Liquid drop micro-fluidic device for cell co-culture and use method

Abstract

The invention discloses a droplet micro-fluidic device for cell co-culture and a use method, belongs to the field of micro-fluidic analysis, and particularly relates to a droplet micro-fluidic device which comprises a chip main body material, an oily liquid substance covering the surface of the chip main body material, and a vertical porous membrane material arranged in the oily liquid substance. The porous membrane material comprises at least one of a polycarbonate porous membrane, a PVDF (Polyvinylidene Fluoride) porous membrane and a porous composite membrane, and the porosity of the porous membrane material is more than 1%. The droplet microfluidic device has the advantages of high flux, low consumption, multi-step operation, direct observation and the like, and has strong compatibility with a classical biochemical mechanism analysis method. The liquid drops are separated by the porous membrane material and are connected with each other, and the liquid drops on the two sides can be used as a cell, organoid or tissue culture chamber and also can be used as a liquid storage tank for sample adding. The liquid drops are used as a culture chamber, so that co-culture of cells can be realized, and support is provided for cell co-culture, drug screening and cell microenvironment simulation.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidic analysis, and in particular relates to a droplet microfluidic device for cell co-culture and a method of use. Background Art

[0002] Cell co-culture techniques that mimic the in vivo microenvironment are of great significance in cell biology research and drug development. However, traditional cell co-culture methods, such as the Transwell system or multi-well plate culture, have many limitations: first, low experimental throughput, making it difficult to perform parallel testing of multiple parameters and conditions; second, insufficient control over the microenvironment, making it impossible to accurately simulate in vivo biochemical gradients or mechanical stimulation; and third, high sample and reagent consumption, which is not conducive to the study of precious cell samples.

[0003] Advances in microfluidics have provided new solutions for cell co-culture. Numerous microfluidic systems can generate drug concentration gradients within microchannels for studying cell migration mechanisms, the effects of chemokines, and drug screening. However, these systems often require complex fluid control mechanisms and cumbersome operation, making high-throughput experiments difficult to implement on a single chip. Microfluidic chips based on closed microchannel designs also often suffer from channel clogging, inflexible operation, and difficulty in real-time sampling. Droplet microfluidics has attracted considerable attention due to its unique advantages. Compared with conventional methods, droplet microfluidics systems offer significant high-throughput capabilities, enabling large-scale studies of cell interactions and drug screening. Furthermore, their microliter-scale reaction volumes significantly reduce sample and reagent consumption, which is particularly important for studying rare cell samples. Designing simple and flexible semi-open microfluidic chips that possess these advantages is a key goal for the development of microfluidic chips in cell biology. Droplet microfluidics offers the advantages of low-volume, high-efficiency, and high-throughput technology, but its application in cell biology is still in its infancy. Summary of the Invention

[0004] The object of the present invention is to provide a droplet microfluidic device for cell co-culture with controllable material diffusion speed, good reset droplet consistency, low membrane pore blockage rate, high porosity and good material exchange effect, and a method for use.

[0005] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are: A droplet microfluidic device comprises: a chip main body material, an oily liquid substance covered on the surface of the chip main body material, and an upright porous membrane material arranged in the oily liquid substance; the porous membrane material comprises at least one of a polycarbonate porous membrane, a PVDF porous membrane and a porous composite membrane, and the porosity of the porous membrane material is above 1%. The droplet microfluidic device of the present invention has the advantages of high throughput, low consumption, multi-step operation, direct observation, etc., and is highly compatible with classical biochemical mechanism analysis methods. The droplets are separated by a porous membrane material and are interconnected at the same time. The droplets on both sides serve as organoid and tissue culture chambers and can also serve as liquid storage tanks for sample addition. This provides support for simulating the concentration changes of drugs entering the human body, and the droplets as culture chambers can realize the co-culture of cells.

[0006] Preferably, grooves are provided on the surface of the chip main body material.

[0007] Preferably, the chip main body material includes PDMS material, PE material or plastic material; or, the oily liquid includes FC40 fluoro oil.

[0008] Preferably, the porous composite membrane is prepared from a composite of PVDF and a polyalkenyl compound, wherein the polyalkenyl compound is polymerized from alkenyl compound monomers, wherein the alkenyl compound monomers include at least one of methyl methacrylate, tetraallylsilane, and diethylene glycol divinyl ether. In the present invention, the porous composite membrane prepared using at least one of methyl methacrylate, tetraallylsilane, and diethylene glycol divinyl ether can be applied to a droplet microfluidic device for cell co-culture. When methyl methacrylate, tetraallylsilane and diethylene glycol divinyl ether are used together to prepare polyalkenyl compounds, tetraallylsilane has multiple alkenyl structures, and a polyalkenyl compound with a cross-linked structure can be prepared. Diethylene glycol divinyl ether can provide a linear long-chain structure for the polyalkenyl compound. When the linear long-chain, cross-linked polyalkenyl compound is compounded with PVDF, a porous composite membrane with a stable pore structure can be obtained under the action of the molecular chains of the polyalkenyl compound and PVDF. The use of tetraallylsilane and diethylene glycol divinyl ether in the porous composite membrane can reduce protein adsorption and blockage, thereby not hindering the material exchange of the porous composite membrane.

[0009] Preferably, in the preparation of the porous composite membrane, a polyalkenyl compound is first prepared from an alkenyl compound monomer under the action of an initiator, and then the polyalkenyl compound is mixed with PVDF in a solvent to prepare a casting solution, and then a porous composite membrane is prepared from the casting solution.

[0010] More preferably, the amount of the polyalkenyl compound used is 10-40 wt % of PVDF; or, the amount of tetraallylsilane used is 10-40 wt % of methyl methacrylate; or, the amount of diethylene glycol divinyl ether used is 5-20 wt % of methyl methacrylate.

[0011] Preferably, the porosity of the porous membrane material is 90% or less, and the porosity may also be 80% or less, 70% or less, and 60% or less.

[0012] Preferably, the porosity of the porous membrane material is greater than 3%, and the porosity may also be greater than 5%, greater than 8%, and greater than 10%.

[0013] The present invention discloses a method for using the above-mentioned droplet microfluidic device, wherein cell microenvironment droplets and tumor cell droplets are respectively added to both sides of a porous membrane material.

[0014] Preferably, the cell microenvironment droplets and the tumor cell droplets have overlapping areas on both sides of the porous membrane material.

[0015] Preferably, the cell microenvironment droplets include immune cell droplets; or, the tumor cell droplets include colon cancer cell droplets.

[0016] Preferably, in the preparation of the polyalkenyl compound, the alkenyl compound monomer and the initiator are added to ethylene glycol butyl ether and mixed, and the mixture is reacted at 120-160° C. for 2-10 hours. After the reaction is completed, the temperature is lowered to 60-80° C., an alkali neutralizer is added and mixed for 10-60 minutes, and then deionized water is added and mixed for 10-60 minutes. The mixture is cooled and filtered to obtain the polyalkenyl compound.

[0017] More preferably, in the preparation of the polyalkenyl compound, the alkenyl compound monomers include methyl methacrylate, tetraallylsilane and diethylene glycol divinyl ether.

[0018] More preferably, in the preparation of the polyalkenyl compound, the amount of methyl methacrylate used is 10-20 wt % of ethylene glycol butyl ether.

[0019] More preferably, in the preparation of the polyalkenyl compound, the amount of tetraallylsilane used is 10-40 wt % of methyl methacrylate.

[0020] More preferably, in the preparation of the polyalkenyl compound, the amount of diethylene glycol divinyl ether used is 5-20 wt % of methyl methacrylate.

[0021] More preferably, in the preparation of the polyalkenyl compound, the initiator is benzoyl peroxide, and the amount of benzoyl peroxide used is 0.2-1 wt % of ethylene glycol butyl ether.

[0022] More preferably, in the preparation of the polyalkenyl compound, the alkaline neutralizing agent is N,N-dimethylethanolamine, and the amount of N,N-dimethylethanolamine used is 20-60 wt % of ethylene glycol butyl ether.

[0023] More preferably, in the preparation of the polyalkenyl compound, the amount of deionized water used is 100-300 wt % of ethylene glycol butyl ether.

[0024] Preferably, in the preparation of the porous composite membrane, PVDF and the polyolefin compound are added to N, N-dimethylacetamide and mixed and stirred for 24-72 hours, degassed under reduced pressure for 10-60 minutes, and then coated on a substrate to form a solution film. The ethanol solution is then sprayed on the plane of the solution film, allowed to stand for 20-60 seconds, and then immersed in a coagulation bath of deionized water for solidification. After solidification, the residual solvent is washed with deionized water and dried to obtain a porous composite membrane.

[0025] More preferably, in the preparation of the porous composite membrane, the amount of PVDF used is 10-30 wt % of N,N-dimethylacetamide.

[0026] More preferably, in the preparation of the porous composite membrane, the amount of the polyalkenyl compound used is 10-40 wt % of PVDF.

[0027] More preferably, in the preparation of the porous composite membrane, the ethanol solution is prepared by mixing ethanol and water, the ethanol content in the ethanol solution is 30-70 wt%, and the amount of the ethanol solution used is 0.01-0.04 mL / cm 2 .

[0028] More preferably, in the preparation of the porous composite membrane, the size of the porous composite membrane is 1 μm-5 mm.

[0029] Preferably, allyltriethoxysilane can be added to the preparation of the polyalkenyl compound, and the amount of allyltriethoxysilane used is 5-20wt% of methyl methacrylate. When preparing the polyalkenyl compound, allyltriethoxysilane can also be added to provide triethoxysilyl groups in the polyalkenyl compound. Under the interaction of tetraallylsilane and diethylene glycol divinyl ether, the porosity of the porous composite membrane is increased, and the adsorption and clogging of the porous composite membrane by proteins can also be reduced.

[0030] Preferably, in constructing the droplet microfluidic device, the surface of the chip main body material is covered with an oily liquid, and then the porous membrane material is vertically placed in the oily liquid to obtain the droplet microfluidic device.

[0031] More preferably, in the construction of the droplet microfluidic device, grooves are present in the chip main body material, and the oily liquid is loaded in the grooves.

[0032] More preferably, in the construction of the droplet microfluidic device, the oily liquid is FC40 fluoro oil.

[0033] More preferably, in the construction of the droplet microfluidic device, multiple porous membrane materials can be placed. Depending on the space of the groove in the chip main body material, the number of porous membrane materials can be selected to be 1-20 or more.

[0034] Preferably, when the droplet microfluidic device is used, the cell microenvironment droplets and the tumor cell droplets are respectively added to both sides of the porous membrane material, and the cell microenvironment droplets and the tumor cell droplets have overlapping areas on both sides of the porous membrane material.

[0035] More preferably, during use of the droplet microfluidic device, multiple cell microenvironment droplets and tumor cell droplets can be placed. Depending on the length of the porous membrane material, the number of cell microenvironment droplets and tumor cell droplets can be selected to be 1-20 pairs or more.

[0036] The present invention first obtains a polyalkenyl compound by initiating polymerization of an alkenyl compound monomer through an initiator, then adopts PVDF and the polyalkenyl compound to mix in N, N-dimethylacetamide to prepare a casting solution, then coats it into a solution film, sprays an ethanol solution on the plane of the solution film, and finally treats it in a coagulation liquid to prepare a porous composite membrane, that is, the present application adopts a mixture of PVDF and a polyalkenyl compound to prepare a porous composite membrane, thus having the following beneficial effects: the porous composite membrane has a high porosity, a good material exchange effect, and a low membrane pore blockage rate. Therefore, the present invention is a droplet microfluidic device and a method for using the cell co-culture with controllable material diffusion rate, good consistency of reset droplets, low membrane pore blockage rate, high porosity, and good material exchange effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the droplet microfluidic device.

[0038] Figure 2 Schematic diagram of the positions of droplets on both sides of the porous membrane material.

[0039] Figure 3 This is the SEM image of the porous composite membrane.

[0040] Figure 4 The porosity diagram.

[0041] Figure 5 This is the congestion rate diagram. DETAILED DESCRIPTION

[0042] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0043] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0044] Example 1: A method for constructing a droplet microfluidic device Construction of droplet microfluidic device: Cover the surface of the chip main material with an oily liquid, and then place the porous membrane material vertically in the oily liquid to obtain a droplet microfluidic device. There is a groove in the chip main material, and the oily liquid is loaded in the groove. The oily liquid is FC40 fluoro oil. The porous membrane material is a polycarbonate porous membrane. Multiple porous membrane materials can be placed, depending on the space of the groove in the chip main material. The schematic diagram of the droplet microfluidic device is shown in the figure below. Figure 1 As shown, 1 is the porous membrane material, 2 is FC40 fluorine oil, and 3 is the chip main body material.

[0045] Example 2: Method for using a droplet microfluidic device Use of the droplet microfluidic device: Cell microenvironment droplets and tumor cell droplets are added to both sides of the porous membrane material, and the cell microenvironment droplets and tumor cell droplets have overlapping areas on both sides of the porous membrane material. Multiple cell microenvironment droplets and tumor cell droplets can be placed, depending on the length of the porous membrane material. The droplet microfluidic device is taken from Example 1. The schematic diagram of the cell microenvironment droplets and tumor cell droplets being added to both sides of the porous membrane material is shown in FIG. Figure 2 As shown, Figure 2 The a in the middle represents one-to-one comparison. Figure 2 In the middle, b represents interlacing. The side of the porous membrane material where the cell microenvironment droplets and the tumor cell droplets are located can be determined based on experimental needs. The cell microenvironment droplets and the tumor cell droplets can be placed one-to-one or interlaced on the porous membrane material. However, there must be an overlapping area so that the substances of the cell microenvironment droplets and the tumor cell droplets can be exchanged.

[0046] Example 3: A method for constructing a droplet microfluidic device The difference between this embodiment and embodiment 1 is that the porous membrane material is a PVDF porous membrane.

[0047] Preparation of a polyalkenyl compound: Add an alkenyl compound monomer and an initiator to ethylene glycol butyl ether, mix, and react at 140°C for 5 hours. After the reaction is complete, cool to 70°C, add an alkaline neutralizer, and mix for 30 minutes. Then, add deionized water and mix for another 30 minutes. After cooling, filter to obtain the polyalkenyl compound. The alkenyl compound monomer includes methyl methacrylate, the initiator is benzoyl peroxide, the amount of methyl methacrylate used is 15wt% of the ethylene glycol butyl ether, the amount of benzoyl peroxide used is 0.6wt% of the ethylene glycol butyl ether, the alkaline neutralizer is N,N-dimethylethanolamine, the amount of N,N-dimethylethanolamine used is 40wt% of the ethylene glycol butyl ether, and the amount of deionized water used is 200wt% of the ethylene glycol butyl ether.

[0048] Preparation of porous composite membrane: PVDF and polyolefin compound are added to N, N-dimethylacetamide and mixed for 48 hours, degassed under reduced pressure for 30 minutes, and then coated on a substrate to form a solution film. Then, ethanol solution is sprayed on the surface of the solution film, allowed to stand for 40 seconds, and then immersed in a coagulation bath of deionized water for solidification. After solidification, the residual solvent is washed with deionized water and dried to obtain a porous composite membrane. The amount of PVDF used is 20wt% of N, N-dimethylacetamide, the amount of polyolefin compound used is 36wt% of PVDF, the ethanol solution is a mixture of ethanol and water, the ethanol content in the ethanol solution is 70wt%, and the amount of ethanol solution used is 0.02mL / cm 2 The size of the porous composite membrane is 5 mm.

[0049] Example 4: Method for using a droplet microfluidic device The difference between this embodiment and embodiment 2 is that the droplet microfluidic device is taken from embodiment 3.

[0050] Example 5: A method for constructing a droplet microfluidic device The difference between this embodiment and embodiment 1 is that the porous membrane material is a porous composite membrane.

[0051] Preparation of a polyalkenyl compound: Add an alkenyl compound monomer and an initiator to ethylene glycol butyl ether, mix, and react at 140°C for 5 hours. After completion of the reaction, cool to 70°C, add an alkaline neutralizer, and mix for 30 minutes. Then, add deionized water and mix for another 30 minutes. Cool and filter to obtain the polyalkenyl compound. The alkenyl compound monomer includes methyl methacrylate, tetraallylsilane, and diethylene glycol divinyl ether. The initiator is benzoyl peroxide. The amount of methyl methacrylate is 15wt% of the ethylene glycol butyl ether, the amount of tetraallylsilane is 20wt% of the methyl methacrylate, the amount of diethylene glycol divinyl ether is 10wt% of the methyl methacrylate, and the amount of benzoyl peroxide is 0.6wt% of the ethylene glycol butyl ether. The alkaline neutralizer is N,N-dimethylethanolamine. The amount of N,N-dimethylethanolamine is 40wt% of the ethylene glycol butyl ether. The amount of deionized water is 200wt% of the ethylene glycol butyl ether.

[0052] Preparation of porous composite membrane: PVDF and polyolefin compound are added to N, N-dimethylacetamide and mixed for 48 hours, degassed under reduced pressure for 30 minutes, and then coated on a substrate to form a solution film. Then, ethanol solution is sprayed on the surface of the solution film, allowed to stand for 40 seconds, and then immersed in a coagulation bath of deionized water for solidification. After solidification, the residual solvent is washed with deionized water and dried to obtain a porous composite membrane. The amount of PVDF used is 20wt% of N, N-dimethylacetamide, the amount of polyolefin compound used is 36wt% of PVDF, the ethanol solution is a mixture of ethanol and water, the ethanol content in the ethanol solution is 70wt%, and the amount of ethanol solution used is 0.02mL / cm 2 The size of the porous composite membrane is 5 mm.

[0053] Example 6: Method for using a droplet microfluidic device The difference between this embodiment and embodiment 2 is that the droplet microfluidic device is taken from embodiment 5.

[0054] Example 7: A method for constructing a droplet microfluidic device Compared with Example 5, this embodiment differs in the preparation of the porous composite membrane.

[0055] Preparation of porous composite membrane: PVDF and polyolefin compound are added to N, N-dimethylacetamide and mixed for 48 hours, degassed under reduced pressure for 30 minutes, and then coated on a substrate to form a solution film. Then, ethanol solution is sprayed on the surface of the solution film, allowed to stand for 40 seconds, and then immersed in a coagulation bath of deionized water for solidification. After solidification, the residual solvent is washed with deionized water and dried to obtain a porous composite membrane. The amount of PVDF used is 20wt% of N, N-dimethylacetamide, the amount of polyolefin compound used is 15wt% of PVDF, and the ethanol solution is a mixture of ethanol and water. The ethanol content in the ethanol solution is 70wt%, and the amount of ethanol solution used is 0.02mL / cm 2 The size of the porous composite membrane is 5 mm.

[0056] Example 8: Method for using a droplet microfluidic device The difference between this embodiment and embodiment 2 is that the droplet microfluidic device is taken from embodiment 7.

[0057] Example 9: A method for constructing a droplet microfluidic device Compared with Example 5, this example differs in the preparation of the polyalkenyl compound.

[0058] Preparation of polyalkenyl compound: Add alkenyl compound monomer and initiator to ethylene glycol butyl ether, mix, react at 140°C for 5 hours, after the reaction is completed, cool to 70°C, add alkaline neutralizer and mix for 30 minutes, then add deionized water and mix for 30 minutes, cool and filter to obtain polyalkenyl compound. The alkenyl compound monomers include methyl methacrylate, tetraallylsilane, diethylene glycol divinyl ether and allyl triethoxysilane, the initiator is benzoyl peroxide, the amount of methyl methacrylate used is 15wt% of ethylene glycol butyl ether, the amount of tetraallylsilane used is 20wt% of methyl methacrylate, the amount of diethylene glycol divinyl ether used is 10wt% of methyl methacrylate, the amount of allyl triethoxysilane used is 10wt% of methyl methacrylate, the amount of benzoyl peroxide used is 0.6wt% of ethylene glycol butyl ether, the alkaline neutralizer is N,N-dimethylethanolamine, the amount of N,N-dimethylethanolamine used is 40wt% of ethylene glycol butyl ether, and the amount of deionized water used is 200wt% of ethylene glycol butyl ether.

[0059] Example 10: Method for using a droplet microfluidic device The difference between this embodiment and embodiment 2 is that the droplet microfluidic device is taken from embodiment 9.

[0060] Example 11: A method for constructing a droplet microfluidic device Compared with Example 9, this example differs in the preparation of the porous composite membrane.

[0061] Preparation of porous composite membrane: PVDF and polyolefin compound are added to N, N-dimethylacetamide and mixed for 48 hours, degassed under reduced pressure for 30 minutes, and then coated on a substrate to form a solution film. Then, ethanol solution is sprayed on the surface of the solution film, allowed to stand for 40 seconds, and then immersed in a coagulation bath of deionized water for solidification. After solidification, the residual solvent is washed with deionized water and dried to obtain a porous composite membrane. The amount of PVDF used is 20wt% of N, N-dimethylacetamide, the amount of polyolefin compound used is 15wt% of PVDF, and the ethanol solution is a mixture of ethanol and water. The ethanol content in the ethanol solution is 70wt%, and the amount of ethanol solution used is 0.02mL / cm 2 The size of the porous composite membrane is 5 mm.

[0062] Example 12: Method for using a droplet microfluidic device The difference between this embodiment and embodiment 2 is that the droplet microfluidic device is taken from embodiment 11.

[0063] Comparative Example 1: A method for constructing a droplet microfluidic device The difference between this comparative example and Example 5 is that the amount of the polyolefin compound used is 5 wt % of PVDF.

[0064] Comparative Example 2: A method for constructing a droplet microfluidic device The difference between this comparative example and Example 5 lies in the preparation of the polyalkenyl compound.

[0065] Preparation of a polyalkenyl compound: Add an alkenyl compound monomer and an initiator to ethylene glycol butyl ether, mix, and react at 140°C for 5 hours. After the reaction is complete, cool to 70°C, add an alkaline neutralizer, and mix for 30 minutes. Then, add deionized water and mix for another 30 minutes. After cooling, filter, and obtain the polyalkenyl compound. The alkenyl compound monomer includes methyl methacrylate and tetraallylsilane. The initiator is benzoyl peroxide. The amount of methyl methacrylate is 15wt% of the ethylene glycol butyl ether, the amount of tetraallylsilane is 20wt% of the methyl methacrylate, and the amount of benzoyl peroxide is 0.6wt% of the ethylene glycol butyl ether. The alkaline neutralizer is N,N-dimethylethanolamine. The amount of N,N-dimethylethanolamine is 40wt% of the ethylene glycol butyl ether. The amount of deionized water is 200wt% of the ethylene glycol butyl ether.

[0066] Comparative Example 3: A method for constructing a droplet microfluidic device The difference between this comparative example and Example 5 lies in the preparation of the polyalkenyl compound.

[0067] Preparation of a polyalkenyl compound: Add an alkenyl compound monomer and an initiator to ethylene glycol butyl ether, mix, and react at 140°C for 5 hours. After the reaction is complete, cool to 70°C, add an alkaline neutralizer, and mix for 30 minutes. Then, add deionized water and mix for another 30 minutes. After cooling, filter, and obtain the polyalkenyl compound. The alkenyl compound monomer includes methyl methacrylate and diethylene glycol divinyl ether. The initiator is benzoyl peroxide, with the amount of methyl methacrylate being 15wt% of the ethylene glycol butyl ether, the amount of diethylene glycol divinyl ether being 10wt% of the methyl methacrylate, and the amount of benzoyl peroxide being 0.6wt% of the ethylene glycol butyl ether. The alkaline neutralizer is N,N-dimethylethanolamine, with the amount of N,N-dimethylethanolamine being 40wt% of the ethylene glycol butyl ether. The amount of deionized water is 200wt% of the ethylene glycol butyl ether.

[0068] Comparative Example 4: A method for constructing a droplet microfluidic device The difference between this comparative example and Example 5 lies in the preparation of the polyalkenyl compound.

[0069] Preparation of a polyalkenyl compound: Add an alkenyl compound monomer and an initiator to ethylene glycol butyl ether, mix, and react at 140°C for 5 hours. After the reaction is complete, cool to 70°C, add an alkaline neutralizer, and mix for 30 minutes. Then, add deionized water and mix for another 30 minutes. Cool and filter to obtain the polyalkenyl compound. The alkenyl compound monomer includes methyl methacrylate, tetraallylsilane, and diethylene glycol divinyl ether. The initiator is benzoyl peroxide. The amount of methyl methacrylate is 15wt% of the ethylene glycol butyl ether, the amount of tetraallylsilane is 5wt% of the methyl methacrylate, the amount of diethylene glycol divinyl ether is 2wt% of the methyl methacrylate, and the amount of benzoyl peroxide is 0.6wt% of the ethylene glycol butyl ether. The alkaline neutralizer is N,N-dimethylethanolamine, the amount of N,N-dimethylethanolamine is 40wt% of the ethylene glycol butyl ether, and the amount of deionized water is 200wt% of the ethylene glycol butyl ether.

[0070] Test example: 1. Surface morphology of porous membranes The present invention uses SEM to characterize the surface morphology of the porous composite membrane. The surface morphology of the porous composite membrane prepared in Example 5 is as follows: Figure 3 As shown, there are micropores on the surface of the porous composite membrane, and the micropores are of different sizes. The micropores on the porous composite membrane penetrate the upper surface and can provide a path for the exchange of microenvironmental substances in cell culture.

[0071] 2. Porosity of porous membrane The present invention uses the density method to measure the porosity of the porous composite membrane in the embodiment and the comparative example. The results are as follows: Figure 4As shown, A is Example 3, B is Example 5, C is Example 7, D is Example 9, E is Example 11, F is Comparative Example 1, G is Comparative Example 2, H is Comparative Example 3, and I is Comparative Example 4. In this application, the porous composite membrane needs to have a certain porosity so that the droplets on both sides of the porous composite membrane can carry out a certain material exchange to simulate the microenvironment required for tumor experiments. Finally, as the industry continues to grow, PVDF is becoming increasingly popular and is a popular choice for film manufacturers looking to expand market share through its low cost, low maintenance, and long-lasting film capabilities. The polyalkenyl compound is obtained by polymerizing methyl methacrylate, tetraallylsilane and diethylene glycol divinyl ether, and the porosity of the obtained porous composite membrane cannot be significantly improved when the amount of the polyalkenyl compound used is too low; when preparing the polyalkenyl compound, if only methyl methacrylate and tetraallylsilane are used, the porosity of the finally prepared porous composite membrane cannot be effectively improved; when preparing the polyalkenyl compound, if only methyl methacrylate and diethylene glycol divinyl ether are used, the porosity of the finally prepared porous composite membrane cannot be effectively improved; when preparing the polyalkenyl compound, if only tetraallylsilane and diethylene glycol divinyl ether are used in low amounts, the porosity of the finally prepared porous composite membrane cannot be effectively improved either; the present invention can further use allyltriethoxysilane as an alkenyl compound monomer to prepare the polyalkenyl compound, and then apply the obtained polyalkenyl compound to the preparation of the porous composite membrane, which can improve the porosity of the porous composite membrane.

[0072] 3. Congestion rate In this application, the droplets on both sides of the porous composite membrane are cell cultures, which will produce proteins. The proteins may clog the porous composite membrane and prevent material exchange between the droplets. The lower the protein blocking rate of the porous composite membrane, the better the effect.

[0073] The present invention uses deionized water containing BSA to perform a clogging test on the porous composite membranes prepared in the examples and comparative examples. First, deionized water is used to run at 0.1 MPa for 30 minutes to measure the water flux. Then, the deionized water is replaced with an aqueous solution containing 0.5 g / L BSA, and the water flux is measured at 0.1 MPa for 30 minutes. The ratio of the water flux of the BSA aqueous solution to the water flux of the deionized water is used as the pass rate, and the pass rate is subtracted from 100% as the clogging rate of the porous composite membrane. The results are shown in FIG. Figure 5 As shown, wherein A is Example 3, B is Example 5, C is Example 7, D is Example 9, E is Example 11, F is Comparative Example 1, G is Comparative Example 2, H is Comparative Example 3, and I is Comparative Example 4. The present invention first initiates polymerization of an alkenyl compound monomer with an initiator to obtain a polyalkenyl compound, then mixes PVDF and the polyalkenyl compound in N, N-dimethylacetamide to prepare a casting solution, then coats the solution film, sprays an ethanol solution on the surface of the solution film, and finally treats the porous composite membrane in a coagulation solution. That is, in this application, PVDF and polyalkenyl are used. The present invention relates to a mixture of methyl methacrylate and tetraallylsilane to prepare a porous composite membrane; in the preparation of the polyalkenyl compound, in addition to methyl methacrylate, tetraallylsilane and diethylene glycol divinyl ether can also be added to prepare a polyalkenyl compound obtained by polymerization of methyl methacrylate, tetraallylsilane and diethylene glycol divinyl ether, and the polyalkenyl compound is prepared together with PVDF to obtain a porous composite membrane. Due to the composite effect of the polyalkenyl compound and PVDF, the clogging rate of the porous composite membrane by the protein is reduced. When the polyalkenyl compound and PVDF are used together, the polyalkenyl compound is When the dosage is reduced, the effect of reducing the clogging rate of the porous composite membrane by the protein is small after the porous composite membrane is obtained; if the polyalkenyl compound obtained by polymerization of methyl methacrylate, tetraallylsilane and diethylene glycol divinyl ether is further reduced, and the dosage of the polyalkenyl compound is too low, the clogging rate of the porous composite membrane obtained by the protein cannot be significantly reduced; when preparing the polyalkenyl compound, if only methyl methacrylate and tetraallylsilane are used, the clogging rate of the porous composite membrane finally prepared by the protein cannot be effectively reduced; when preparing the polyalkenyl compound, if only methyl methacrylate and tetraallylsilane are used, the clogging rate of the porous composite membrane finally prepared by the protein cannot be effectively reduced. Even when using methyl methacrylate and diethylene glycol divinyl ether, the clogging rate of the protein on the porous composite membrane finally prepared cannot be effectively reduced; when preparing polyalkenyl compounds, if only tetraallylsilane and diethylene glycol divinyl ether are used in low amounts, the clogging rate of the protein on the porous composite membrane finally prepared cannot be effectively reduced; the present invention can further use allyltriethoxysilane as an alkenyl compound monomer to prepare a polyalkenyl compound, and then apply the obtained polyalkenyl compound to the preparation of a porous composite membrane, which can reduce the clogging rate of the protein on the porous composite membrane.

[0074] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0075] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A droplet microfluidic device comprising: The chip main body material, the surface of the chip main body material is covered with an oily liquid, and an upright porous membrane material is arranged in the oily liquid; it is characterized in that the porous membrane material includes at least one of a polycarbonate porous membrane, a PVDF porous membrane and a porous composite membrane, and the porosity of the porous membrane material is above 1%.

2. A droplet microfluidic device according to claim 1, characterized in that: A groove is provided on the surface of the chip main body material.

3. A droplet microfluidic device according to claim 1, characterized in that: The chip main body material includes PDMS material, PE material or plastic material; or the oily liquid includes FC40 fluoro oil.

4. A droplet microfluidic device according to claim 1, characterized in that: The porous composite membrane is prepared by compounding PVDF and a polyalkenyl compound. The polyalkenyl compound is polymerized by alkenyl compound monomers. The alkenyl compound monomers include at least one of methyl methacrylate, tetraallylsilane and diethylene glycol divinyl ether.

5. The droplet microfluidic device according to claim 1, characterized in that: In the preparation of the porous composite membrane, firstly, a polyalkenyl compound is prepared from an alkenyl compound monomer under the action of an initiator, then the polyalkenyl compound is mixed with PVDF in a solvent to prepare a casting solution, and then a porous composite membrane is prepared from the casting solution.

6. A droplet microfluidic device according to claim 4 or 5, characterized in that: The polyalkenyl compound is used in an amount of 10-40 wt % of PVDF; or, the tetraallylsilane is used in an amount of 10-40 wt % of methyl methacrylate; or, the diethylene glycol divinyl ether is used in an amount of 5-20 wt % of methyl methacrylate.

7. The droplet microfluidic device according to claim 1, characterized in that: The porosity of the porous membrane material is less than 90%.

8. The method for using the droplet microfluidic device according to claim 1, characterized in that: Cell microenvironment droplets and tumor cell droplets are added on both sides of the porous membrane material respectively.

9. The method for using the droplet microfluidic device according to claim 8, characterized in that: The cell microenvironment droplets and the tumor cell droplets have overlapping areas on both sides of the porous membrane material.

10. The method for using the droplet microfluidic device according to claim 8, characterized in that: The cell microenvironment droplets include immune cell droplets; or, the tumor cell droplets include colon cancer cell droplets.

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