Micro-droplet generation chip for cell wrapping

Abstract

The utility model provides a micro-droplet generating chip for wrapping cells, which comprises a chip body, a cell phase inlet, an oil phase inlet, a magnetic bead phase inlet and a reagent collecting port, a first flow channel communicated with the cell phase inlet, a second flow channel communicated with the magnetic bead phase inlet and two third flow channels simultaneously communicated with the oil phase inlet are formed in the chip body, and the outflow ends of the first flow channel and the second flow channel are converged at the inflow end of a fourth flow channel; the two third flow channels and the fourth flow channel are communicated in a crossed mode to form a cross-shaped liquid drop generating structure, the outflow end of the fourth flow channel is communicated with the reagent collecting opening, and piezoelectric ceramic brakes are correspondingly arranged on the first flow channel, the second flow channel and the third flow channels. The braking ends of the piezoelectric ceramic brakes are used for conducting controllable connection and disconnection on fluid in the first flow channel, the second flow channel and the third flow channel. According to the utility model, uniform liquid drops with higher cell wrapping rate are obtained, and cell membrane damage or protein denaturation caused by an electric field or heating is completely eradicated.

Description

Technical Field

[0001] The utility model belongs to the technical field of biochip manufacturing, and particularly relates to a micro-droplet generation chip for cell encapsulation. Background Art

[0002] Based on the characteristics of high precision and repeatability in operating on tiny fluid volumes, microfluidic devices are widely used. However, with the reduction of the size of these devices and the increase of the surface area to volume ratio, reagent adsorption and diffusion limitations cannot be ignored. Therefore, many recent studies have focused on droplet / capsule-based microfluidics, where picoliters to microliters of reagents are encapsulated in immiscible carrier fluids. In addition, through considerations of electric fields, thermal activation, or hydrodynamics, the operation and monitoring of capsules in microfluidic networks become possible. Droplet-based microfluidics has a variety of key applications, such as high-throughput screening, single-cell analysis and encapsulation, protein crystallization, polymer / gel particle synthesis, vesicle production, and chemical microreactors. These devices typically involve the flow of two immiscible fluids, a continuous flow (usually mineral oil) and a segmented flow (usually water). At the intersection of the two flows, the continuous phase shears the dispersed phase into ordered and uniform droplets.

[0003] Many applications require controlling the coalescence of droplets for biological or chemical reactions in daughter droplets. Two or more droplet generators are used on the same chip, each generating droplets with different contents. These droplets fuse downstream in a designated chamber. To achieve effective coalescence, synchronization between the droplets arriving at the chamber is necessary, and this synchronization can only be achieved by electronically controlling droplet generation.

[0004] Some droplet generators are used to solve the above problems. It is reported that bubble / droplet generators are based on temporarily heating the air-water interface to locally reduce the surface tension. This technique has several disadvantages: firstly, heat conduction and heat capacity limit the generation rate to the order of a few hertz; secondly, heating may not be suitable for live cell analysis or protein crystallization. Another method of generating droplets is to apply high electric field pulses at the oil-water interface to charge them and generate electrostatic forces. This method requires a high on-chip electric field of about 1 kV, which is impractical for low-cost simple devices and is also not suitable for cell encapsulation or diagnosis.

[0005] In many applications, cells must be locally contained to control cell-cell interactions and cell signaling. 3D tissue printing requires careful control of the cellular environment to direct cell growth and cell fate, typically by using hydrogel capsules and altering the mechanical or chemical properties of the extracellular matrix, although two-phase bioprinting offers an alternative way to prevent cell-cell interactions. For example, an artificial pancreas uses encapsulated islet cells to limit the immune system response after implantation. Conversely, it can also interact with specific cells, such as in cell fusion for hybridoma formation, cell reprogramming, and antibiotic drug discovery, activities that require precise control of cell location and environment, and are also needed in studies of protein expression and antibody production. An evolving method of controlling the cellular environment is to utilize droplet-based microfluidics principles, where aqueous fluids are segmented into individual droplets in an immiscible carrier fluid (usually a mineral or fluorinated oil) to encapsulate cells, organic molecules, and reagents.

[0006] Some of the advantages of encapsulating cells within droplets are evident. First, since the oil-water interface provides a natural barrier to diffusion, cell products remain in their vicinity, and thus dilution can be minimized and maintained even when there are large concentration gradients between droplets. Second, compared to single-phase microfluidic systems, the reaction volume is significantly reduced, which is an important factor when using high-value reagents such as enzymes or DNA. Third, the ability to control the position and duration of discrete fluid volumes is enhanced, and long-term cell culture is simplified by preventing adhesion between encapsulated cells or between device features. Summary of the Utility Model

[0007] Therefore, the technical problem to be solved by the present utility model is to provide a microdroplet generation chip for cell encapsulation, which can eliminate the technical problems of cell membrane damage or protein denaturation caused by using an electric field or heating in the prior art.

[0008] To solve the above problems, the present utility model provides a microdroplet generation chip for cell encapsulation, comprising: a chip body, on which a cell phase inlet, an oil phase inlet, a magnetic bead phase inlet, and a reagent collection port are constructed; a first flow channel communicated with the cell phase inlet, a second flow channel communicated with the magnetic bead phase inlet, and two third flow channels simultaneously communicated with the oil phase inlet are constructed inside the chip body; the outflow ends of the first flow channel and the second flow channel converge at the inflow end of a fourth flow channel; the two third flow channels intersect and communicate with the fourth flow channel to form a cross droplet generation structure; the outflow end of the fourth flow channel is communicated with the reagent collection port; piezoelectric ceramic brakes are respectively arranged on the first flow channel, the second flow channel, and each third flow channel in a one-to-one correspondence, and the braking ends of the piezoelectric ceramic brakes are respectively used for controlling the on-off of the fluids in the first flow channel, the second flow channel, and the third flow channel.

[0009] In some embodiments, accommodation grooves are formed in series on the first flow channel, the second flow channel, and each of the third flow channels, and the braking ends of the piezoelectric ceramic brakes are respectively accommodated in the accommodation grooves.

[0010] In some embodiments, a flexible film is provided at the notch of the accommodation groove, and the flexible film can deform following the expansion and contraction of each braking end.

[0011] In some embodiments, the flexible film is a silica gel sleeve sleeved on each braking end.

[0012] In some embodiments, a micro-pit array is further constructed in the chip body. The micro-pit array is connected in series to the fourth flow channel and is located between the cross-shaped droplet generation structure and the reagent collection port.

[0013] In some embodiments, the micro-pit array is formed by arranging a plurality of micro-pits at intervals in the horizontal and vertical directions.

[0014] In some embodiments, the diameter of the micro-pits is 50 microns to 200 microns.

[0015] In some embodiments, both the diameter and the depth of the micro-pits are 200 microns.

[0016] In some embodiments, two of the third flow channels form a rectangle symmetrically left and right, wherein the oil phase inlet and the cross-shaped droplet generation structure are respectively located at the midpoints of two parallel sides of the rectangle, and the first flow channel and the second flow channel are located inside the rectangle.

[0017] In some embodiments, the flow channel widths of the first flow channel, the second flow channel, and the third flow channels are the same and are d, and the flow channel width of the fourth flow channel is D, and d / D ≤ 0.5.

[0018] A micro-droplet generation chip for cell encapsulation provided by the present utility model uses a piezoelectric ceramic brake to accurately convert voltage into mechanical translation that directly determines the volume of the fluid segment. Uniform droplets with a high cell encapsulation rate can be obtained through any combination of the size, distance, and frequency of the droplets encapsulating cells. The micro-droplet generation chip in this technical solution is particularly suitable for biological applications because it does not use an electric field or heating, thereby being able to prevent the destruction of cell membranes or protein denaturation caused by the electric field or heating. In addition, the technically mature piezoelectric ceramic brake can be easily integrated into any droplet-based microfluidic device using a miniature, inexpensive, commercially available actuator with low power consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic three-dimensional structure diagram of the micro-droplet generation chip for cell encapsulation according to an embodiment of the present utility model;

[0020] Figure 2 is Figure 1 a schematic three-dimensional structure diagram of the chip body in

[0021] Figure 3 is Figure 1 a schematic three-dimensional structure diagram (partially disassembled) of the piezoelectric ceramic brake in

[0022] The reference numerals are shown as:

[0023] 1. Chip body; 11. Cell phase inlet; 12. Oil phase inlet; 13. Magnetic bead phase inlet; 14. Reagent collection port; 20. Accommodating groove; 21. First flow channel; 22. Second flow channel; 23. Third flow channel; 24. Fourth flow channel; 25. Cross droplet generation structure; 3. Piezoelectric ceramic brake; 31. Flexible film; 4. Micro-pit array. Specific embodiments

[0024] Referring to Figures 1 to 3 as shown, according to an embodiment of the present invention, there is provided a micro-droplet generation chip for cell encapsulation, including: a chip body 1, on which a cell phase inlet 11, an oil phase inlet 12, a magnetic bead phase inlet 13 and a reagent collection port 14 are constructed, and a first flow channel 21 communicated with the cell phase inlet 11, a second flow channel 22 communicated with the magnetic bead phase inlet 13 and two third flow channels 23 communicated with the oil phase inlet 12 at the same time are constructed inside the chip body 1. The outflow ends of the first flow channel 21 and the second flow channel 22 converge at the inflow end of the fourth flow channel 24, and the two third flow channels 23 are cross-communicated with the fourth flow channel 24 to form a cross droplet generation structure 25. The outflow end of the fourth flow channel 24 is communicated with the reagent collection port 14. Piezoelectric ceramic brakes 3 are respectively arranged on the first flow channel 21, the second flow channel 22 and each third flow channel 23 in a one-to-one correspondence. The braking ends of the piezoelectric ceramic brakes 3 are respectively used for controlling the on-off of the fluid in the first flow channel 21, the second flow channel 22 and the third flow channel 23, so as to control the feeding speed of the fluid in each flow channel. It can be understood that the aforementioned chip body 1 specifically includes a substrate and a cover plate bonded together as a whole, wherein the first flow channel 21, the second flow channel 22 and the third flow channel 23 are all constructed on one side surface of the substrate, and the cover plate can seal the opening sides of the first flow channel 21, the second flow channel 22 and the third flow channel 23. It can be understood that the aforementioned piezoelectric ceramic brake 3 is a conventional device that converts voltage into the expansion and contraction of its braking end, and an appropriate structure can be selected according to the volume control requirements of the fluid.

[0025] In this technical solution, the piezoelectric ceramic actuator 3 adopted precisely converts voltage into mechanical translation that directly determines the volume of the fluid segment. Uniform droplets with a high cell encapsulation rate can be obtained through any combination of the droplet size, droplet distance, and frequency of the droplets encapsulating cells. The micro-droplet generation chip in this technical solution is particularly suitable for biological applications because no electric field or heating is used, thus eliminating the destruction of cell membranes or protein denaturation caused by the electric field or heating. In addition, the well-developed piezoelectric ceramic actuator 3 can be easily integrated into any droplet-based microfluidic device using a miniature, inexpensive, commercially available actuator with low power consumption.

[0026] In this technical solution, the liquid dispersed phase materials flowing out from the magnetic bead phase, oil phase, and cell phase inlets are blocked by the structure of the piezoelectric ceramic actuator 3. After starting the piezoelectric ceramic actuator 3 under certain frequency conditions, the on-off situation of the liquid paths of the magnetic bead phase, oil phase, and cell phase changes with the change of its frequency, thereby realizing the control of the feeding speed of each fluid material. For example, by controlling the frequency of the piezoelectric ceramic actuator 3 in the cell phase and the area of the circular region in the cell phase channel in the microfluidic chip, the flow situation of the cell phase can be controlled, and the content of the cell phase in the droplet can be controlled, so as to obtain an ultra-high cell encapsulation rate.

[0027] In this technical solution, the piezoelectric ceramic actuators 3 correspondingly arranged on each flow channel can be separately controlled. The frequency, the area of the contact surface between the piezoelectric ceramic actuator 3 and the chip, and the driving force of the dispersed phase affect the content of the dispersed phase flowing into the cross region (i.e., the aforementioned cross-droplet generation structure 25). Under the influence of the structure of the cross region, droplets encapsulating cells are formed under the shearing action. In a specific embodiment, using the micro-droplet generation chip of the present invention can preferably achieve the encapsulation of a single cell in a single droplet, and the encapsulation rate of a single cell is greater than 90%.

[0028] The micro-droplet generation chip of the present invention can also be used for magnetic beads in cell sorting, which are mostly immunolabeled magnetic beads, that is, antibodies are directly or indirectly coupled to the magnetic beads to specifically recognize the surface antigens of target cells. Commonly used indirectly labeled magnetic beads include anti-immunoglobulin magnetic beads, anti-biotin magnetic beads, anti-streptavidin magnetic beads, and anti-fluorescein magnetic beads, etc. In addition, the micro-droplet generation chip of the present invention can also be used for the generation of emulsion droplets and multiple emulsion droplets. With the above structure, various emulsion droplets can be relatively easily manufactured.

[0029] In some embodiments, the aforementioned cross-droplet generation structure 25 can also be a T-shaped droplet generation structure.

[0030] The aforementioned chip body 1 can be fabricated by combining two of various materials such as PDMS, glass, silicon wafer, COC, COP, PMMA, etc. For different materials, relevant surface treatments are required according to their material properties to enable normal droplet generation. The bonding method of the chip body 1 can be selected according to the material needs. For example, PDMS, silicon wafer, glass, etc. in the silicon-based system can be bonded after plasma treatment; the bonding method of adhesive bonding can be selected; or bonding methods such as hot pressing, laser welding, ultrasonic welding, etc. can also be selected.

[0031] In some embodiments, accommodation grooves 20 are respectively formed in series on the first flow channel 21, the second flow channel 22, and each third flow channel 23, and the braking ends of each piezoelectric ceramic actuator 3 are respectively accommodated in each accommodation groove 20.

[0032] In some embodiments, a flexible film 31 is provided at the notch of the accommodation groove 20. The flexible film 31 can deform following the expansion and contraction of each braking end. Specifically, the flexible film 31 is a silica gel sleeve sleeved on each braking end, and is used for sealing between the chip body 1 and the braking end of the piezoelectric ceramic actuator 3 within the position of the accommodation groove 20.

[0033] In some embodiments, a micro-pit array 4 is further constructed in the chip body 1. The micro-pit array 4 is connected in series to the fourth flow channel 24 and is located between the cross-droplet generation structure 25 and the reagent collection port 14. The micro-pit array 4 and the reagent collection port 14 are jointly used to collect the generated droplets.

[0034] In some embodiments, the micro-pit array is formed by arranging a plurality of micro-pits at intervals in the horizontal and vertical directions. In some embodiments, the diameter of the micro-pits is 50 microns to 200 microns. In a specific embodiment, the diameter and depth of the micro-pits are both 200 microns, and the number is 30*40, which is used for single-cell culture.

[0035] In a specific embodiment, refer to Figure 1 and Figure 2 As shown, the two third flow channels 23 are symmetrically formed into a rectangle on the left and right, wherein the oil phase inlet 12 and the cross-droplet generation structure 25 are respectively located at the midpoints of two parallel sides of the rectangle. The first flow channel 21 and the second flow channel 22 are located within the rectangle. The relatively symmetrical flow channel structure design can make the cutting effect of the oil phase better.

[0036] In some embodiments, the flow channel widths of the first flow channel 21, the second flow channel 22, and the third flow channel 23 are the same and are d, and the flow channel width of the fourth flow channel 24 is D, where d / D ≤ 0.5, that is, the ratio of the dispersed phase inlet to the main channel width in the droplet generation chip is not greater than 0.5, thereby ensuring the generation of single droplets. At the same time, a higher ratio can be used for the burst generation of droplets.

[0037] It is easy for those skilled in the art to understand that, on the premise of no conflict, the above advantageous ways can be freely combined and superimposed.

[0038] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention. The above is only the preferred implementation manner of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and variations can be made, and these improvements and variations should also be regarded as the protection scope of the present invention.

Claims

1. A micro-droplet generation chip for cell encapsulation, characterized in that: include: A chip body (1), wherein the chip body (1) is provided with a cell phase inlet (11), an oil phase inlet (12), a magnetic bead phase inlet (13) and a reagent collection port (14); wherein the chip body (1) is provided with a first flow channel (21) connected to the cell phase inlet (11), a second flow channel (22) connected to the magnetic bead phase inlet (13) and two third flow channels (23) connected to the oil phase inlet (12); the outflow ends of the first flow channel (21) and the second flow channel (22) are combined in a fourth flow channel (24). The second flow channel (22) and the third flow channel (23) are connected to the inlet end of the second flow channel (23), the two third flow channels (23) are cross-connected with the fourth flow channel (24) to form a cross droplet generation structure (25), the outlet end of the fourth flow channel (24) is connected to the reagent collection port (14), and piezoelectric ceramic brakes (3) are respectively arranged on the first flow channel (21), the second flow channel (22) and each third flow channel (23) in a one-to-one correspondence, and the braking end of each piezoelectric ceramic brake (3) is used for controllably opening and closing the fluid in the first flow channel (21), the second flow channel (22) and the third flow channel (23).

2. The micro-droplet generation chip according to claim 1, characterized in that: The first flow channel (21), the second flow channel (22) and each third flow channel (23) are respectively connected in series to form a receiving groove (20), and the braking end of each piezoelectric ceramic brake (3) is respectively accommodated in each of the receiving grooves (20).

3. The micro-droplet generation chip according to claim 2, characterized in that: A flexible membrane (31) is provided at the notch of the accommodation groove (20), and the flexible membrane (31) can deform following the expansion and contraction of each braking end.

4. The micro-droplet generation chip according to claim 3, characterized in that: The flexible membrane (31) is a silicone sleeve sleeved on each of the braking ends.

5. The micro-droplet generation chip according to claim 1, characterized in that: A micro-pit array (4) is also constructed in the chip body (1), and the micro-pit array (4) is connected in series to the fourth flow channel (24) and is located between the cross droplet generation structure (25) and the reagent collection port (14).

6. The micro-droplet generation chip according to claim 5, characterized in that: The micro-pit array is formed by a plurality of micro-pits arranged in a horizontal and vertical interval.

7. The micro-droplet generation chip according to claim 6, characterized in that: The diameter of the micropit is 50 micrometers to 200 micrometers.

8. The micro-droplet generation chip according to claim 7, characterized in that: The diameter and depth of the micro-pits are both 200 microns.

9. The micro-droplet generation chip according to claim 1, characterized in that: The two third flow channels (23) are symmetrically arranged to form a rectangle, wherein the oil phase inlet (12) and the cross droplet generation structure (25) are respectively located at the midpoints of two parallel sides of the rectangle, and the first flow channel (21) and the second flow channel (22) are located within the rectangle.

10. The micro-droplet generation chip according to claim 1, characterized in that: The flow channel widths of the first flow channel (21), the second flow channel (22) and the third flow channel (23) are all the same and are d, and the flow channel width of the fourth flow channel (24) is D, where d / D≤0.5.

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