Inertia micro-fluidic chip for cross-scale packaging
By designing an asymmetric sinusoidal aqueous phase channel and a closed-loop oil phase channel in an inertial microfluidic chip, efficient mixing of Agrobacterium and soybean cells is achieved, solving the problem of low soybean conversion efficiency in the existing Agrobacterium-mediated method, simplifying operation and reducing costs.
Patent Information
- Application Number
- CN202520264977.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-19
- Publication Date
- 2026-01-09
- Estimated Expiration
- 2035-02-19
AI Technical Summary
Existing Agrobacterium-mediated transformation methods for soybeans have low efficiency, are complex to operate, require sophisticated equipment, and are costly, making it difficult to efficiently infect soybean cells.
Using a cross-scale packaging inertial microfluidic chip, an asymmetric sinusoidal aqueous phase channel and a closed-loop oil phase channel are designed. By focusing Agrobacterium cells through fluid dynamics principles, droplets are formed at the junction of the oil and aqueous phases, achieving efficient mixing of Agrobacterium and soybean cells.
It significantly improves the contact efficiency between Agrobacterium and soybean cells and the gene conversion efficiency, simplifies the operation process, and reduces equipment costs.
Smart Images
Figure CN223780237U_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The utility model relates to micro -fluidic technique field, concretely relates to a cross -scale packaging inertia micro -fluidic chip. BACKGROUND
[0002] Agrobacterium is a natural plant genetic engineering vector, which can transfer and integrate T-DNA on its Ti plasmid or Ri plasmid into the plant genome. Using this characteristic, Agrobacterium-mediated gene transfer method has significant advantages: high gene transfer efficiency, stable insertion fragment, large DNA fragment transfer and accurate integration, which makes Agrobacterium-mediated method particularly suitable for obtaining stable transgenic plants.
[0003] In the transgenic research of soybean, Agrobacterium-mediated method can effectively introduce pest-resistant, disease-resistant, stress-resistant and quality-improved genes, so as to cultivate excellent new soybean varieties. However, as a crop with low transformation efficiency, Agrobacterium-mediated transgenic process faces many challenges, so further research and optimization of experimental conditions are needed to improve the transformation efficiency.
[0004] At present, the commonly used methods of Agrobacterium infection of soybean include Agrobacterium co-culture method, Agrobacterium pollen tube method and Agrobacterium injection method. However, these methods have some defects: (1) long screening and regeneration process is needed; (2) high technical requirements are needed, and special equipment is needed; (3) low transformation efficiency, especially for some soybean varieties; (4) complex operation process, high technical difficulty, such as pollen tube injection method. UTILITY MODEL CONTENT
[0005] The utility model aims at overcoming the deficiencies in the prior art, providing a cross-scale packaging inertia micro -fluidic chip for the method of Agrobacterium infection of soybean cells, simplifying the operation of promoting Agrobacterium infection, improving the efficiency of infection, and reducing the cost of equipment.
[0006] To achieve the above object, the utility model adopts the technical scheme that a cross-scale packaging inertia micro -fluidic chip, including first water phase channel, second water phase channel, mixing channel and oil phase channel, the first water phase channel adopts asymmetric sine shape structure, and the output end of the first water phase channel and the second water phase channel all are communicated with the mixing channel; The oil phase channel adopts closed loop structure, and the mixing channel penetrates the output end of the oil phase channel and communicates with it.
[0007] Optionally, the first water phase channel, the second water phase channel, the mixing channel and the oil phase channel are all opened on the cover plate, and the bottom of the cover plate is provided with a bottom plate.
[0008] Optionally, an outlet is formed on the cover plate, one end of the mixing channel is communicated with the outlet, and the other end of the mixing channel is communicated with the output ends of the first water phase channel and the second water phase channel.
[0009] Optionally, a first inlet and a second inlet are further formed on the cover plate, the input end of the first water phase channel is communicated with the first inlet, and the input end of the second water phase channel is communicated with the second inlet.
[0010] Optionally, an oil phase inlet is further formed on the cover plate, and the input end of the oil phase channel is communicated with the oil phase inlet.
[0011] Optionally, the first inlet, the second inlet, the outlet and the oil phase inlet all penetrate the cover plate.
[0012] Optionally, the oil phase channel adopts a symmetrical closed loop structure, and the oil phase inlet, the outlet and the mixing channel are all distributed on the symmetry line of the oil phase channel.
[0013] Optionally, the first water phase channel, the second water phase channel and the mixing channel are distributed in a Y-shaped structure, and the first water phase channel and the second water phase channel are both located inside the closed loop structure of the oil phase channel.
[0014] Compared with the prior art, the utility model has the advantages that:
[0015] (1) In the utility model, the first water phase channel for injecting agrobacterium culture solution adopts an asymmetric sinusoidal structure channel, the channel can focus the agrobacterium cells, so that the agrobacterium cells can enter the next reaction channel in a regular arrangement state. The accumulation of this change along the channel can lead to new inertial migration behavior of the agrobacterium, compared with the straight channel, the number of aggregation positions and the required channel length can be effectively reduced, and the agrobacterium is focused into a single beam, so that the agrobacterium can be combined with soybean cells in a regular arrangement state, which lays a foundation for generating uniform agrobacterium-soybean cell group mixed liquid drops.
[0016] (2) Compared with the traditional infection method, the utility model directly generates a combination liquid drop of agrobacterium and soybean cells through the physical structure of the microfluidic chip, which is more conducive to the efficient infection of the agrobacterium. At the same time, since the multiple processing and transfer operations are reduced, the loss in the experimental process can be reduced, and the transformation efficiency is significantly improved. BRIEF DESCRIPTION OF DRAWINGS
[0017] Figure 1 is the structure schematic view of the inertial microfluidic chip for cross-scale packaging in the embodiment of the utility model;
[0018] Figure 2This is a schematic diagram showing the position and structure of the base plate and cover plate in an embodiment of this utility model;
[0019] Figure 3 This is a schematic diagram of the structure of the first aqueous phase channel in an embodiment of this utility model;
[0020] Figure 4 This is a schematic diagram of the liquid flow direction in each channel in an embodiment of this utility model;
[0021] Among them, 1. cover plate; 2. bottom plate; 3. first aqueous phase channel; 31. first inlet; 4. second aqueous phase channel; 41. second inlet; 5. mixing channel; 51. outlet; 6. oil phase channel; 61. oil phase inlet. Detailed Implementation
[0022] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. These drawings are simplified schematic diagrams, which are only used to illustrate the basic structure of the present invention in a schematic manner, and therefore only show the components related to the present invention.
[0023] Example 1, as Figure 1 and Figure 2 As shown, an inertial microfluidic chip for cross-scale packaging includes a cover plate 1 and a base plate 2. The bottom of the cover plate 1 has a first aqueous phase channel 3, a second aqueous phase channel 4, a mixing channel 5, and an oil phase channel 6. The base plate 2 is fixedly installed on the bottom of the cover plate 1 to seal the bottom surface of each channel.
[0024] As described above, the output ends of the first aqueous phase channel 3 and the second aqueous phase channel 4 are connected to the mixing channel 5. The mixing channel 5 passes through and connects to the output end of the oil phase channel 6. The first aqueous phase channel 3 adopts an asymmetric sinusoidal structure, and the oil phase channel 6 adopts a closed-loop structure.
[0025] In the experiment of Agrobacterium infection of soybeans, Agrobacterium culture medium was injected into the first aqueous channel 3, plant cell solution was injected into the second aqueous channel 4, and PDMS-silicone oil mixture (mass ratio of 1:1) was injected into the oil channel 6.
[0026] The first aqueous phase channel 3, used for injecting Agrobacterium culture medium, employs a special micro-asymmetric sinusoidal structure that focuses the incoming Agrobacterium cells, allowing them to enter the next reaction channel in a regularly arranged manner. This accumulation along the channel leads to a new inertial migration behavior in the Agrobacterium, effectively reducing the number of aggregation sites and the required channel length compared to a direct current channel. Simultaneously, it focuses the Agrobacterium into a single bundle, enabling it to merge with soybean cells in a regularly arranged manner, laying the foundation for the subsequent generation of a uniform Agrobacterium-soybean cell cluster droplet.
[0027] Compared with a traditional infection method, the physical structure of the microfluidic chip directly generates the combined droplet of the agrobacterium and the soybean cell, which is more conducive to efficient infection of the agrobacterium.
[0028] In order to facilitate injection of the solution into the channel and guide the generated target droplet out, the cover plate 1 is further provided with a first inlet 31, a second inlet 41, an oil phase inlet 61 and an outlet 51, and the first inlet 31, the second inlet 41, the oil phase inlet 61 and the outlet 51 all penetrate through the cover plate 1.
[0029] The input end of the first water phase channel 3 is in communication with the first inlet 31, the input end of the second water phase channel 4 is in communication with the second inlet 41, and the input end of the oil phase channel 6 is in communication with the oil phase inlet 61; the output ends of the first water phase channel 3 and the second water phase channel 4 are both in communication with the input end of the mixing channel 5 and meet at the same place, and the output end of the mixing channel 5 is in communication with the outlet 51.
[0030] The oil phase channel 6 adopts a symmetrical closed loop structure, such as a rectangle, a circle or the like, the mixing channel 5 penetrates through one side thereof, and the oil phase inlet 61, the outlet 51 and the mixing channel 5 are all distributed on the symmetry line of the oil phase channel 6. That is, the first water phase channel 3, the second water phase channel 4 and the mixing channel 5 are distributed in a Y-shaped structure, so as to ensure that the agrobacterium can orderly and efficiently contact the target plant cell; and the first water phase channel 3 and the second water phase channel 4 are both located inside the closed loop structure of the oil phase channel 6, so as to ensure that only the mixing channel 5 intersects with the oil phase channel 6, so as to facilitate shearing of the droplet.
[0031] Specifically, after the oil phase is injected into the oil phase channel 6 from the oil phase inlet 61, it is divided into two paths and synchronously moves to the output end, shears the mixed liquid in the mixing channel 5 into small droplets, and then moves to the outlet 51 together. In order to ensure liquid shear balance, the lengths of the oil phase channels 6 on both sides of the mixing channel 5 are equal, and the shapes are consistent.
[0032] Before the experiment starts, a mixed solution of PDMS and silicone oil with a mass ratio of 1:1 needs to be prepared as the oil phase solution. At the same time, agrobacterium cell culture solution and soybean cell culture solution are prepared as water phase solutions. Each solution is transferred to a different syringe, and they are connected to the corresponding inlets of the chip through a hose.
[0033] Firstly, the oil phase is injected into the chip through the micro pump, and after the oil phase is stably injected, the two water phase solutions, i.e. the soybean cell culture solution and the agrobacterium culture solution, are injected.
[0034] When injecting the aqueous phase, the flow rate of the syringe pump needs to be precisely adjusted to ensure that the aqueous phase forms stable droplets in the oil phase. Adjusting the flow rate also controls the droplet size, allowing selection of a droplet size that meets experimental requirements. Once the droplets have stably formed at the water-oil interface, flow rate control should continue to ensure the uniformity and stability of the droplets.
[0035] A collection tube is connected to the chip's outlet to guide the generated target droplets into a collection container, such as a petri dish or other suitable equipment. The droplets can then be cultured in the collection container for subsequent experimental procedures. Throughout this process, the flow rate, solution ratio, and chip structure all require precise control to ensure optimal droplet generation and Agrobacterium-soybean cell contact.
[0036] like Figure 1 and Figure 3 As shown, the Agrobacterium channel (i.e., the first aqueous phase channel 3) is an asymmetric sinusoidal channel, containing multiple "S"-shaped structures connected sequentially along a straight line (50 "S"-shaped structures are set here). Unlike traditional symmetric sinusoidal channels, asymmetric sinusoidal channels do not have symmetry along a certain axis or plane in shape. The peaks and troughs of its sinusoidal curves differ in position, amplitude, or period, which is manifested here as different heights of the peaks and troughs.
[0037] The asymmetric sinusoidal channel has an inner radius of R1 and an outer radius of R2 at its peak, and an inner radius of R3 and an outer radius of R4 at its trough. The channel's height is H. This channel is used to focus the incoming Agrobacterium cells, allowing them to enter the next part of the reaction channel in a regularly arranged manner.
[0038] Specifically, the first aqueous phase channel 3 has R1 of 0.02mm, R2 of 0.07mm, R3 of 0.11mm, R4 of 0.14mm, and a height H of 0.2mm. The diameters of the first inlet 31, the second inlet 41, the oil phase inlet 61, and the outlet 51 are all 4mm. The heights of the first aqueous phase channel 3, the second aqueous phase channel 4, the mixing channel 5, and the oil phase channel 6 are all 1.5mm, and the widths are all 1.2mm.
[0039] The first aqueous channel 3 adopts an asymmetric sinusoidal structure, while the other channels have ordinary cuboid structures. The cross-sectional area of the two types of channels is on the order of 10 cubed. Through this differentiated design, cross-scale encapsulation of Agrobacterium and plant cells is achieved.
[0040] The micro-fluidic chip is composed of two materials: a glass bottom plate 2 and a cover plate 1 containing a micro-channel structure, and PDMS material is adopted, wherein the design of the micro-channel is the core of the whole chip structure. In particular, a sinusoidal structure is adopted in the design of the agrobacterium injection channel. The chip front end is provided with three fluid inlet channels, which are respectively used for injecting oil phase, agrobacterium liquid and soybean cell group liquid; and the tail end is provided with a fluid outlet channel, which is used for collecting the liquid drops after the agrobacterium combines with the plant cells.
[0041] Operation flow:
[0042] First, the PDMS-silicon oil mixed liquid is injected through the oil phase inlet 61, and then the agrobacterium liquid and the soybean cell group liquid are injected through the first inlet 31 and the second inlet 41 respectively; the oil phase and the water phase form liquid drops under the shearing (as shown in Figure 4 ) effect in the channel, and then flow into the subsequent channel; in this process, the agrobacterium effectively reduces the number of aggregation points through the sinusoidal structure channel, ensures the uniform dispersion of the agrobacterium without damage; at the same time, the agrobacterium and the soybean cell group are wrapped in the liquid drops, creating a safe and stable environment, and significantly improving the infection efficiency of the agrobacterium.
[0043] As shown in Figure 1 , Figure 2 , Figure 3 and Figure 4 , the experimental principle of the cross-scale packaging inertial micro-fluidic chip in the process of the agrobacterium infection of soybeans is as follows.
[0044] (1) Injection setting of oil phase and water phase: in the experiment, the oil phase uses the PDMS-silicon oil mixed liquid with a mass ratio of 1:1, and the oil phase channel 6 is injected with the oil phase as the fluid medium through the oil phase inlet 61; the water phase has two types of agrobacterium culture liquid and soybean cell solution, the former is injected into the first water phase channel 3 through the first inlet 31, and the latter is injected into the second water phase channel 4 through the second inlet 41; the injection of the two liquids is controlled by flow distribution and flow rate, which ensures the effective distribution and mixing of different fluids in the micro-flow channel, and provides an ideal fluid environment for the subsequent experiment.
[0045] (2) Focusing agrobacterium cells: the first water phase channel 3 adopts an asymmetric sinusoidal structure with different curvature radii of the curved shape, which aims to focus the injected agrobacterium cells into a single beam flow through the principle of fluid mechanics. In this way, the agrobacterium cells can be arranged in order in the flow channel, and finally efficiently converge with the plant cells.
[0046] Due to the uneven distribution of cells at the entrance of the agrobacterium channel, the state of the cells is mixed, and such uneven distribution may cause part of the agrobacterium cells to fail to effectively contact the plant cells, thereby affecting the efficiency of gene transformation.
[0047] Therefore, it is essential to set up the asymmetric sinusoidal flow channel structure with focusing function before the agrobacterium cells and the soybean cells are combined. The structure can accurately arrange and concentrate the agrobacterium cells through the regulation of fluid mechanics, and ensure that the agrobacterium cells can orderly and efficiently contact the target plant cells, thereby significantly improving the success rate and efficiency of gene transformation.
[0048] (3) Forming agrobacterium-soybean cell group mixed liquid droplets: at the channel where the oil phase and the water phase are combined, the mixed liquid of the agrobacterium and the soybean cells is sheared into small droplets by using the fluid shearing principle. In this process, the interaction between the oil phase and the water phase creates a closed and stable environment, which further promotes the infection of the agrobacterium to the soybean cells.
[0049] Through the formation of the droplets, not only the contact efficiency of the agrobacterium and the soybean cells is improved, but also more ideal reaction conditions are provided for the subsequent gene transformation process, thereby maximizing the transformation effect and stability.
[0050] On the basis of example one, the application further provides a manufacturing method of the chip, comprising the following steps.
[0051] (1) 3D printing processing channel mold: due to the narrow and complex structure of the asymmetric sinusoidal channel, 3D printing processing is not suitable, and other channel molds can be completed by 3D printing processing.
[0052] (2) Channel mold processed by photolithography technology: as described above, the asymmetric sinusoidal channel is relatively narrow and is not suitable for 3D printing processing, so photolithography technology is needed to manufacture the channel mold, and the specific steps are as follows:
[0053] S1, film pasting: cut a piece of Dupont photosensitive dry film with a size similar to that of the glass block, remove the protective film on one side, paste it on the glass sheet after being wetted with water, and then use a plastic packaging machine to press it tightly. After the first layer of film is pasted, remove the protective layer on the surface and wet it with water, then paste the second layer of dry film. After the plastic packaging machine is pressed tightly, repeat the previous steps until the same thickness as the sinusoidal microchannel is obtained.
[0054] S2, exposure: tightly paste the side of the glass sheet with the dry film to the side of the mask with ink, and then place the mask side upward under the UV lamp for exposure.
[0055] S3, development: Dupont photosensitive dry film is a kind of photolithography negative glue. The exposed part is the required channel structure, and the unexposed part will be washed away by sodium carbonate solution later. After the dry film glass sheet is exposed, it is placed in a culture dish, sodium carbonate solution is poured into the culture dish, and the culture dish is shaken manually until the development is completed. Then, the surface moisture is gently washed with deionized water and dried with nitrogen. Finally, the microchannel mold structure is obtained by placing it in a drying oven.
[0056] (3) Process the PDMS cover sheet (i.e. cover plate 1) with channel structure:
[0057] (A) Silanization treatment: facilitate subsequent PDMS peeling from the template surface;
[0058] (B) PDMS pouring: match and fix the positions of the aforementioned two molds, then mix the PDMS and the PDMS curing agent uniformly according to the mass ratio, pour into the vessel containing the channel template, then put into the vacuum kettle to perform vacuum extraction until the bubbles in the PDMS completely disappear, finally take it out and place it in the oven for curing;
[0059] (C) PDMS cover sheet processing: after drying, remove the PDMS channel from the glass sheet, cut off the excess part, and punch holes at the inlet and outlet of the channel using a punch, to obtain a PDMS cover sheet with channel structure.
[0060] (4) Hydrophobic treatment of the channel: Since the chip in this experiment involves oil phase injection, the microchannel needs to be hydrophobic before the experiment. First, inject glass anti-fog agent into the microchannel and soak for a certain period of time. Then, clean the channel with acetone and place it in a constant temperature drying oven for drying, finally obtain a hydrophobic microchannel.
[0061] (5) Bonding of the PDMS cover sheet and the glass bottom sheet:
[0062] (a) To ensure cleanliness and flatness, first clean the glass bottom sheet with deionized water several times, then use adhesive tape to adsorb and clean the dust on the surface of the PDMS several times;
[0063] (b) Add a small amount of deionized water on the glass bottom sheet to facilitate the movement of the PDMS on the glass surface and alignment;
[0064] (c) Place the side of the PDMS with channels on the wet glass surface, observe under a microscope and ensure that the channels are aligned according to the predetermined structure. Let it stand for a few minutes to ensure that the two are well bonded;
[0065] (d) Finally, transfer the bonded structure to a hot plate and gently press it with a weight on the surface, heat it at 80°C for 1-2 hours to obtain a bonded microfluidic chip.
[0066] In summary, the utility model discloses in order to fill the technical vacancy of high-efficiency realization agrobacterium infection plant cell in prior art, design a kind of inertia microfluidic chip based on sinusoidal channel structure, for the method for agrobacterium infection soybean cell, simplify operation and improve the efficiency of infection, reduce equipment cost.
[0067] In the description of the utility model, it is necessary to understand that the orientation or positional relation indicated by the terms "center", "longitudinal", "transverse", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like is the orientation or positional relation based on the orientation or positional relation shown in the drawings, which is only for the convenience of describing the utility model and simplifying the description, and does not indicate or imply that the device or element indicated must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the utility model. In addition, the terms "first", "second" and the like are only for the purpose of description, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features limited by "first", "second" and the like can explicitly or implicitly include one or more features. In the description of the utility model, unless otherwise specified, the meaning of "a plurality of" is two or more.
[0068] In the description of the utility model, it should be explained that, unless otherwise specified and limited, the terms "mounting", "connection", "connection" should be understood broadly, for example, it can be fixed connection, can also be detachable connection, or integrally connected, can be mechanical connection, can also be electrical connection, can be directly connected, can also be indirectly connected through an intermediate medium, can be the communication inside two elements. For ordinary skilled in the art, the specific meaning of the above terms in the utility model can be understood through specific circumstances.
[0069] According to the ideal embodiments of the utility model, the above description, relevant personnel can make various changes and modifications without deviating from the technical idea of the utility model. The technical scope of the utility model is not limited to the contents in the specification, and the technical scope must be determined according to the scope of claims.
Claims
1. An inertial microfluidic chip for cross-scale packaging, characterized by: The first water phase channel (3), the second water phase channel (4), the mixing channel (5) and the oil phase channel (6) are arranged on the cover plate (1), and the bottom of the cover plate (1) is provided with the bottom plate (2).
2. The inertial microfluidic chip for cross-scale packaging according to claim 1, characterized in that: The cover plate (1) is provided with an outlet (51), one end of the mixing channel (5) is communicated with the outlet (51), and the other end is communicated with the output ends of the first water phase channel (3) and the second water phase channel (4).
3. The inertial microfluidic chip for cross-scale packaging according to claim 2, characterized in that: The cover plate (1) is provided with a first inlet (31) and a second inlet (41), the input end of the first water phase channel (3) is communicated with the first inlet (31), and the input end of the second water phase channel (4) is communicated with the second inlet (41).
4. The inertial microfluidic chip for cross-scale packaging according to claim 3, characterized in that: The cover plate (1) is provided with an oil phase inlet (61), and the input end of the oil phase channel (6) is communicated with the oil phase inlet (61).
5. The inertial microfluidic chip for cross-scale packaging according to claim 4, characterized in that: The first inlet (31), the second inlet (41), the outlet (51) and the oil phase inlet (61) all penetrate the cover plate (1).
6. The inertial microfluidic chip for cross-scale packaging according to claim 5, characterized in that: The oil phase channel (6) adopts a symmetrical closed loop structure, and the oil phase inlet (61), the outlet (51) and the mixing channel (5) are all distributed on the symmetry line of the oil phase channel (6).
7. The inertial microfluidic chip for cross-scale packaging according to claim 5, wherein: The first water phase channel (3), the second water phase channel (4) and the mixing channel (5) are distributed in a Y-shaped structure, and the first water phase channel (3) and the second water phase channel (4) are both located inside the closed loop structure of the oil phase channel (6).
8. The inertial microfluidic chip for cross-scale packaging according to claim 7, characterized in that: