Establishment method and application of intestinal wall cell mucous layer model

Abstract

The invention relates to the technical field of surface plasma resonance, and discloses an establishment method and application of an intestinal wall cell mucous layer model. The method comprises the following steps: carrying out bionic treatment on the surface of a metasurface plasmon resonance chip, inputting a culture solution containing intestinal wall cells into the surface of the metasurface plasmon resonance chip through a micro-fluidic chip to carry out intestinal wall cell culture, inputting an intestinal tract simulation solution into the surface of the metasurface plasmon resonance chip, and carrying out intestinal tract simulation on the surface of the metasurface plasmon resonance chip. And detecting the refractive index change of the surface of the metasurface plasmon resonance chip, and monitoring the forming process of the mucus layer of the intestinal wall cell in real time according to the refractive index change. According to the intestinal wall cell mucous layer model obtained through the construction method, unmarked real-time continuous monitoring of the intestinal wall cell mucous layer forming process, dynamic observation of physiological changes of cells and simulation of the real intestinal environment can be achieved, and the method has high sensitivity and accuracy, is low in detection cost and has remarkable market prospects.

Description

Technical Field

[0001] This invention relates to the field of surface plasmon resonance technology, specifically to a method for establishing a mucus layer model of intestinal wall cells and its application. Background Technology

[0002] The intestinal mucus layer is a sticky substance on the surface of intestinal epithelial cells, mainly composed of mucins, and has multiple functions, including protecting intestinal epithelial cells, regulating the gut microbiota, and participating in immune responses. The gut microbiome is a complex ecosystem with intricate interactions between probiotics and other gut microbes. Studying the structure and function of the intestinal mucus layer is crucial for understanding the mechanisms of intestinal diseases, developing novel drugs, and evaluating drug absorption and transport in the gut. However, a rapid and accurate model and detection method for analyzing the intestinal cell mucus layer is currently lacking.

[0003] Currently, the main technologies used to monitor the secretion of mucus layer by cells are fluorescent biosensors. However, these technologies suffer from significant detection interference and require fluorescent labeling of samples, increasing both operation time and cost, and potentially affecting cell state. Traditional surface plasmon resonance (SPR) technology uses a two-dimensional planar chip surface, requiring additional optical coupling elements, resulting in large and expensive equipment. Furthermore, most existing research models are based on single-cell culture, making it difficult to realistically simulate the complex physiological environment of the human gut. While intestinal organoids can better mimic the structure and function of the intestine, efficient mucus layer analysis methods suitable for them are lacking. Therefore, there is an urgent need to provide a method for constructing an intestinal cell mucus layer model that can reduce detection interference and detection costs, and enable label-free real-time continuous monitoring of the formation process of the intestinal wall cell mucus layer, simulating the real intestinal environment. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems of existing intestinal cell mucus layer model construction, such as large detection interference, high cost, and difficulty in simulating the real intestinal environment, and to provide a method for constructing an intestinal wall cell mucus layer model and its application. The intestinal wall cell mucus layer model obtained by this method can achieve label-free real-time continuous monitoring of the intestinal wall cell mucus layer formation process, dynamically observe physiological changes of cells, and simulate the real intestinal environment. This method has high sensitivity and accuracy, low detection cost, and significant market potential.

[0005] In order to achieve the above object, the present application provides a method for constructing an intestinal cell mucus layer model, which comprises the following steps: carrying out biomimetic treatment on the surface of a surface plasmon resonance chip, inputting a culture solution containing intestinal wall cells into the surface of the surface plasmon resonance chip through a microfluidic chip to culture the intestinal wall cells, inputting an intestinal simulation solution into the surface of the surface plasmon resonance chip through the intestinal simulation solution, detecting the refractive index change of the surface of the surface plasmon resonance chip, and monitoring the formation process of the mucus layer of the intestinal wall cells in real time according to the refractive index change.

[0006] Preferably, the surface plasmon resonance chip comprises a surface plasmon resonance chip plate and a MetaSPR sensor chip integrated on the surface plasmon resonance chip plate; the surface plasmon resonance chip plate is provided with a plurality of chip micropores, the MetaSPR sensor chip comprises a substrate, a plurality of nanopores imprinted on the substrate and corresponding to the chip micropores, and a metal film covering the surface of the nanopores, and the metal film comprises a transition metal film layer and a noble metal film layer arranged from inside to outside.

[0007] Preferably, the substrate is made of PET material; the transition metal film layer is a titanium film layer with a thickness of 4-6 nm, and the noble metal film layer comprises a gold film layer with a thickness of 15-25 nm and / or a silver film layer with a thickness of 50-70 nm.

[0008] Preferably, the nanopores are frustum-shaped, and the diameter of the large opening of the nanopores is 100-400 nm and the depth is 400-600 nm; the spacing between two adjacent chip micropores is 200-600 nm.

[0009] Preferably, the microfluidic chip comprises a bottom plate and a microfluidic channel structure arranged on the bottom plate, the microfluidic channel structure comprises a first microfluidic channel suitable for the flow of the culture solution containing intestinal wall cells and a second microfluidic channel suitable for the flow of the intestinal simulation solution, the first microfluidic channel and the second microfluidic channel are connected in communication, and the surface plasmon resonance chip is arranged at the communication position of the first microfluidic channel and the second microfluidic channel.

[0010] Preferably, the flow direction of the culture solution in the first microfluidic channel and the flow direction of the intestinal simulation solution in the second microfluidic channel are opposite.

[0011] Preferably, the bottom plate is made of PDMS material.

[0012] Preferably, the process of biomimetic treatment comprises: adding an adhesion agent into the nanopores to form a film, and then removing the adhesion agent, washing and drying.

[0013] Preferably, the adhesion agent is at least one selected from sericin, recombinant human fibroin, collagen type IV and polyethylene imine, preferably polyethylene imine.

[0014] Preferably, the adhesion agent is added in a solution with a concentration of 8-12 μg / mL and an added amount of 50-100 μL in each of the micro-holes.

[0015] Preferably, the film coating conditions include a temperature of 4-6 ℃ and a time of 14-16 h.

[0016] Preferably, the intestinal wall cell culture conditions include a cell inoculation amount of 0.1-20 thousand in each of the micro-holes, a temperature of 35-40 ℃ and a CO2 concentration of 4-6 %.

[0017] Preferably, the intestinal wall cells are Caco-2 cells and / or HT29-MTX cells, preferably Caco-2 cells and HT29-MTX cells.

[0018] Preferably, the density ratio of Caco-2 cells to HT29-MTX cells in the culture solution containing intestinal wall cells is 4-10:1.

[0019] Preferably, the intestinal fluid simulation solution contains 10-50 μg / L epidermal factor and 5-10 μg / L N-2 additive.

[0020] Preferably, the flow rate of the intestinal fluid simulation solution is 0.4-0.6 μL / min.

[0021] The second aspect of the present application provides an application of the intestinal cell mucus layer model obtained by the construction method of the first aspect in evaluating the interaction between mucus proteins and probiotics.

[0022] Preferably, the probiotics are at least one selected from Bifidobacterium longum BL21, Bifidobacterium breve BBr60, Bifidobacterium lactis BLa80, Lactobacillus gasseri LG08 and Lactobacillus rhamnosus LRa05.

[0023] By the above technical solution, the present application has the following advantages: The method for establishing an intestinal wall cell mucus layer model provided by this invention involves introducing a culture medium containing intestinal wall cells onto the surface of a metasurface plasmon resonance chip via a microfluidic chip for intestinal cell culture, and introducing a simulated intestinal fluid onto the surface of the metasurface plasmon resonance chip. Using MetaSPR technology, the formation process of the intestinal wall cell mucus layer is dynamically monitored in real time by detecting changes in the refractive index of the metasurface plasmon resonance chip surface. This method enables label-free continuous monitoring of the intestinal wall cell mucus layer formation process, dynamically observing physiological changes in cells, and simulating the real intestinal environment. It has high sensitivity and accuracy, low detection cost, and significant market potential. Attached Figure Description

[0024] Figure 1 This is one of the structural schematic diagrams of MetaSPR chip I in this invention; Figure 2 This is the second schematic diagram of the structure of MetaSPR chip I in this invention; Figure 3 This is a schematic diagram of the cross-sectional structure of MetaSPR chip I in this invention; Figure 4 This is a schematic diagram of the cross-sectional structure of the MetaSPR chip II in this invention; Figure 5 These are the real-time cell growth curves of MetaSPR chips I and II in Embodiment 1 of this invention; Figure 6 These are real-time curves of cell adhesion and growth at different cell concentrations in Example 2 of this invention; Figure 7 These are the real-time curves of cell adhesion and growth under different modifiers in Example 2 of this invention; Figure 8 This is one of the structural schematic diagrams of the microfluidic chip in Embodiment 4 of the present invention; Figure 9 This is the second schematic diagram of the microfluidic chip in Embodiment 4 of the present invention; Figure 10 Real-time curves of probiotic screening using a microfluidic chip in Embodiment 5 of this invention. Detailed Implementation

[0025] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0026] In a first aspect, the present application provides a method for constructing an intestinal cell mucus layer model, which comprises the following steps: biomimetic treatment is performed on the surface of a surface plasmon resonance chip, a culture solution containing intestinal wall cells is input into the surface of the surface plasmon resonance chip through a microfluidic chip for intestinal wall cell culture, an intestinal simulation solution is input into the surface of the surface plasmon resonance chip through the microfluidic chip, the refractive index change of the surface of the surface plasmon resonance chip is detected, and the formation process of the mucus layer of the intestinal wall cells is monitored in real time according to the refractive index change.

[0027] The inventors of the present application found in the research process that, by inputting a culture solution containing intestinal wall cells into the surface of a surface plasmon resonance chip through a microfluidic chip for intestinal cell culture, inputting an intestinal simulation solution into the surface of the surface plasmon resonance chip, and using MetaSPR technology to dynamically monitor the formation process of the mucus layer of the intestinal wall cells by detecting the refractive index change of the surface of the surface plasmon resonance chip, the formation process of the mucus layer of the intestinal wall cells can be continuously monitored without labeling, the physiological changes of the cells can be dynamically observed, and the real intestinal environment can be simulated. The method has high sensitivity and accuracy, low detection cost, and significant market prospects.

[0028] According to the present application, preferably, the surface plasmon resonance chip comprises a surface plasmon resonance chip plate and a MetaSPR sensor chip integrated on the surface plasmon resonance chip plate; the surface plasmon resonance chip plate is provided with a plurality of chip micropores, the MetaSPR sensor chip comprises a substrate, a plurality of nanopores stamped on the substrate and corresponding to the chip micropores, and a metal film covering the surface of the nanopores, the metal film comprises a transition metal film layer and a noble metal film layer arranged from inside to outside. The inventors found that, in this preferred embodiment, the sensitivity and stability of the SPR signal of the chip can be improved, the small changes of the cells can be more accurately detected, and thus the sensitivity and accuracy of the intestinal wall cell barrier model can be improved.

[0029] According to the present application, in order to further improve the sensitivity and accuracy of the intestinal wall cell barrier model, preferably, the substrate is made of PET material; the transition metal film layer is a titanium film layer with a thickness of 4-6 nm, and the noble metal film layer comprises a gold film layer with a thickness of 15-25 nm and / or a silver film layer with a thickness of 50-70 nm. The transition metal film layer and the noble metal film layer can be covered on the surface of the nanopores by evaporation.

[0030] According to the application, preferably, the noble metal film layer is a gold film layer with a thickness of 15-25 nm. The inventor has found that in the preferred embodiment, the above noble metal film layer is more conducive to the adherent growth of the intestinal wall cells, thereby improving the sensitivity and accuracy of the intestinal wall cell mucus layer model and simulating the intestinal environment.

[0031] According to the application, in order to further improve the sensitivity and accuracy of the intestinal wall cell mucus layer model, preferably, the nano-pores are frustum-shaped, the diameter of the large opening is 100-400 nm, the depth is 400-600 nm, and the distance between two adjacent chip micro-holes is 200-600 nm.

[0032] According to the application, as a preferred embodiment of the micro-fluidic chip, the micro-fluidic chip comprises a bottom plate and a micro-fluid channel structure arranged on the bottom plate, the micro-fluid channel structure comprises a first micro-fluid channel suitable for the flow of the culture solution containing intestinal wall cells and a second micro-fluid channel suitable for the flow of the intestinal tract simulation solution, the first micro-fluid channel and the second micro-fluid channel are communicated, and the surface plasmon resonance chip is arranged at the communication position of the first micro-fluid channel and the second micro-fluid channel. By arranging the surface plasmon resonance chip at the communication position of the first micro-fluid channel and the second micro-fluid channel, the surface plasmon resonance chip is exposed to the micro-fluid channel structure, which facilitates the input of the culture solution containing intestinal wall cells to the surface of the surface plasmon resonance chip through the micro-fluidic chip, and also facilitates the input of the intestinal tract simulation solution to the surface of the surface plasmon resonance chip, thereby simulating the real intestinal environment.

[0033] According to the application, preferably, referring to Figure 7 and Figure 8 , the cross section of the communication position of the first micro-fluid channel and the second micro-fluid channel in the horizontal direction is hexagonal. In the preferred embodiment, the cross section is hexagonal, which is conducive to the uniform distribution of fluid shear stress, simulates the intestinal physiological environment, possibly increases the cell adhesion area, promotes cell culture, and is also conducive to micro-fabrication and integration with other micro-structures.

[0034] In the application, the micro-fluid channel structure can be one or multiple, which can be designed by the tester as needed. Exemplarily, when the micro-fluid channel structure is multiple, the MetaSPR technology can simultaneously detect the interaction of multiple biological molecules, which lays a foundation for the study of the complex physiological functions of the intestinal cell mucus layer and probiotics.

[0035] According to the present application, preferably, the flow direction of the culture solution in the first microfluidic channel and the flow direction of the intestinal tract simulation solution in the second microfluidic channel are opposite. In this preferred embodiment, a counter-current material exchange environment can be formed, facilitating more efficient transmission of nutrients and metabolic waste, while simulating the dynamic fluid shear force in the intestinal tract, maintaining the epithelial cell barrier function, also helping to build a stable oxygen concentration gradient, supporting the co-culture of anaerobic microorganisms and cells, and improving the simulation accuracy of the model on the intestinal physiological environment.

[0036] According to the present application, in order to further improve the sensitivity and accuracy of the intestinal wall cell mucus layer model and improve the authenticity of the simulated intestinal tract, preferably, the bottom plate is made of PDMS material.

[0037] According to the present application, the construction method can use a WeSPR multifunctional molecular detector with the surface plasmon resonance biochip to detect the refractive index change on the surface of the surface plasmon resonance biochip in real time. For example, after the surface plasmon resonance biochip is assembled with the microfluidic chip, it is placed in the WeSPR™ CellAnalyzer SP real-time label-free cell analyzer to detect the light refractive index change of the surface plasmon resonance biochip. The adhesion state of the intestinal wall cells is different, the light refractive index of the surface plasmon resonance biochip is different, and the signal response value caused by the difference is also different. Through the WeSPR™ CellAnalyzer SP real-time label-free cell analyzer, a real-time change corresponding platform can be formed, which can monitor the dynamic process of intestinal wall cells at different stages and observe the formation process of the intestinal wall cell mucus layer.

[0038] According to the present application, preferably, the process of the biomimetic treatment comprises: adding an adhesion agent to coat the film in the nanopore, and then removing the adhesion agent, washing and drying. The inventors have found that in this preferred embodiment, it is beneficial to the culture and fixation of intestinal wall cells on the MetaSPR chip surface, promotes the normal growth of intestinal wall cells and the formation of mucus layer, and further improves the sensitivity and accuracy of the intestinal wall cell mucus layer model.

[0039] According to the present application, preferably, the adhesion agent is at least one selected from the group consisting of sericin, recombinant human fibroin, collagen IV and polyethyleneimine (PEI), and is preferably polyethyleneimine. The inventors have found that in this preferred embodiment, it is beneficial to improve the adhesion effect of intestinal wall cells on the chip plate, promote the normal growth of intestinal wall cells and the formation of mucus layer, and further improve the sensitivity and accuracy of the intestinal wall cell mucus layer model.

[0040] According to the present application, preferably, the adherent agent is added in the form of a solution with a concentration of 8-12 μg / mL in the single nano-pore, and the added amount is 50-100 μL. The concentration of the adherent agent refers to the total concentration of all adherent agent components in the solution.

[0041] According to the present application, preferably, the conditions of the coating film at least include: the temperature is 4-6 ℃, specifically, 4 ℃, 5 ℃, 6 ℃, or any value between the aforementioned two values; the time is 14-16 h, specifically, 14 h, 15 h, 16 h, or any value between the aforementioned two values. The inventors have found that, in the preferred embodiment, it is beneficial for the adherent agent to adhere to the surface plasmon resonance biochip to form a coating film, thereby improving the adhesion effect of the intestinal wall cells.

[0042] According to the present application, preferably, the conditions of the intestinal wall cell culture at least include: the number of cells inoculated in the single chip micro-well is 0.1-20 thousand, specifically, 0.1 thousand, 10 thousand, 20 thousand, or any value between the aforementioned two values; the temperature is 35-40 ℃, specifically, 35 ℃, 37 ℃, 40 ℃, or any value between the aforementioned two values; the CO2 concentration is 4-6%, specifically, 4%, 5%, 6%, or any value between the aforementioned two values, and the CO2 concentration of 4-6% refers to the volume content of CO2 in the air being 4-6%.

[0043] In the present application, the intestinal wall cells refer to various cells that constitute the intestinal wall, and preferably, the intestinal wall cells are Caco-2 cells and / or HT29-MTX cells, preferably Caco-2 cells and HT29-MTX cells. Caco-2 cells are derived from human colon adenocarcinoma cells, and when cultured in vitro, they can spontaneously differentiate into cells with intestinal epithelial cell characteristics, forming a single-layer cell structure similar to the small intestinal epithelium, with typical intestinal epithelial features such as microvilli, and can simulate the intestinal barrier function; HT29-MTX cells are derived from the human colon adenocarcinoma HT29 cell line, and are obtained by long-term culture in a culture medium containing methotrexate (MTX). Differentiated HT29-MTX cells synthesize and secrete mucin, forming a mucus layer similar to the intestinal mucosa, simulating the mucus barrier environment in the intestinal tract.

[0044] According to the present application, in order to further improve the sensitivity and accuracy of the intestinal wall cell mucus layer model, preferably, the density ratio of Caco-2 cells to HT29-MTX cells in the culture solution containing intestinal wall cells is 4-10:1, specifically, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or any value between the aforementioned two values.

[0045] In the present application, the culture solution containing the intestinal wall cells can be any culture solution suitable for the culture of intestinal wall cells. Preferably, the culture solution comprises DMEM medium, fetal bovine serum (FBS) and penicillin-streptomycin (P / S). Exemplarily, the culture solution comprises DMEM medium, 8-12 wt% FBS and 0.5-2 wt% P / S.

[0046] According to the present application, in order to further improve the authenticity of the intestinal environment, preferably, the intestinal simulation solution contains 10-50 μg / L epidermal factor and 5-10 μg / L N-2 additive. The intestinal simulation solution can be dissolved in deionized water.

[0047] According to the present application, in order to further improve the authenticity of the intestinal environment, preferably, the flow rate of the intestinal simulation solution is 0.4-0.6 μL / min, and can be specifically 0.4 μL / min, 0.5 μL / min, 0.6 μL / min, or any value between the aforementioned two values.

[0048] According to the present application, preferably, the method further comprises: fixing the intestinal wall cells before the intestinal wall cells are cultured. The inventors have found that, in this preferred embodiment, the stability of the cell structure can be improved, and thus the stability of the intestinal wall cell mucus layer model can be improved.

[0049] According to the present application, the conditions for fixing the intestinal wall cells at least comprise: a time of 24 h-48 h, and can be specifically 24 h, 36 h, 48 h, or any value between the aforementioned two values.

[0050] In the present application, after the intestinal wall cells are fixed, the intestinal wall cells are washed, and the solvent used for washing can be a PBS solution.

[0051] According to a particularly preferred embodiment of the present application, a method for constructing an intestinal wall cell mucus layer model is provided, which comprises: performing a biomimetic treatment on the surface of a surface plasmon resonance chip, inputting a culture solution containing intestinal wall cells into the surface of the surface plasmon resonance chip through a microfluidic chip to culture the intestinal wall cells, inputting an intestinal simulation solution into the surface of the surface plasmon resonance chip through the microfluidic chip, detecting the change in the refractive index of the surface of the surface plasmon resonance chip, and monitoring the formation process of the mucus layer of the intestinal wall cells in real time according to the change in the refractive index. The super surface plasmon resonance chip comprises a super surface plasmon resonance chip plate and a MetaSPR sensor chip integrated on the super surface plasmon resonance chip plate, the super surface plasmon resonance chip plate is provided with a plurality of chip micropores, the MetaSPR sensor chip comprises a substrate, a plurality of nanopores which are imprinted on the substrate and correspond to the chip micropores, and a metal film which covers the surface of the nanopores, the metal film comprises a titanium film layer with a thickness of 4-6 nm and a gold film layer with a thickness of 15-25 nm which are arranged from inside to outside, the substrate is made of PET material, the nanopores are in the shape of a truncated cone, the diameter of the large opening of the nanopores is 100-400 nm, the depth of the nanopores is 400-600 nm, and the spacing between two adjacent chip micropores is 200-600 nm; the microfluidic chip comprises a bottom plate and a microchannel structure arranged on the bottom plate, the microchannel structure comprises a first microchannel suitable for the flow of the culture solution containing intestinal wall cells and a second microchannel suitable for the flow of the intestinal tract simulation solution, the first microchannel and the second microchannel are connected in communication, the super surface plasmon resonance chip is arranged at the communication position of the first microchannel and the second microchannel, the flow direction of the culture solution in the first microchannel is opposite to the flow direction of the intestinal tract simulation solution in the second microchannel, and the bottom plate is made of PDMS material; The process of the biomimetic treatment comprises: adding a cell adhesion agent into the nanopore, coating for 14-16 h at a temperature of 4-6 DEG C, and then removing the cell adhesion agent, washing and drying, the cell adhesion agent is polyethyleneimine, the cell adhesion agent in a single nanopore is added in the form of a solution with a concentration of 8-12 mu g / mL and an addition amount of 50-100 mu L; the culture conditions of the intestinal wall cells at least comprise: the number of cells inoculated in a single chip micropore is 0.1-20 thousand, the temperature is 35-40 DEG C, and the CO2 concentration is 4-6%; the intestinal wall cells are Caco-2 cells and HT29-MTX cells, and the density ratio of the Caco-2 cells to the HT29-MTX cells in the culture solution containing the intestinal wall cells is 4-10:1; the intestinal tract simulation solution contains 10-50 mu g / L epidermal factor and 5-10 mu g / L N-2 additive, and the flow rate of the intestinal tract simulation solution is 0.4-0.6 mu L / min.

[0052] The intestinal wall cell mucus layer model constructed by the above particularly preferred embodiment can realize label-free continuous monitoring of the formation process of the intestinal wall cell mucus layer, dynamically observe the physiological changes of the cells, and simulate the real intestinal tract environment, and the method has high sensitivity and accuracy and low detection cost.

[0053] The second aspect of the present application provides the application of the intestinal wall cell mucus layer model constructed by the construction method of the first aspect in the evaluation of the interaction between mucus proteins and probiotics.

[0054] In the present application, the intestinal cell mucus layer model obtained can screen probiotic strains with stronger interaction with mucus proteins and better colonization ability on the intestinal mucosal surface, thereby improving the effect and stability of the probiotic preparation.

[0055] According to the present application, preferably, the probiotic is selected from at least one of Bifidobacterium longum BL21, Bifidobacterium breve BBr60, Bifidobacterium lactis BLa80, Lactobacillus gasseri LG08 and Lactobacillus rhamnosus LRa05.

[0056] The present application will be described in detail below through examples.

[0057] The experimental methods and equipment described in the following examples are all conventional methods and equipment unless otherwise specified. The experimental materials used in the following examples are all commercially available unless otherwise specified.

[0058] Caco-2 cells were purchased from Promocell, model CL-0050; HT29-MTX cells were purchased from Zhijiao Xin Zhou Company, model ZQ1181; polyethyleneimine (PEI) was purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.; recombinant human fibronectin was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., product model S234010; silk fibroin was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., product model S571100; type IV collagen was purchased from Corning, model C5533; complete medium was purchased from Jiangsu Kaigi Biotechnology Co., Ltd. Biology, product model KGL1206-500; epidermal growth factor was purchased from Yixing Biological Technology (Shanghai) Co., Ltd., model 92708ES60; N-2 additive was purchased from Zhikopu Risheng Company, model PRS-N2S.

[0059] Preparation Example 1 Example 1 Preparation of MetaSPR chip 1.1 MetaSPR chip I The MetaSPR sensor chip comprises a substrate (PET material), a plurality of nanopores imprinted on the substrate, and a metal film covering the surface of the nanopores, the metal film comprising a transition metal film layer and a noble metal film layer arranged from inside to outside, the nanopores are frustum-shaped and have a large opening diameter of 200 nm and a depth of 500 nm, the spacing between adjacent two nanopores is 400 nm, the transition metal film layer comprises a titanium film layer with a thickness of 5 nm, and the noble metal film layer comprises a silver film layer with a thickness of 60 nm and a gold film layer with a thickness of 20 nm arranged from inside to outside; the specific preparation process is as follows: the surface of the PET substrate is pre-pressed to obtain a matrix array of nanopores, and then a metal film is evaporated on the substrate.

[0060] The MetaSPR sensor chip is cut into 13 cm x 8.5 cm fragments and pasted onto a 96-well super surface plasmon resonance chip plate, which has chip micropores corresponding to the nanopores, each chip micropore has a height of 500 nm, an upper circular radius of 200 nm at the opening, and a bottom circular diameter of 100 nm, to serve as the MetaSPR chip I. The structure of the MetaSPR chip I is shown in Figures 1 to 3 .

[0061] 1.2 MetaSPR chip II The MetaSPR chip II is prepared according to the method of 1.1, except that the noble metal film layer is a 60 nm gold film layer. The cross-sectional structure diagram of the obtained MetaSPR chip II is shown in Figure 4 .

[0062] In the experiment, the MetaSPR chip I and the MetaSPR chip II are used respectively, under the same conditions, according to the above experimental operation for screening cell adhesion conditions, and only 10 μg / mL polyethyleneimine (PEI) is selected as the chip modification condition; Caco-2 cells 10000 cells / well.

[0063] Figure 5 The real-time curves of cell growth of the MetaSPR chips I and II prepared for Example 1 are shown. The results show that the cell growth state of the pure gold chip is better than that of the gold-silver chip, which is more conducive to the adhesion growth of cells.

[0064] Example 2 Screening cell adhesion condition experiment Chip selection: the MetaSPR chip II (5 nm titanium film and 60 nm gold film) prepared in 1.2 of Example 1.

[0065] Chip modification condition: 10 μg / mL silk glue protein, 10 μg / mL recombinant human fibrin; 10 μg / mL type IV collagen; 10 μg / mL polyethyleneimine (PEI); no treatment.

[0066] Modification method: use ultrapure water to dilute the silk glue protein, recombinant human fibrin, and type IV collagen to the corresponding concentrations (i.e. the same as the above chip modification conditions) respectively to obtain modification reagents, 50 μL of modification reagents are added to each well, the chip modification condition without treatment means that only 50 μL of ultrapure water is added; seal the plate with a sealing film, and stand overnight (more than 14 h) at 4°C; before use, pour out the liquid in the well plate, wash once with 100 μL of ultrapure water, and dry for standby.

[0067] Cell plating: Caco-2 cells and HT29-MTX cells were cultured to more than 80% confluence, and the complete medium (DMEM + 10% FBS (v / v) + 1% P / S (v / v) was discarded. The cells were washed once with sterile PBS, and the cells were digested with 0.25% (w / v) trypsin, and the digestion was terminated with complete medium. The cells were collected at 200g for 7 min, and the supernatant was discarded. The cells were resuspended with complete medium (DMEM + 10% FBS (v / v) + 1% P / S (v / v). Different ratios of mixed cells (Caco-2 cells and HT29-MTX cells with density ratios of 9:1, 7:3, and 6:1) were added to the chip plate at 150 μL / well of cell suspension (i.e., 1250 cells / well, 2500 cells / well, 5000 cells / well, and 10000 cells / well). The negative control wells were added with 150 uL of complete medium (DMEM + 10% FBS (v / v) + 1% P / S (v / v), and the plate was sealed with a sealing film and placed in a 37°C, 5% CO2 incubator.

[0068] Experimental detection: The real-time curve of cell adhesion growth was detected by MetaSPR technology.

[0069] Experimental results: The optimal cell plating number for real-time detection of cell growth curve was 20000 cells / well. Figure 6 The real-time curve of cell adhesion growth for different cell concentrations (i.e., 5000 cells / well, 10000 cells / well, and 20000 cells / well) was detected. The negative control wells were added with 150 uL of complete medium. 10 ug / mL polyethyleneimine (PEI) was the optimal cell adhesion modification condition. Figure 7 The real-time curve of cell adhesion growth for different modification agents was detected.

[0070] Example 3: Microfluidic chip design experiment (1) The MetaSPR chip II prepared in Example 1.2 was used to modify the chip surface with 10 ug / mL polyethyleneimine (PEI) solution; (2) A microfluidic chip was prepared by soft lithography technology using polydimethylsiloxane (PDMS) material, see Figure 7 and Figure 8, the microfluidic chip comprises a bottom plate and a microfluid channel structure arranged on the bottom plate, the microfluid channel structure comprises a first microfluid channel and a second microfluid channel, the first microfluid channel and the second microfluid channel are communicated, the MetaSPR chip II is arranged at the communication position of the first microfluid channel and the second microfluid channel, the cross section of the communication position of the first microfluid channel and the second microfluid channel in the horizontal direction is a hexagon, and the surface of the MetaSPR chip II is ensured to be exposed in the microfluid channel structure; one end of the first microfluid channel is provided with a cell sample inlet, and the other end is provided with a cell sample outlet; one end of the second microfluid channel is provided with a microfluidic sample inlet, and the other end is provided with a microfluidic sample outlet; the culture solution containing intestinal wall cells is input to the surface of the MetaSPR chip II through the first microfluid channel for cell culture, and the intestinal tract simulation solution is input to the surface of the MetaSPR chip II through the second microfluid channel, the flow state of the fluid in the intestinal tract is simulated, and the flow directions of the liquids in the first microfluid channel and the second microfluid channel are opposite.

[0071] Example 4: Monitoring the formation of mucus layer of cells by microfluidic technology combined with MetaSPR chip (1) Preparation of cell suspension: Caco-2 cells and HT29-MTX cells with a confluence of 80% were taken, digested with 0.25wt% trypsin, resuspended in DMEM complete culture medium (10wt% FBS, 1wt% double-antibiotic), mixed according to a density ratio of 6:1, and adjusted to a cell density of 1×10 5 / mL; (2) Cell inoculation and adhesion: the microfluidic chip and the MetaSPR chip II with a surface modified by PEI provided in Example 3 were used, the cell suspension was injected into the first microfluid channel through the cell sample inlet of the microfluidic chip at a flow rate of 0.1μL / min, so that the cells were uniformly distributed on the surface of the MetaSPR chip II, and the cells were placed in a 37℃, 5% CO2 incubator for 4h, and the adhesion state of the cells was monitored in real time by MetaSPR technology; when the cell density reached 10000 cells per well, the inoculation was stopped (at this time, the adhesion rate was >90%); (3) Dynamic culture of mucus layer: after the adhesion was completed, the culture solution (containing 10μg / L epidermal factor and 5μg / L N-2) was continuously input from the second microfluid channel through the microfluidic sample inlet at a flow rate of 0.5μL / min, so as to simulate the intestinal fluid environment; (4) The culture solution was replaced every 2 days, and during the 21-day culture period, the refractive index change (wavelength 633nm, incident angle 40-70°) in the mucus layer formation process was monitored in real time by MetaSPR, and when the refractive index fluctuation was stable, the mucus layer was determined to be mature (thickness about 12.5±1.8μm).

[0072] Example 5: Evaluation of the interaction between mucus protein and probiotics by using the intestinal cell mucus layer model The formation of the cell mucus layer was monitored according to the method of Example 4, except that in step (3), after the cells were attached, the co-culture was performed for 1-7 days, and after the mucus layer was formed, the activated probiotic culture solution was taken from Suzhou Weikang Probiotics Co., Ltd. (Bifidobacterium longum BL21, Bifidobacterium breve BBr60, Bifidobacterium lactis BLa80, Lactobacillus gasseri LG08 or Lactobacillus rhamnosus LRa05), centrifuged at 5000 rpm for 10 minutes at 4°C, the supernatant was discarded, the precipitate was resuspended with sterile PBS and the bacterial concentration was adjusted to 10 7 -10 11 CFU / mL. The injection pump flow rate (0.2-0.5 μL / min) was set in the first microfluidic channel for continuous injection for 24 h, so that the strains were uniformly attached to the inner wall of the channel or suspended in the channel. The second microfluidic channel was continuously fed with cell complete medium at a flow rate of 0.5 μL / min through the microfluidic sampling port to provide the required nutrients for the cells and to remove cell metabolites. The adhesion effect of different probiotics on the mucus layer was monitored in real time for 2 h by MetaSPR. The real-time curve of screening probiotics in the microfluidic chip in Example 5 is shown in Figure 10 .

[0073] The preferred embodiments of the application are described in detail above, but the application is not limited thereto. Within the technical concept of the application, various simple modifications can be made to the technical solutions of the application, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be considered as disclosed by the application and fall within the protection scope of the application.

Claims

1. A method of constructing an intestinal cell mucus layer model, characterized by, The method comprises the following steps: biomimetic treatment is performed on the surface of a surface plasmon resonance chip; a culture solution containing intestinal wall cells is input into the surface of the surface plasmon resonance chip through a microfluidic chip to culture the intestinal wall cells; an intestinal tract simulation solution is input into the surface of the surface plasmon resonance chip through the microfluidic chip; the refractive index change of the surface of the surface plasmon resonance chip is detected; and the formation process of the mucus layer of the intestinal wall cells is monitored in real time according to the refractive index change.

2. The construction method of claim 1, wherein, The surface plasmon resonance chip comprises a surface plasmon resonance chip plate and a MetaSPR sensor chip integrated on the surface plasmon resonance chip plate; the surface plasmon resonance chip plate is provided with a plurality of chip micropores; the MetaSPR sensor chip comprises a substrate, a plurality of nanopores which are imprinted on the substrate and correspond to the chip micropores, and a metal film which covers the surface of the nanopores; and the metal film comprises a transition metal film layer and a noble metal film layer which are arranged from inside to outside. Preferably, the substrate is made of PET material; the transition metal film layer is a titanium film layer with a thickness of 4-6 nm; and the noble metal film layer comprises a gold film layer with a thickness of 15-25 nm and / or a silver film layer with a thickness of 50-70 nm. Preferably, the nanopores are frustum-shaped, the diameter of the large opening of the nanopores is 100-400 nm, the depth of the nanopores is 400-600 nm, and the spacing between two adjacent chip micropores is 200-600 nm.

3. The construction method of claim 2, wherein, The microfluidic chip comprises a bottom plate and a microfluid channel structure arranged on the bottom plate; the microfluid channel structure comprises a first microfluid channel suitable for the flow of the culture solution containing intestinal wall cells and a second microfluid channel suitable for the flow of the intestinal tract simulation solution; the first microfluid channel and the second microfluid channel are connected in communication; and the surface plasmon resonance chip is arranged at the communication position of the first microfluid channel and the second microfluid channel. Preferably, the flow direction of the culture solution in the first microfluid channel and the flow direction of the intestinal tract simulation solution in the second microfluid channel are opposite. Preferably, the bottom plate is made of PDMS material.

4. The construction method of claim 2, wherein, The process of the biomimetic treatment comprises the following steps: adding an adhesion agent into the nanopores to form a film, removing the adhesion agent, washing, and drying.

5. The construction method according to claim 4, characterized in that, The adhesion agent is selected from at least one of sericin, recombinant human fibrin, collagen IV, and polyethyleneimine, and is preferably polyethyleneimine. Preferably, the adhesion agent is added in the form of a solution with a concentration of 8-12 μg / mL and an addition amount of 50-100 μL in each nanopore. Preferably, the film forming conditions comprise the following: a temperature of 4-6 ℃ and a time of 14-16 h.

6. The method of construction of claim 2, wherein, The culture conditions of the intestinal wall cells comprise the following: the number of cells inoculated in each chip micropore is 0.1-20 thousand, the temperature is 35-40 ℃, and the CO2 concentration is 4-6 %.

7. The construction method of claim 6, wherein, The intestinal wall cells are Caco-2 cells and / or HT29-MTX cells, and are preferably Caco-2 cells and HT29-MTX cells. Preferably, the density ratio of Caco-2 cells to HT29-MTX cells in the culture solution containing intestinal wall cells is 4-10:

1.

8. The construction method according to any one of claims 1 to 7, wherein, The intestinal fluid simulation solution contains 10-50 μg / L epidermal factor and 5-10 μg / L N-2 additive; Preferably, the flow rate of the intestinal fluid simulation solution is 0.4-0.6 μL / min.

9. Use of the intestinal cell mucus layer model obtained by the construction method of any one of claims 1 to 8 in evaluating the interaction of mucin with probiotics.

10. Use according to claim 9, characterized in that, The probiotics are selected from at least one of Bifidobacterium longum BL21, Bifidobacterium breve BBr60, Bifidobacterium lactis BLa80, Lactobacillus gasseri LG08 and Lactobacillus rhamnosus LRa05.

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