold after silane treatment, evacuate for another 20 min, ensure there are no bubbles, and then place it in an oven and heat it at 80 °C for 2 h to cure, obtaining a cured PDMS layer with PDMS channels on it.

[0097] 2) PDMS channel treatment: Slowly peel the cured PDMS layer from the mold, and cut it into a regular shape with a blade. According to the design structure of the microfluidic chip, use a punch to punch holes at both ends of the PDMS channel to form the inlets and outlets of the PDMS channel.

[0098] S3. Bond the cured PDMS to the PC film so that the PDMS channels face the PC film, and inject liquid metal into the ports of the PDMS channels to form metal electrodes. Specifically, it includes the following steps:

[0099] 1) Bonding of the PDMS channels to the PC film: Take a PC film of appropriate size, tear off the plastic film on one side of the PC film, place the PDMS channels with holes punched above and the PC film side by side in the chamber of the plasma machine, perform plasma treatment according to the corresponding steps of the plasma machine, then take it out, and directly press the PDMS channels on the PC film so that the PDMS channels face the PC film, and press it firmly once.

[0100] 2) Injection of liquid metal: Place the bonded PDMS channels and PC film, liquid metal, and metal syringe in an oven and heat at 80 °C. After the liquid metal melts, use the metal syringe to inject the liquid metal from the inlet of the PDMS channel to fill the entire PDMS channel with liquid metal, and a small amount of liquid metal overflows at the outlet end; Take out the PDMS channels (liquid metal electrodes) with liquid metal injected and the PC film from the oven and wait for cooling to form metal electrodes.

[0101] S4. Separate the PDMS from the PC film to form the first PDMS layer 1 or the third PDMS layer 3.

[0102] Specifically, after cooling, add a small amount of water between the interface of the PC film and the PDMS, and then separate the PDMS layer from the PC film to form the first PDMS layer 1 or the third PDMS layer 3 with PDMS channels.

[0103] Step 2. Processing of the PDMS stamp. Specifically, it includes the following steps:

[0104] S1. Pour the prepared PDMS solution on the silane-treated glass substrate and cure it to form a PDMS stamp, and modify the PDMS stamp to obtain a modified PDMS stamp. Specifically, it includes the following steps:

[0105] 1) Clean the glass substrate, and the specific steps are the same as the pretreatment steps of the glass substrate in the processing of liquid metal electrodes.

[0106] 2) Pouring PDMS: Mix PDMS and the curing agent in a mass ratio of 10:1, stir with a clean glass rod for 15 - 20 min, and evacuate for 30 min to ensure that the bubbles in the evenly stirred mixture completely disappear. Then, subject the glass substrate to silanization treatment so that a layer of silane is deposited on the surface of the glass substrate, which helps prevent PDMS from adhering to the glass substrate and makes it easy to detach the PDMS channels from the glass substrate. Finally, pour the prepared PDMS solution onto the silane-treated glass substrate, evacuate for another 20 min, and after ensuring no bubbles, place it in an oven and heat at 80 °C for 2 h to cure it.

[0107] 3) Treatment of the PDMS stamp: Slowly peel the cured PDMS from the glass substrate and cut it into a regular shape with a blade to form the PDMS stamp.

[0108] 4) Modification of the stamp: Use a pipette to take 20 μL of 1H,1H,2H,2H - perfluorooctyltrichlorosilane solution, place the PDMS block and the pipette tip together in a petri dish, put it in a vacuum chamber, evacuate to 0.09 MPa to allow the 1H,1H,2H,2H - perfluorooctyltrichlorosilane solution to volatilize and thus achieve the modification of the PDMS block. Take it out after treatment for 3 - 4 hours to obtain the modified PDMS stamp.

[0109] Step Three: Processing of the second PDMS layer 2. Specifically, it includes the steps:

[0110] S1. Cover the glass substrate with photoresist and perform exposure and development using a channel mask to form a mold with a channel pattern. Specifically, it includes the steps:

[0111] 1) Pretreatment of the glass substrate: First, wash it by hand with a cleaning agent, then ultrasonically clean it in acetone and isopropyl alcohol for 10 min in sequence, then rinse with ionized water and dry with nitrogen; finally, place the dried glass substrate in an oven and heat at 80 °C for 15 min.

[0112] 2) Covering of the photoresist: To obtain channels with a depth of 75 μm, the photoresist of the model DuPont photosensitive dry film ST925 is used. Since the thickness of a single layer of photoresist is 25 μm, three layers of dry film are selected for covering here.

[0113] First, select a washed glass substrate, drop a small amount of water droplets on the surface to facilitate the removal of air bubbles between the photoresist and the glass substrate; then, cover the photoresist through a thermal laminator. After completion, perform pre - baking first, that is, place the glass substrate with the photoresist attached on a hot plate at 60 °C for 30 min for pre - baking.

[0114] 3) Exposure: Place the channel mask (MASK) on top of the photoresist under a UV lamp. Note that the side of the MASK with ink should be in close contact with the photoresist. Then, press it tightly with a transparent plate and place it under the UV lamp for exposure.

[0115] 4) Development: Before development, post-baking is required, that is, heating on a hot plate, increasing the temperature from 60 °C to 95 °C and then maintaining it at 95 °C for 35 min. Next, place the cooled mold in a 1 wt% concentration of Na2CO3 developer solution for development. After 10 min of development, take it out, wash it with deionized water, dry it with nitrogen, and then bake it in an oven at 80 °C for 10 - 20 min to obtain a mold with a channel pattern.

[0116] S2. Pour the prepared PDMS solution into the mold with a channel pattern and press the modified PDMS stamp on the channel pattern to form a second PDMS layer 2 with a PDMS stamp.

[0117] Specifically, it includes the steps of: preparing a small amount of solution by mixing PDMS and a curing agent in a mass ratio of 10:1, stirring with a clean glass rod for 15 - 20 min, and evacuating for 30 min to ensure that the bubbles in the evenly stirred mixture completely disappear. Then pour the prepared solution into the mold with a channel pattern, and then press the modified PDMS stamp on the channel pattern, press to drive out the bubbles in the middle PDMS solution, let it stand at room temperature for 20 min, and then place it on a hot plate, increasing the temperature from room temperature to 90 °C. After standing for 1 h, PDMS combines with the PDMS stamp to form a PDMS fluid channel layer with a PDMS stamp, that is, the second PDMS layer 2, and remove it from the glass substrate.

[0118] Step 3. Bonding of the chip. Specifically, it includes the steps of:

[0119] S1. Align and bond the side of the third PDMS layer 3 with liquid metal with the side of the second PDMS layer 2 with the third PDMS channel 6, and then remove the PDMS stamp.

[0120] Specifically, place the side of the third PDMS layer 3 with liquid metal and the side of the PDMS fluid channel layer of the second PDMS layer 2 with a PDMS stamp facing up and place them side by side in the chamber of the plasma machine, and perform plasma treatment according to the corresponding steps of the plasma machine; then take it out, and under a microscope, align the side of the third PDMS layer 3 with liquid metal with the side of the second PDMS layer 2 with the third PDMS channel 6. A small amount of water can be dropped on the PDMS fluid channel layer with a PDMS stamp to facilitate fine adjustment; after alignment, press firmly for a few minutes, and then place it in an oven and heat it at 60 °C for 40 min; then remove the PDMS stamp.

[0121] S2. Punch holes at both ends of the third PDMS channel 6 in the second PDMS layer 2 so that the holes penetrate the second PDMS layer 2 and the third PDMS layer 3, forming a channel inlet 601 and a channel outlet 602 on the second PDMS layer 2, and forming a fluid inlet 9 and a fluid outlet 10 on the third PDMS layer 3.

[0122] Specifically, use a punch to punch holes at the inlet and outlet of the third PDMS channel 6 on the second PDMS layer 2, and the holes should penetrate the third PDMS layer 3, thereby forming a channel inlet 601 and a channel outlet 602 on the second PDMS layer 2, and forming a fluid inlet 9 and a fluid outlet 10 on the third PDMS layer 3.

[0123] S3. Align and bond the side of the first PDMS layer 1 with liquid metal to the second PDMS layer 2.

[0124] Specifically, place the side of the first PDMS layer 1 with liquid metal and the side of the bonded liquid metal electrode and PDMS fluid channel layer with the PDMS fluid channel layer facing up side by side in the chamber of the plasma machine, and perform plasma treatment according to the corresponding steps of the plasma machine; then take it out, align the side of the first PDMS layer 1 with liquid metal to the second PDMS layer 2 under the microscope. A small amount of water can be dropped on the PDMS fluid channel layer to facilitate fine adjustment; after alignment, press firmly for a few minutes, and then place it in an oven and heat at 60 °C for 40 min to obtain a well-bonded chip.

[0125] It should be noted that when fine adjustment is required during the alignment process, do not press firmly, and handle it as gently as possible to avoid being bonded together and unable to move. Bonding is a very crucial step. The quality of bonding directly affects the sealing effect of the channels in the final chip, and thus affects the reliability and accuracy of the experimental results.

[0126] The preparation method of the cell three-dimensional rotation and electroporation microfluidic chip based on AC electric field regulation in this embodiment is simple to process, requires a small applied voltage, does not require external modification of cells, and has precise manipulation.

[0127] Example Three

[0128] Based on Embodiment One and Embodiment Two, this embodiment conducts experimental verification on the cell three-dimensional rotation and electroporation microfluidic chip based on AC electric field regulation. For the size data of each structure of the cell three-dimensional rotation and electroporation microfluidic chip based on AC electric field regulation in this embodiment, refer to Figure 5As shown in Table 1, further, the dimensional data of the second PDMS channel thickness 5 is the same as that of the first PDMS channel thickness 4, and the dimensional data of the fifth PDMS channel 8 is the same as that of the fourth PDMS channel 7.

[0129] Dimensional data of the chip structure in Table 1

[0130] Symbol Physical meaning Value Unit <![CDATA[H1]]> The thickness of the first PDMS channel is 4 50 [μm] <![CDATA[W1]]> The width of the first PDMS channel is 4 150 [μm] <![CDATA[H2]]> The thickness of the third PDMS channel is 6 75 [μm] <![CDATA[W2]]> The width of the third PDMS channel is 6 75 [μm] <![CDATA[H3]]> The thickness of the fourth PDMS channel is 7 50 [μm] <![CDATA[W3]]> The width of the fourth PDMS channel is 7 150 [μm]

[0131] During the three-dimensional cell rotation experiment: First, prepare the buffer solution. Add a certain amount of KCl particles and deionized water to a beaker and stir well to obtain a buffer solution with a conductivity of 38 mS / m; then use the buffer solution to prepare a yeast cell solution of about 100 cells / μL.

[0132] Secondly, configure absolute ethanol and Tween solution in a volume ratio of 9:1 to obtain Solution A. The main function of Solution A is to reduce the adhesion of particles on the channel or substrate surface. Configure Solution A and the prepared yeast cell solution in a volume ratio of 1:99 to obtain Solution D.

[0133] Then, perform the experimental operations. The specific steps include:

[0134] Step 1: Turn on the computer, signal generator, microscope, and CCD switch, and observe whether the equipment is operating normally; then turn on the ImageView image acquisition software on the computer and observe the scene on the microscope stage in real time.

[0135] Step 2: Fix the microfluidic chip for three-dimensional cell rotation and electroporation based on AC electric field regulation provided in this embodiment on the stage, and adjust the chip position and focal length; then fix a 25-μL microinjector on the syringe pump, suck in a certain amount of Solution D, and insert the injection head of the syringe pump that has sucked in a certain amount of Solution D into the metal connector at the channel inlet 601, and ensure good sealing.

[0136] Step 3: Connect the first excitation electrode 401, the second excitation electrode 501, the third excitation electrode 701, and the fourth excitation electrode 801 to the signal generator through electrode leads respectively, and adjust the signal voltage, phase difference, and frequency parameters on the signal generator as well as the flow control parameters on the syringe pump.

[0137] Step 4: Start the syringe pump and let Solution D flow into the second PDMS channel 6 from the channel inlet 601 at the controlled flow rate. When the fluid flow in the channel is stable, press the signal application button on the signal generator.

[0138] Step 5: Observe under the microscope, and readjust the focal length and the position of the chip again, and select the height at which the yeast is clearest and most stable for video detection and recording.

[0139] Step 6: Repeat Steps 3 - 5, continuously adjust the voltage, frequency, and flow rate, observe the experimental phenomena and record them.

[0140] Step 7: Rotate the yeast to the required angle, turn off the signal generator, and use a power amplifier to apply a square wave signal with a frequency of 10 kHz, an amplitude of approximately 65 Vpp, and a duration of 5 ms on the liquid metal electrode. Use this signal to conduct an electroporation experiment on yeast cells and record.

[0141] At Figure 1 the above - mentioned experiment was carried out on the cell three - dimensional rotation and electroporation microfluidic chip based on AC electric field regulation as shown. In the experiment, the signal voltage amplitude was 6 Vpp and the frequency was 100 kHz. For the experimental results, refer to Figure 6 and Figure 7 . Figure 6 This is a graph showing the experimental results of three - dimensional rotation control of yeast provided by an embodiment of the present invention. Figure 6 These are images of yeast after applying the electric field for 0 s, 0.07 s, 0.14 s, 0.21 s, and 0.28 s. It can be seen from the figure that the yeast performs three - dimensional rotational motion along the direction perpendicular to the plane. Figure 7 This is a graph showing the experimental results of electroporation of yeast provided by an embodiment of the present invention. Since the yeast cells were pre - stained with propidium iodide, once electroporation is completed, the cell wall and cell membrane of the yeast will be temporarily opened, and the propidium iodide that could not originally enter the living cells will enter the yeast, causing the yeast to emit red fluorescence under the excitation light of 535 nm. Figure 7 These are fluorescence images of yeast after applying 0, 1, and 2 electroporation signals. It can be seen from Figure 7 the figure that after applying a pulse signal to the yeast, electroporation occurred to the yeast, and the more times of electroporation, the more propidium iodide entered the cells, and the stronger the fluorescence intensity.

[0142] The above content is a further detailed description of the present invention in combination with specific preferred embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, which should all be regarded as belonging to the protection scope of the present invention.

Claims

1. A cell three-dimensional rotation and electroporation microfluidic chip based on AC electric field regulation, characterized in that: include: A first PDMS layer (1), a second PDMS layer (2) and a third PDMS layer (3) are stacked in sequence, wherein: The first PDMS layer (1) is provided with a first PDMS channel (4) and a second PDMS channel (5), the second PDMS layer (2) is provided with a third PDMS channel (6), and the third PDMS layer (3) is provided with a fourth PDMS channel (7), a fifth PDMS channel (8), a fluid inlet (9) and a fluid outlet (10); The first PDMS channel (4) and the second PDMS channel (5) are arranged opposite to each other, and both the first PDMS channel (4) and the second PDMS channel (5) have sides parallel to the third PDMS channel (6) and located below the third PDMS channel (6); the first PDMS channel (4) and the second PDMS channel (5) are both filled with liquid metal; one end of the first PDMS channel (4) forms a first excitation electrode (401), and the other end is used to connect an electrode lead; one end of the second PDMS channel (5) forms a second excitation electrode (501), and the other end is used to connect an electrode lead; One end of the third PDMS channel (6) forms a channel inlet (601) for injecting a solution containing cells, and the other end forms a channel outlet (602) for discharging the solution containing cells, and the channel inlet (601) is connected to the fluid inlet (9), and the channel outlet (602) is connected to the fluid outlet (10); The fourth PDMS channel (7) and the fifth PDMS channel (8) are arranged opposite to each other, and both the fourth PDMS channel (7) and the fifth PDMS channel (8) have sides parallel to the third PDMS channel (6) and located below the third PDMS channel (6); both the fourth PDMS channel (7) and the fifth PDMS channel (8) are filled with liquid metal; one end of the fourth PDMS channel (7) forms a third excitation electrode (501), and the other end is used to connect an electrode lead; one end of the fifth PDMS channel (8) forms a fourth excitation electrode (801), and the other end is used to connect an electrode lead; The inner end of the first PDMS channel (4), the inner end of the second PDMS channel (5), the inner end of the fourth PDMS channel (7) and the inner end of the fifth PDMS channel (8) are all located within the third PDMS channel (6).

2. The cell three-dimensional rotation and electroporation microfluidic chip based on AC electric field regulation according to claim 1, characterized in that: The first PDMS channel (4), the second PDMS channel (5), the fourth PDMS channel (7) and the fifth PDMS channel (8) all include a first vertical segment (110), a parallel segment (120) and a second vertical segment (130), wherein the parallel segment (120) is connected between the first vertical segment (110) and the second vertical segment (130).

3. The cell three-dimensional rotation and electroporation microfluidic chip based on AC electric field regulation according to claim 1, characterized in that: The phases of the electric fields applied to the first excitation electrode (401), the second excitation electrode (501), the third excitation electrode (501) and the fourth excitation electrode (801) increase by 90° in sequence.

4. The cell three-dimensional rotation and electroporation microfluidic chip based on AC electric field regulation according to claim 1, characterized in that: The phases of the electric field applied to the first excitation electrode (401), the second excitation electrode (501), the third excitation electrode (501) and the fourth excitation electrode (801) are 0°, 90°, 180° and 270° respectively.

5. The cell three-dimensional rotation and electroporation microfluidic chip based on AC electric field regulation according to claim 1, characterized in that: When an electric field is applied, the time-averaged dielectrophoretic force acting on the cell is: The time-averaged electrical rotation torque is: Where K(ω) is the CM factor, Im[K(ω)] is the imaginary part of the CM factor, Re[K(ω)] is the real part of the CM factor, r is the cell radius, E is the electric field intensity, * is the conjugate complex number, ~ is the complex amplitude, is the gradient operator; in, is the complex dielectric constant of the cell, is the complex dielectric constant of the solution; e * =ε-j(σ / ω) Among them, ε is the dielectric constant, σ is the conductivity, ω is the angular frequency, and j is the imaginary unit.

6. The cell three-dimensional rotation and electroporation microfluidic chip based on AC electric field regulation according to claim 1, characterized in that: The liquid metal includes bismuth-based liquid metal.

7. The cell three-dimensional rotation and electroporation microfluidic chip based on AC electric field regulation according to claim 1, characterized in that: The fluid inlet (9) and the fluid outlet (10) are both provided with metal connectors.

8. The cell three-dimensional rotation and electroporation microfluidic chip based on AC electric field regulation according to claim 1, characterized in that: The first PDMS channel (4) has a thickness of 50 μm and a width of 150 μm; The second PDMS channel (5) has a thickness of 50 μm and a width of 150 μm; The third PDMS channel (6) has a thickness of 75 μm and a width of 75 μm; The fourth PDMS channel (7) has a thickness of 50 μm and a width of 150 μm; The fifth PDMS channel (8) has a thickness of 50 μm and a width of 150 μm.

9. A method for preparing a cell three-dimensional rotation and electroporation microfluidic chip based on AC electric field regulation, characterized in that: The method is used to prepare a cell three-dimensional rotation and electroporation microfluidic chip based on AC electric field regulation as described in any one of claims 1 to 8, comprising the steps of: processing the first PDMS layer (1) and the third PDMS layer (3); processing the PDMS stamp; processing the second PDMS layer (2); bonding the chip, wherein: The processing of the first PDMS layer (1) and the third PDMS layer (3) comprises the steps of: S1, covering a glass substrate with photoresist, and performing exposure and development using a channel mask to form a channel mold of a first PDMS layer (1) and a third PDMS layer (3); S2, pouring the prepared PDMS solution on the channel mold after silane treatment and curing it to form a PDMS channel, and punching holes on the cured PDMS to form ports of the PDMS channel; S3, bonding the cured PDMS to the PC membrane so that the PDMS channel faces the PC membrane, and injecting liquid metal from the port of the PDMS channel to form a metal electrode; S4, separating the PDMS and the PC film to form the first PDMS layer (1) or the third PDMS layer (3); The processing of the PDMS stamp includes the steps of: S1, pouring the prepared PDMS solution on the glass substrate treated with silane and curing it to form a PDMS stamp, and modifying the PDMS stamp to obtain a modified PDMS stamp; The processing of the second PDMS layer (2) comprises the steps of: S1, covering the glass substrate with photoresist, and performing exposure and development using a channel mask to form a mold with a channel pattern; S2, pouring the prepared PDMS solution into the mold with the channel pattern and pressing the modified PDMS stamp on the channel pattern to form a second PDMS layer (2) with the PDMS stamp; The bonding of the chip comprises the steps of: S1, aligning and bonding the side of the third PDMS layer (3) with the liquid metal and the side of the second PDMS layer (2) with the third PDMS channel (6), and then peeling off the PDMS stamp; S2, drilling holes at both ends of the third PDMS channel (6) of the second PDMS layer (2) so that the holes penetrate the second PDMS layer (2) and the third PDMS layer (3), forming a channel inlet (601) and a channel outlet (602) on the second PDMS layer (2), and forming a fluid inlet (9) and a fluid outlet (10) on the third PDMS layer (3); S3, aligning and bonding the side of the first PDMS layer (1) with the liquid metal to the second PDMS layer (2).

Images