A detachable microfluidic chip and its application method

Abstract

The present invention discloses a detachable microfluidic chip and an application method thereof, belonging to the technical field of microfluidic chips. It includes a cover plate, an intermediate layer and a limiting base arranged from top to bottom; the limiting base is provided with a groove, the intermediate layer is located in the groove, the cover plate is covered on the intermediate layer, and is detachably connected to the limiting base; the intermediate layer includes a transparent cover plate, a flow channel layer one, a flow channel layer two, a flow channel layer three and a transparent bottom plate placed from top to bottom; the microfluidic chip of the present invention has a layered template structure, which is easy to replace, convenient to clean the flow channel and reuse. By replacing the flow channel layer with different flow channel patterns, different concentration gradients can be generated after assembly, eliminating the complicated manufacturing process. The flow channel layer is provided with narrow and thin flow channels, and the cytokines in the solution can diffuse into the culture chamber through the narrow and thin flow channels to form a concentration gradient in the culture chamber; the influence of shear force on cell growth can be avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of microfluidic chips, and in particular relates to a detachable microfluidic chip and an application method thereof. Background Art

[0002] In the human body, the concentration gradient of biomolecules controls many basic cell functions. For example, biological processes such as development, immune response, wound healing, embryogenesis and cancer metastasis all rely on biomolecule gradients to increase and regulate cell signaling pathways. In order to understand the effects of chemical stimulation on cell signaling pathways, biological researchers seek methods that can simulate the in vivo cell microenvironment in vitro. In biological research, it is usually necessary to prepare samples and reagent solutions with concentration gradients to examine the effects of reagents of different concentrations on experimental subjects. For example, in cell differentiation experiments, different cytokine concentrations have a large difference in cell development. Therefore, when examining the effects of molecular concentrations on cell development, it is necessary to prepare a series of cytokines with concentration gradients. Microfluidic chip technology plays an important role in the field of cell analysis. By making two fluids with concentration differences repeat the process of diversion-mixing-diversion in a microchannel with a special structure, solutions of different concentrations are finally dispersed into multiple branch channels to form a series of solutions with concentration gradients. In order to study the effects of biochemical concentration gradients on cell functions, various microfluidic concentration gradient chips have been developed and applied. However, most of the current microfluidic chips are made by bonding polydimethylsiloxane (PDMS) and glass, and the bonding between PDMS and glass is irreversible. This structure makes it difficult to clean the internal channels, and a chip can only form a fixed concentration gradient. If different concentration gradients need to be generated, it is necessary to prepare microfluidic chips with different flow channels. The traditional production process includes the production of photoresist molds on silicon wafers (cleaning silicon wafers, coating, pre-baking, exposure, mid-baking, developing, and hardening molds), configuring PDMS mixed glue, making inverted mold containers, pouring molds, heating and curing, demolding, cutting, punching, bonding and packaging, and a series of steps, and the process is complicated. For this reason, it is necessary to design a detachable microfluidic chip. When different concentration gradients need to be generated, only the flow channel layer with different flow channel styles needs to be replaced. After assembly, different concentration gradients can be generated, thereby improving the practicality and flexibility of use of the microfluidic chip.

[0003] Chinese patent CN206502830U discloses a solution gradient generation and cell culture microfluidic chip, including a gradient generation layer and a cell culture layer stacked up and down, a concentration gradient generation microchannel is formed in the gradient generation layer, and a plurality of cell culture chambers are formed in the cell culture layer, and each of the cell culture chambers is connected to a concentration gradient outlet pool through a vertical channel. The utility model places the concentration gradient generator and the cell culture area in different planes, so that when the cells are sampled, the cells will not enter the concentration gradient generator channel, avoid blocking the microchannel in the concentration gradient generator, and thus ensure that a stable concentration gradient solution can be generated. However, the concentration gradient solution of the microfluidic chip flows directly into the culture chamber, and the shear force generated by the solution convection will directly act on the cells, causing greater damage to the cells, which is not conducive to cell growth; and the chip can only form a fixed concentration gradient. On the other hand, the gradient generation layer and the cell culture layer in the microfluidic chip do not overlap, and the cell culture layer also includes an inlet, a branched microchannel and an outlet, which occupies a large area, resulting in a large volume of the chip. Summary of the invention

[0004] In view of the deficiencies of the above prior art, the present invention provides a detachable microfluidic chip, which has a multi-layer flow channel layer, which is convenient for changing the flow channel configuration to form different concentration gradients. When different concentration gradients need to be generated, it is only necessary to replace the flow channel layer with different flow channel patterns, and different concentration gradients can be obtained after reassembly. In addition, the microfluidic chip of the present invention can not only generate a stable concentration gradient, but also avoid the influence of the shear force generated during solution convection on cell growth; in addition, the microfluidic chip of the present invention can generate concentration gradients in two directions at the same time, which is convenient for observing the growth of cells under the superposition of the concentration gradients of two solutions at the same time.

[0005] In order to achieve the above-mentioned invention object, the following technologies are specifically used:

[0006] A detachable microfluidic chip comprises a cover plate, an intermediate layer and a limiting base arranged from top to bottom, wherein a groove is provided at the center of the limiting base, and the intermediate layer is located in the groove; the groove is used to place the intermediate layer and limit the intermediate layer; the cover plate is covered on the intermediate layer and is detachably connected to the limiting base.

[0007] The middle layer is composed of a transparent cover plate, a flow channel layer 1, a flow channel layer 2, a flow channel layer 3 and a transparent bottom plate placed from top to bottom, and the transparent cover plate is provided with a first liquid inlet, a culture cavity and a first liquid outlet; the flow channel layer 1 is provided with a first liquid inlet, a first transverse flow channel, a second transverse flow channel, a third transverse flow channel, a culture cavity and a first liquid outlet; the flow channel layer 2 is provided with a first through hole, a second through hole, a third through hole, a second culture cavity, a narrow flow channel and a first liquid outlet; the flow channel layer 3 is provided with a first curved flow channel, a second curved flow channel, a third curved flow channel, a culture cavity and a first liquid outlet.

[0008] Preferably, the first transverse flow channel is connected to the first curved flow channel through a first through hole; the second transverse flow channel is connected to the first curved flow channel and the second curved flow channel through a second through hole; the third transverse flow channel is connected to the second curved flow channel and the third curved flow channel through a third through hole. The third curved flow channel is connected to the culture chamber through the narrow flow channel.

[0009] Preferably, circular through holes are provided at positions corresponding to the first transverse flow channel, the second transverse flow channel, the third transverse flow channel, the first curved flow channel, the second curved flow channel, the third curved flow channel and the first through hole, the second through hole, and the third through hole.

[0010] Preferably, the groove is square, the side length of which is greater than the side length of the middle layer, and the depth of the groove is less than the thickness of the middle layer. More preferably, the side length of the groove is equal to the side length of the middle layer.

[0011] Preferably, the transparent cover plate, flow channel layer one, flow channel layer two, flow channel layer three and transparent bottom plate are squares with equal sides, and when placed on the square groove, they are positioned by the first liquid outlet provided on the transparent cover plate, flow channel layer one, flow channel layer two, flow channel layer three and transparent bottom plate.

[0012] Preferably, the side lengths of the transparent cover plate, the flow channel layer 1, the flow channel layer 2, the flow channel layer 3 and the transparent bottom plate are 35-40 mm.

[0013] Preferably, light-transmitting holes are provided at the center positions of the cover plate and the limiting base, so as to facilitate direct observation of the growth status of cells under a microscope; the light-transmitting hole is square or circular, and if the light-transmitting hole is square, the side length of the square is smaller than the side length of the middle layer; if the light-transmitting hole is circular, the diameter of the circle is smaller than the side length of the middle layer.

[0014] Preferably, the thickness of the transparent cover plate is 1.0-2.0 mm, the thickness of the transparent bottom plate is 0.2-0.5 mm, the thickness of the flow channel layer 1 and the flow channel layer 3 is 0.1-0.3 mm, and the thickness of the flow channel layer 2 is 0.1-0.5 mm.

[0015] The cover plate and the limiting base are made of metal; the transparent cover plate and the transparent bottom plate are made of transparent glass or transparent acrylic plate; the flow channel layer one and the flow channel layer three are made of silicone film; the flow channel layer two is made of one of silicone film, transparent glass or transparent acrylic plate.

[0016] Preferably, the cover plate and the limiting base have the same side length, and the cover plate and the limiting base are connected by threaded hole bolts.

[0017] Preferably, a vertical concentration gradient generating module is provided above the culture chamber, for generating a longitudinal concentration gradient perpendicular to the plane direction of the microfluidic chip in the culture chamber.

[0018] More preferably, the vertical concentration gradient generating module is made of transparent acrylic material, which includes a liquid tank, a concentration gradient generating component, a second liquid inlet, a second liquid outlet, and a top cover; the shape and size of the concentration gradient generating component are the same as those of the culture chamber, and a narrow through hole is provided thereon, and the diameter of the narrow through hole is 0.2-0.4 mm.

[0019] The present invention also provides an application method of the detachable microfluidic chip, wherein the microfluidic chip is used to study the response of cells to a cytokine concentration gradient, and specifically comprises the following steps:

[0020] S1. Inject solutions of different concentrations into the two first liquid inlets and continue to perfuse at the same flow rate;

[0021] S2. Prepare gel and evenly encapsulate cells inside the three-dimensional gel;

[0022] S3. Observe the movement of the solution in the flow channel under a microscope. When the flow rate reaches a stable level, start injecting the gel into the culture chamber so that the gel adheres to the bottom of the culture chamber.

[0023] S4. Continuously passing liquid to the first liquid inlet;

[0024] S5. Observe the growth of cells under the action of concentration gradient under a microscope;

[0025] S6. Injecting fluorescent dye into the microfluidic chip to stain the target protein in the cell;

[0026] S7. Observe the protein expression of cells under concentration gradient.

[0027] Preferably, a method for applying a detachable microfluidic chip further comprises the following steps after step S4: embedding a vertical concentration gradient generating module into the culture chamber, continuously passing liquid to the second liquid inlet at a uniform speed, and continuously drawing the solution out from the second liquid outlet.

[0028] Compared with the prior art, the present invention is beneficial in that:

[0029] (1) The detachable microfluidic chip of the present invention can generate concentration gradients and has a layered template structure, which is easy to replace and convenient to clean the flow channel. The preparation process is simple, and only needs to make the flow channel layer by laser punching according to the drawing and then assemble it. By replacing the flow channel layer with different flow channel patterns, different concentration gradients can be generated after assembly, avoiding the need to prepare a new microfluidic chip to generate different concentration gradients, thereby eliminating the complicated production process.

[0030] (2) A culture chamber is provided on the middle layer, which enables cells to be cultured directly on the chip; and the culture chamber is connected to the external environment, which can effectively ensure the environment required for cell growth.

[0031] (3) The second flow channel layer is provided with narrow flow channels, through which the cytokines in the solution can diffuse into the culture chamber, forming a concentration gradient in the culture chamber; this can avoid the influence of the shear force generated by the convection of the solution on the cell growth when the solution directly flows into the culture chamber.

[0032] (4) Wrapping cells with gel can limit the activity area of ​​cells and prevent cells from entering the flow channel during the culture process, causing cells to block the channel and affect the experimental results. Gel is a porous material with very small pores, which allows cytokines to diffuse freely in the gel to generate a concentration gradient in a single culture chamber. It can also effectively reduce the effect of shear force on cell growth.

[0033] (5) The microfluidic chip of the present invention can generate a concentration gradient not only between culture chambers, but also within a single culture chamber.

[0034] (6) The flow channel layers one to three generate a concentration gradient in the horizontal direction, and the vertical concentration gradient generating module generates a concentration gradient in a direction perpendicular to the plane of the microfluidic chip; that is, the microfluidic chip of the present invention can generate concentration gradients in two directions, which is convenient for observing the growth of cells under the concentration gradients of two solutions at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a schematic structural diagram of a detachable microfluidic chip of the present invention;

[0036] Figure 2 It is a structural schematic diagram of the cover plate and the limiting base;

[0037] Figure 3 It is a schematic diagram of the structure of the middle layer;

[0038] Figure 4 This is an exploded view of the middle layer;

[0039] Figure 5is a schematic diagram of the structure of a transparent cover;

[0040] Figure 6 Schematic diagram of the structure of the flow channel layer 1;

[0041] Figure 7 It is a schematic diagram of the structure of the flow channel layer 2;

[0042] Figure 8 Schematic diagram of the structure of the flow channel layer three;

[0043] Fig. 9 It is a schematic diagram of the structure in which the flow channel layer 3 and the flow channel layer 2 are stacked from bottom to top;

[0044] Fig.10 It is a schematic diagram of the structure of a detachable microfluidic chip equipped with a vertical concentration gradient generating module;

[0045] Fig.11 It is a structural schematic diagram of the vertical concentration gradient generation module;

[0046] Fig.12 Schematic diagram of the structure of the top cover of the vertical concentration gradient generating module.

[0047] Wherein: 1-cover plate, 2-middle layer, 3-limiting base, 4-groove, 5-transparent cover plate, 6-flow channel layer one, 7-flow channel layer two, 8-flow channel layer three, 9-transparent bottom plate, 10-first liquid inlet, 11-culture chamber, 12-first liquid outlet, 13-first lateral flow channel, 14-second lateral flow channel, 15-third lateral flow channel, 16-first through hole, 17-second through hole, 18-third through hole, 19-narrow flow channel, 20-second culture chamber, 21-first curved flow channel, 22-second curved flow channel, 23-third curved flow channel, 24-light-transmitting hole, 25-threaded hole, 26-vertical concentration gradient generating module, 27-liquid tank, 28-concentration gradient generating component, 29-second liquid inlet, 30-second liquid outlet, 31-narrow through hole, 32-top cover. DETAILED DESCRIPTION

[0048] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] In the description of the present invention, it should be noted that the directions or positional relationships indicated by the terms "middle", "upper", "lower", "left", "right", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", "third", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0050] The application principle of the present invention is described in detail below in conjunction with the accompanying drawings.

[0051] Example 1

[0052] like Figure 1 As shown, this embodiment provides a detachable microfluidic chip, which includes a cover plate 1, an intermediate layer 2, and a limiting base 3 arranged from top to bottom.

[0053] like Figure 2 As shown, a groove 4 is provided at the center of the limiting base 3, and the function of the groove 4 is to place the middle layer 2 and limit the middle layer 2; the groove 4 is square, and the side length is equal to the side length of the middle layer 2, and the depth is less than the thickness of the middle layer 2; the cover plate 1 is sealed and attached to the middle layer 2, so as to achieve a sealed connection of the middle layer; the cover plate 1 and the limiting base 3 are detachably connected. Figure 3-4 As shown, the middle layer 2 is composed of a transparent cover plate 5, a flow channel layer 1 6, a flow channel layer 2 7, a flow channel layer 3 8 and a transparent bottom plate 9 placed from top to bottom; the transparent cover plate 5, the flow channel layer 1 6, the flow channel layer 2 7, the flow channel layer 3 8 and the transparent bottom plate 9 are all squares with a side length of 35-40 mm; Figure 5 As shown, the transparent cover plate 5 is provided with a first liquid inlet 10, a culture chamber 11 and a first liquid outlet 12. Figure 6 As shown, the flow channel layer 16 is provided with a first liquid inlet 10, a first lateral flow channel 13, a second lateral flow channel 14, a third lateral flow channel 15, a culture chamber 11 and a first liquid outlet 12. Figure 7 As shown, the flow channel layer 2 7 is provided with a first through hole 16, a second through hole 17, a third through hole 18, a second culture chamber 20, a narrow flow channel 19 and a first liquid outlet 12. Figure 8 As shown, the flow channel layer 3 8 is provided with a first curved flow channel 21, a second curved flow channel 22, a third curved flow channel 23, a culture chamber 11 and a first liquid outlet 12. Figure 4In the order shown, the transparent bottom plate 9, the flow channel layer 3 8, the flow channel layer 2 7, the flow channel layer 1 6 and the transparent cover plate 5 are placed in the groove 4 of the limiting base 3 from bottom to top, and positioned through the first liquid outlet 12. At this time, the first transverse flow channel 13 of the flow channel layer 1 6, the first through hole 16 of the flow channel layer 2 7 and the inlet at the upper part of the first curved flow channel 21 of the flow channel layer 3 8 just overlap in the horizontal direction; that is, the first transverse flow channel 13 of the flow channel layer 1 6 and the inlet at the upper part of the first curved flow channel 21 of the flow channel layer 3 8 are connected through the first through hole 16 of the flow channel layer 2 7. The straight lines formed by the second transverse flow channel 14 of flow channel layer 1 6, the second through hole 17 of flow channel layer 2 7, the outlet at the bottom of the first curved flow channel 21 of flow channel layer 3, and the inlet at the top of the second curved flow channel 22 just overlap in the horizontal direction; that is, the second transverse flow channel 14 of flow channel layer 1 6 is connected with the outlet at the bottom of the first curved flow channel 21 of flow channel layer 3 and the inlet at the top of the second curved flow channel 22 through the second through hole 17 of flow channel layer 2 7. The straight lines formed by the third transverse flow channel 15 of flow channel layer 1 6, the third through hole 18 of flow channel layer 2 7, the outlet at the bottom of the second curved flow channel 22 of flow channel layer 3, and the inlet at the top of the third curved flow channel 23 just overlap in the horizontal direction. That is, the third transverse flow channel 15 of flow channel layer 1 6 is connected with the outlet at the bottom of the second curved flow channel 22 of flow channel layer 3 and the inlet at the top of the third curved flow channel 23 through the third through hole 18 of flow channel layer 2 7. In summary, the first transverse flow channel 13 is connected to the first curved flow channel 21 through the first through hole 16; the second transverse flow channel 14 is connected to the first curved flow channel 21 and the second curved flow channel 22 through the second through hole 17; the third transverse flow channel 15 is connected to the second curved flow channel 22 and the third curved flow channel 23 through the third through hole 18. The third curved flow channel 23 is connected to the culture chamber 11 through the narrow flow channel 19. Fig. 9 As shown, the circular through holes at both ends of the narrow flow channel 19 on the flow channel layer 7 are connected to the culture chamber 11, and half of the circular through holes connecting the culture chamber 11 are located inside the culture chamber 11, and the other half are located outside the culture chamber 11, which can simultaneously ensure the maximization of the cell culture area in the culture chamber 11 and the maximization of the connection area between the through hole and the culture chamber 11; the circular through hole in the middle of the narrow flow channel 19 is connected to the longitudinal flow channel portion of the third curved flow channel 23; that is, the narrow flow channel 19 connects the culture chamber 11 and the longitudinal flow channel portion of the third curved flow channel 23.

[0054] Furthermore, circular through holes are provided at positions corresponding to the first lateral flow channel 13, the second lateral flow channel 14, the third lateral flow channel 15, the first curved flow channel 21, the second curved flow channel 22, the third curved flow channel 23 and the first through hole 16, the second through hole 17, the third through hole 18, so that closed flow channels can be formed between the flow channel layers to prevent the solution from leaking from the flow channels.

[0055] Furthermore, in this embodiment, a square light-transmitting hole 24 is provided at the center position of the cover plate 1 and the limiting base 3. In this embodiment, the side length of the light-transmitting hole is 32 mm, which is smaller than the side length of the groove 4. The first liquid inlet 10 and the culture chamber 11 can be seen in the light-transmitting hole 24, which is convenient for directly observing the growth status of cells under a microscope.

[0056] Furthermore, the shape of the light-transmitting hole is not limited to a square, but may also be a circle, as long as the movement of the solution in the flow channel layer and the growth of the cells in the culture chamber can be seen through the light-transmitting hole.

[0057] Furthermore, the thickness of the transparent cover plate 5 of this embodiment is 1.0-2.0 mm, the thickness of the transparent bottom plate 9 is 0.2-0.5 mm, the thickness of the flow channel layer 1 6 and the flow channel layer 3 8 is 0.1-0.3 mm, and the thickness of the flow channel layer 2 7 is 0.1-0.5 mm.

[0058] The cover plate 1 and the limiting base 3 of the present embodiment are made of stainless steel; the transparent cover plate 5 and the transparent bottom plate 9 are made of transparent glass; the flow channel layer 1 6 and the flow channel layer 3 8 are made of silicone film, and the flow channel 2 7 can be made of materials such as silicone film, transparent glass and transparent acrylic plate. The material of the flow channel layer 2 7 of the present embodiment is silicone film.

[0059] Furthermore, in order to achieve a detachable connection between the cover plate 1 and the limiting base 3 , the cover plate 1 and the limiting base 3 of this embodiment are connected by bolts via threaded holes 25 .

[0060] When in use, a solution containing cytokines of different concentrations is injected from the first liquid inlet 10, and the solution flows into the flow channel layer 1 6 through the first liquid inlet 10 of the transparent cover plate 5, passes through the first transverse flow channel 13 on the flow channel layer 1 6, and flows into the first curved flow channel 21 of the flow channel layer 3 8 through the first through hole 16 on the flow channel layer 2 7, and is decelerated through the first curved flow channel 21, and then flows into the second transverse flow channel 14 on the flow channel layer 1 6 through the second, fourth, and sixth through holes in the second through hole 17 on the flow channel layer 2 7, and then flows into the second transverse flow channel 14 on the flow channel layer 1 6 through the first, third, fifth, and seventh through holes in the second through hole 17 on the flow channel layer 2 7. It flows into the second curved flow channel 22 of the flow channel layer three 8, is decelerated through the second curved flow channel 22, and then flows into the third transverse flow channel 15 on the flow channel layer one 6 through the 2nd, 4th, 6th and 8th through holes in the third through holes 18 on the flow channel layer two 7, and then flows into the third curved flow channel 23 of the flow channel layer three 8 through the 1st, 3rd, 5th, 7th and 9th through holes in the third through holes 18 on the flow channel layer two 7; when passing through the longitudinal flow channel of the third curved flow channel 23, the cytokines in the culture medium diffuse into the culture chamber 11 through the narrow flow channel 19 on the flow channel layer two 7, forming a concentration gradient in the culture chamber 11. Because the fluid repeatedly passes through the channel layer 1 6, the channel layer 2 7 and the channel layer 3 8, and the first curved channel 21, the second curved channel 22 and the third curved channel 23 on the channel layer 3 8 will slow down the fluid, when the fluid finally flows through the longitudinal channel of the third curved channel 23, the flow rate of the fluid is already very slow, and the longitudinal channel part of the third curved channel 23 is connected to the culture chamber 11 through the narrow channel 19, so the cytokines in the fluid can freely diffuse into the culture chamber 11 through the narrow channel 19, and generate a concentration gradient in the culture chamber 11; the cytokines enter the culture chamber 11 through free diffusion, which can reduce the effect of the shear force generated during fluid convection on cell growth. Finally, the waste liquid of the middle layer 2 flows through the longitudinal channel in the third curved channel 23 and then flows out of the middle layer 2 through the first liquid outlet 12.

[0061] Furthermore, the channel layer 1 6, the channel layer 2 7 and the channel layer 3 8 of the present invention may be other channel patterns in addition to the channel patterns in the present embodiment. By replacing the channel layers, a microfluidic chip with different channel patterns can be obtained, and the flow pattern of the fluid can be flexibly changed to produce different concentration gradients. The operation is simple, which improves the practicality of the microfluidic chip.

[0062] This embodiment also provides a method for using the above-mentioned detachable microfluidic chip to study the effect of concentration gradient on cell growth. The detachable microfluidic chip can generate a stable concentration gradient and avoid the adverse effect of shear force on cell growth, thereby being used to study the response of cells to cytokine concentration gradient. Specifically, the method comprises the following steps:

[0063] S1. Use a syringe pump to inject interleukin with a concentration of 0 mol / m into the first liquid inlet 10 on the left. 3 A solution with an interleukin concentration of 0.1 mol / m is injected into the first liquid inlet 10 on the right side. 3 Solution, and keep the same flow rate for continuous perfusion. S2. Prepare dextran hydrogel, and evenly wrap the cells inside the gel; S3. Observe the movement of the solution in the flow channel under a microscope, and after the concentration gradient appears in the longitudinal flow channel part of the curved flow channel 23, start injecting gel into the culture chamber 11, so that the gel fits tightly with the bottom of the culture chamber 11, and the gel reaches 1 / 4 of the narrow flow channel 19; S4. Continue to pass liquid to the first liquid inlet 10; S5. Observe the growth of cells under the concentration gradient under a microscope; S6. Inject fluorescent dye into the microfluidic chip to dye the target protein in the cells; S7. Observe the protein expression of cells under the concentration gradient.

[0064] The concentration gradients in the culture chamber 11 from left to right are: 0-0.025 mol / m 3 ; 0.025-0.05mol / m 3 ; 0.05-0.075mol / m 3 ; 0.075-0.1mol / m 3 Because the culture chamber contains gel, which creates flow resistance and prevents mixing of solutions, a concentration gradient can be generated in a single culture chamber, allowing the study of 0-0.1 mol / m 3 Effects of concentrations on cell growth throughout the range.

[0065] Example 2

[0066] The difference between this embodiment and embodiment 1 is that a vertical concentration gradient generating module 26 is provided on the culture chamber 11 of this embodiment, which is used to generate a longitudinal concentration gradient perpendicular to the plane direction of the microfluidic chip in the culture chamber 11. Fig.10 As shown, the vertical concentration gradient generating module 26 is embedded in the culture chamber 11; Figure 11-12As shown, the vertical concentration gradient generating module 26 includes a liquid tank 27, a concentration gradient generating component 28, a second liquid inlet 29, a second liquid outlet 30 and a top cover 32; the shape and size of the concentration gradient generating component 28 are the same as those of the culture chamber 11, and a narrow through hole 31 is provided thereon, and the diameter of the narrow through hole 31 is 0.2-0.4mm. When in use, a solution containing cytokines is injected from the second liquid inlet 29, and a certain flow rate is maintained for continuous injection, and the solution can freely diffuse into the culture chamber 11 through the narrow through hole 31, and form a concentration gradient in a direction perpendicular to the plane of the microfluidic chip. Since the diameter of the narrow through hole 31 is very small, only 0.2-0.4mm, and the flow resistance is very large, the solution in the liquid tank 27 enters the culture chamber 11 by free diffusion. The gel contained in the culture chamber 11 can prevent the mixing of the solution in the culture chamber 11, and finally form a concentration gradient in a direction perpendicular to the plane of the microfluidic chip; finally, the cytokines diffuse to the liquid outlet 12 through the narrow channel 19.

[0067] This embodiment also provides a method for using the above-mentioned detachable microfluidic chip to study the effect of concentration gradient on cell growth. Studying the growth of cells under the concentration gradient of two solutions specifically includes the following steps:

[0068] S1. Use a syringe pump to inject interleukin with a concentration of 0 mol / m into the first liquid inlet 10 on the left. 3 A solution with an interleukin concentration of 0.1 mol / m is injected into the first liquid inlet 10 on the right side. 3 Solution, and maintain the same flow rate for continuous perfusion. S2. Prepare dextran hydrogel and evenly wrap the cells inside the gel; S3. Observe the movement of the solution in the flow channel under a microscope. After a concentration gradient appears in the longitudinal flow channel portion of the curved flow channel 23, start injecting gel into the culture chamber 11 so that the gel fits tightly to the bottom of the culture chamber 11, and the gel reaches 1 / 4 of the narrow flow channel 19; S4. Continue to pass liquid to the first liquid inlet 10; S5. Embed the vertical concentration gradient generating module 26 into the culture chamber 11, and inject interferon with a concentration of 0.1 mol / m 3 solution and keep injecting it continuously at a certain flow rate; S6. Observe the growth of cells under the concentration gradient under a microscope; S7. Inject fluorescent dye into the microfluidic chip to stain the target protein in the cells; S8. Observe the protein expression of cells under the concentration gradient.

[0069] After the solution is injected from the second liquid inlet 19, the solution can diffuse freely into the culture chamber 11 through the narrow through hole 31. Because the culture chamber 11 contains gel, it can cause flow resistance and prevent the mixing of the solution. After 10-15 minutes of diffusion, a stable concentration gradient can be generated in the direction perpendicular to the plane of the microfluidic chip. The concentration gradient of the area directly below the narrow through hole 31 in the culture chamber 11 is 0.1-0.07 mol / m from top to bottom. 3 After determining the target protein and its specific location in the culture chamber, the COMSOL multi-physics simulation software can be used to accurately simulate the concentration of the target protein in the culture chamber; and study the growth of cells under different concentration gradients.

[0070] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A detachable microfluidic chip, It is characterized in that It comprises a cover plate, an intermediate layer and a limiting base arranged from top to bottom; the limiting base is provided with a groove, the intermediate layer is located in the groove, the cover plate covers the intermediate layer and is detachably connected to the limiting base; The middle layer comprises a transparent cover plate, a flow channel layer 1, a flow channel layer 2, a flow channel layer 3 and a transparent bottom plate placed from top to bottom; the transparent cover plate is provided with a first liquid inlet, a culture cavity and a first liquid outlet; the flow channel layer 1 is provided with a first liquid inlet, a first transverse flow channel, a second transverse flow channel, a third transverse flow channel, a culture cavity and a first liquid outlet; the flow channel layer 2 is provided with a first through hole, a second through hole, a third through hole, a second culture cavity, a narrow flow channel and a first liquid outlet; the flow channel layer 3 is provided with a first curved flow channel, a second curved flow channel, a third curved flow channel, a culture cavity and a first liquid outlet; The first transverse flow channel is connected to the first curved flow channel through a first through hole; the second transverse flow channel is connected to the first curved flow channel and the second curved flow channel through a second through hole; the third transverse flow channel is connected to the second curved flow channel and the third curved flow channel through a third through hole; the third curved flow channel and the culture chamber are connected through the narrow flow channel; Circular through holes are provided at positions corresponding to the first transverse flow channel, the second transverse flow channel, the third transverse flow channel, the first curved flow channel, the second curved flow channel, the third curved flow channel and the first through hole, the second through hole, and the third through hole.

2. A detachable microfluidic chip according to claim 1, It is characterized in that The side length of the groove is equal to the side length of the middle layer, and the depth of the groove is less than the thickness of the middle layer.

3. The detachable microfluidic chip according to claim 1, It is characterized in that The transparent cover plate, flow channel layer 1, flow channel layer 2, flow channel layer 3 and transparent bottom plate are squares with equal sides, and the side length is 35-40 mm.

4. The detachable microfluidic chip according to claim 1, It is characterized in that The thickness of the transparent cover plate is 1.0-2.0 mm, the thickness of the transparent bottom plate is 0.2-0.5 mm, the thickness of the flow channel layer 1 and the flow channel layer 3 is 0.1-0.3 mm, and the thickness of the flow channel layer 2 is 0.1-0.5 mm; The cover plate and the limiting base are made of metal; the transparent cover plate and the transparent bottom plate are made of transparent glass or transparent acrylic plate; the flow channel layer one and the flow channel layer three are made of silicone film; the flow channel layer two is made of one of silicone film, transparent glass or transparent acrylic plate.

5. The detachable microfluidic chip according to claim 1, It is characterized in that A vertical concentration gradient generating module is provided above the culture chamber, which is used to generate a longitudinal concentration gradient perpendicular to the plane direction of the microfluidic chip in the culture chamber; The vertical concentration gradient generating module includes a liquid tank, a concentration gradient generating component, a second liquid inlet, a second liquid outlet, and a top cover; the shape and size of the concentration gradient generating component are the same as those of the culture chamber, and a narrow through hole is provided thereon, and the diameter of the narrow through hole is 0.2-0.4mm.

6. A method for applying a detachable microfluidic chip according to any one of claims 1 to 5, It is characterized in that The specific steps include: S1. Inject solutions of different concentrations into the first liquid inlet and maintain the same flow rate for continuous perfusion; S2. Prepare gel and evenly encapsulate cells inside the three-dimensional gel; S3. Observe the movement of the solution in the flow channel under a microscope. After the flow rate stabilizes, start injecting the gel into the culture chamber so that the gel adheres to the bottom of the culture chamber. S4. Continuously passing liquid to the first liquid inlet; S5. Observe the growth of cells under the action of concentration gradient under a microscope; S6. Injecting fluorescent dye into the microfluidic chip to stain the target protein in the cell; S7. Observe the protein expression of cells under concentration gradient.

7. The application method of the detachable microfluidic chip according to claim 6, It is characterized in that The following steps are included after step S4: embedding the vertical concentration gradient generating module into the culture chamber, continuously passing liquid to the second liquid inlet at a uniform speed, and continuously leading the solution out of the second liquid outlet.

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