CN107497503B - Microfluidic chip for researching tumor single cell invasion and epithelial mesenchymal transition - Google Patents

Abstract

The invention provides a micro-fluidic chip for researching the invasion of single tumor cells and the epithelial-mesenchymal transition process capacity, which comprises four layers of structures which are sequentially stacked together and sealed with each other, and a top cover, a micro-fluidic channel layer, a substrate with a micropore array and a bottom plate with a cavity group from top to bottom. The invention also provides a system matched with the chip and used for single cell detection. The micro-fluidic chip and the corresponding detection system thereof can be used for rapidly, simply and cheaply sorting single cells, researching the invasion capacity and epithelial-mesenchymal transition, and carrying out gene analysis on target single cells.

Description

Microfluidic chip for researching tumor single cell invasion and epithelial mesenchymal transition

Technical Field

The invention belongs to the technical field of microfluidic chips, and particularly relates to a microfluidic chip for researching tumor cell invasion capacity and epithelial-mesenchymal transition

Background

In recent years, the incidence and mortality of cancer has rapidly increased and has become the internationally recognized first disease of mortality. In cancer cases of death, however, the spread and metastasis of cancer are major factors in death. Therefore, the research on the tumor spreading and metastasis mechanism is greatly helpful for understanding the occurrence and development of cancer and overcoming the cancer.

More than 90% of the tumor cells are derived from epithelial cells. The epithelial cells need to be transformed into mesenchymal cells with migration and invasion capacity for the processes of diffusion and metastasis. The physiological phenomenon that epithelial cells with polarity lose polarity and are transformed into mesenchymal cells with migration ability is called epithelial-mesenchymal transition (EMT). With the intensive clinical research on the cancer EMT process, the EMT is found to promote the in-situ cancer to develop into the invasive cancer and simultaneously influence the prognosis and treatment effect of cancer patients. The generation of EMT and Circulating Tumor Cells (CTCs) is closely inseparable; CTCs are currently clinically used in patients with aggressive cancer as one of the indicators for prognosis, recurrence and survival rate prediction. EMT can make cancer cells obtain stem cell characteristics such as self-renewal, and is the core reason of cancer treatment difficulty and recurrence. The EMT promotes the generation of heterogeneity of tumor cells, and the heterogeneous nature makes the morphology and function of tumor cells in a dynamic changing process, which brings great difficulty to the diagnosis and treatment of tumors. Therefore, the research on the EMT process has important clinical significance for resisting tumor metastasis, reducing the mortality of cancer patients and the like.

The presence of tumor cell heterogeneity increases the complexity of studying EMT: different cells respond differently to EMT-inducing agents; cells that respond to EMT may not migrate. Most tumor cells exhibit heterogeneity: the tumor cells have differences in cell phenotype, invasion and proliferation capacity, cell action and the like. The occurrence of heterogeneity makes cells in dynamic change process, which brings difficulties in precise diagnosis and treatment and anti-metastasis. Studies have shown that the initial invasive cells associated with diffusion and metastasis, often a very small population of cells, exhibit heterogeneity. And the population is achieved by individual migration of single cells during the transfer process. If population analysis is performed, information about these rare cells will be missed. Therefore, the method is significant for the research on the invasiveness of the tumor cells and the EMT process on the single cell level, accurate diagnosis and treatment, metastasis resistance and the like.

However, due to the limitations of detection methods and sorting techniques, cell invasiveness and EMT processes have been studied for a long time at the level of research on population cells, such as commercial invasion chambers, and the like.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention aims to provide the microfluidic chip for researching the invasion capacity of single-cell-level tumor cells and epithelial-mesenchymal transition.

Another objective of the invention is to provide a system for detecting the microfluidic chip for studying the invasion capacity of single-cell-level tumor cells and the epithelial-mesenchymal transition of tumors.

The third purpose of the invention is to propose the application of the microfluidic chip.

The technical scheme for realizing the above purpose of the invention is as follows:

a micro-fluidic chip for researching tumor single-cell invasion and epithelial-mesenchymal transition comprises four layers of structures which are sequentially stacked, wherein the four layers of structures are respectively a top cover, a micro-fluidic channel layer, a substrate with a sorting unit array and a bottom plate with a cavity group from top to bottom, and the sorting unit on the substrate and the cavity on the bottom plate are oppositely arranged;

the sorting unit is provided with a square micro-well on the upper surface of the substrate and is used for obtaining single cells; the square micro-well penetrates through the substrate in the vertical direction, the bottom of the square micro-well is a square bottom plate provided with a plurality of small micropores, and the square bottom plate and the substrate are connected into a whole.

Wherein, the top cover is provided with a sample inlet and a sample outlet; the microfluidic channel layer is provided with a flow channel, the flow channel is in a strip shape with a wide middle section and gradually narrowed two ends, the middle section of the flow channel is opposite to the region of the substrate provided with the sorting unit array, and the two ends of the flow channel are respectively opposite to the sample inlet and the sample outlet.

Preferably, the depth of the channel on the microfluidic channel layer is 10-200 microns, the width of the middle section of the flow channel is 1-20 mm, and the length of the microfluidic channel is 10-100 mm.

Furthermore, the cavity on the bottom plate is square, a connecting hole is formed in the bottom of the cavity, the opening of the connecting hole is square, the side length is 5-1000 micrometers, and the length of the connecting hole is 5-10000 micrometers; the side length of the cavity is 15-5000 microns, and the depth is 5-5000 microns.

The connecting hole below each cavity is used for exchanging with the outside or introducing appointed liquid; the connecting holes can be independent and unconnected with each other, and the cavities can be appointed to be connected with each other as required to share one connecting hole.

The number and arrangement mode of the sorting units on the substrate are arranged according to requirements, and the positions of the sorting units correspond to the flow channels. Preferably in a staggered array; and an extracellular matrix layer simulating the tumor cell invasion environment is paved on the square bottom plate of the square micro-well.

Wherein the thickness of the spread extracellular matrix layer is 20-1000 microns. Preferably, the thickness is 50 to 200 micrometers.

One of the preferable technical schemes of the invention is that the side length of the opening of the sorting unit on the substrate is 10-1000 microns, and the depth of the square micro-well is 10-1000 microns; the side length of the micro-hole formed on the square bottom plate is 5-100 micrometers, and the thickness (namely the depth of the micro-hole) of the square bottom plate is 2-100 micrometers. The number of the micropores formed in one square bottom plate is 4-25.

The top cover is made of transparent materials, the microfluidic channel layer is made of insulating polymers or silicon, and the flow channel is made by an etching method; the substrate is made of insulated polymer, glass or silicon, and is made into a sorting unit array by adopting a photoetching and/or etching method; the bottom plate is made of transparent polymer, glass, silicon or stainless steel, and a square cavity is formed by adopting an acid etching and/or photoetching method.

A system for studying tumor single cell invasion and epithelial-mesenchymal transition, comprising the microfluidic chip of any one of claims 1-8, and a fluorescence probe, a fluorescence microscope, an image processing device, a syringe pump, a constant temperature water bath and a Polymerase Chain Reaction (PCR) amplifier;

the fluorescent probe is used for monitoring and analyzing protein expression information of a sample, the fluorescent microscope and the image processing equipment are used for observing the microfluidic chip, and the injection pump is connected with the microfluidic chip and used for sample injection; the PCR amplification instrument is used for analyzing the genome information of the separated cells.

The fluorescent probe is an antibody or polypeptide marked with a fluorescent molecule, can be used for identifying a single protein (such as E-cadherin, epithelial cell cadherin or Vimentin and Vimentin) by using one fluorescent probe independently, and can also be used for identifying a plurality of target proteins by using a plurality of fluorescent probes simultaneously.

The micro-fluidic chip disclosed by the invention is applied to the research of tumor single cell invasion and epithelial mesenchymal transition.

The invention further provides a method for researching and detecting the invasion capacity and epithelial-mesenchymal transition of the single-cell level tumor cells, wherein cell suspension is injected into the microfluidic chip, and the cells fall into the upper hole of the substrate sorting unit in a single-cell form. The upper well is pre-loaded with an extracellular matrix layer (the thickness of which can be adjusted as required) for studying the invasion of cells, and the tumor cells are landed on the extracellular matrix layer in the larger microwell. The tumor cells with invasive ability gradually decompose the extracellular matrix, pass through the substrate through the small holes below to reach the lower layer of the substrate, while the cells without invasive ability cannot decompose the extracellular matrix and thus cannot pass through the substrate, and remain in the microwells, thereby screening the single tumor cells with invasive ability. The screened tumor cells can be continuously cultured or further studied. Meanwhile, different drugs or chemotactic factors can be injected into the chip through the connecting holes of the bottom plate, so that the invasion and migration conditions of tumor cells under different conditions can be researched. Then injecting fluorescent probes with different antibodies, and researching the change and hot spots of the protein expression of the tumor cells by using an immunofluorescence method. Meanwhile, after the cells pass through the substrate and reach the cavity of the bottom plate, lysis solution, gene reverse transcription or gene amplification reagent can be introduced, gene amplification is directly carried out on the chip, and then amplification products are taken out to study the gene expression condition of the cells.

The invention develops a multilayer microstructure chip by introducing a micromachining technology and a microfluidic chip technology, realizes the screening of tumor cells with invasive capability at a single cell level, simultaneously researches the phenotypic change of the tumor cell invasive process and the influence factors of medicines and the like, and detects and analyzes the screened tumor cells on the gene expression level. The invention provides a new method for researching the invasiveness of the tumor cells at the single cell level, and can be used for researching the invasiveness of the cells at the single cell level more efficiently, more accurately and with higher flux.

The invention has the following advantages:

the research on the invasiveness of the tumor cells on the single cell level is realized. Since invasion of in situ tumor cells is often caused by a very small amount of single cells, it is of great significance to perform invasion experimental study at a single cell level. However, the traditional methods for studying tumor cell invasion are all mass studies, and cannot be applied to single cell studies. The invention develops a method for researching the cell invasion at the single cell level by means of a microfluidic chip, and has great significance for the research on the diffusion and the metastasis of cancers.

And (II) the research of the tumor cell EMT on the single cell level is realized. The response of the same cells to EMT-inducing agents is different due to the presence of tumor cell heterogeneity; cells that respond to EMT may not migrate. The current study of EMT remains in a population-based assay, resulting in the loss of cellular information that is partially heterogeneous. The invention develops a method for researching the EMT of the cells on the single cell level by means of the microfluidic chip, and further defines the generation mechanism of the EMT and the generation of the heterogeneity of the EMT process.

And (III) the high integration of a plurality of experimental steps is realized, the invention integrates complicated single cell acquisition, invasive research, protein and gene analysis on one microfluidic chip by utilizing the microfluidic technology, and compared with the prior art, the invention greatly reduces the operation steps, thereby improving the success rate and the reliability of the experiment. Meanwhile, depending on a highly integrated chip and simplified operation steps, the optical identification and the reaction control based on the injection pump can be completed by automatic means such as camera shooting, image analysis, single chip microcomputer processing and programming, the processing speed is improved, the accuracy is greatly improved, the experiment time is shortened, and the sample processing efficiency is improved.

And fourthly, the detection system and the detection method corresponding to the microfluidic chip provided by the invention have multiple detection functions, and are wide in application range and strong in universality. The chip and the detection method realize the screening of tumor cells with invasive ability aiming at the single cell level, simultaneously research the invasive process and influencing factors of the tumor cells, and detect and analyze the screened tumor cells on the protein and gene level, thereby having multiple functions. Besides the function of researching tumor invasiveness, different drugs and induction factors can be injected into the connecting hole of the chip base plate, so that the chip base plate can be used for researching inflammatory reaction and other aspects, and the design of the chip is not required to be changed. The cost is not increased, and the application fields of the chip and the detection system are greatly expanded.

Drawings

Fig. 1 is an exploded view of a microfluidic chip according to the present invention.

Figure 2 is a cross-sectional view a-a of the substrate and base plate combination.

In the figure, 1-sample inlet; 2-a top cover; 3-a sample outlet; 4-a fluid channel; 5-a substrate with a sorting array; 6-a sorting unit; 7-a bottom plate; 8-a bottom plate cavity; 9-cavity connecting groove; 10-cavity intercommunicating pore; 11-square micro-wells; 12-micro holes on a square base plate; 13-extracellular basement membrane position.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Unless otherwise specified, the means used in the examples are all technical means known in the art.

Example 1 microfluidic chip for studying tumor single cell invasion and epithelial-mesenchymal transition and preparation method thereof

The microfluidic chip provided by the embodiment comprises four layers of structures which are sequentially stacked together and sealed with each other, namely a top cover, a microfluidic channel layer, a substrate with a sorting array and a bottom plate with a cavity from top to bottom, wherein the sorting unit on the substrate and the cavity on the bottom plate are arranged oppositely; the sorting unit is provided with a square micro-well on the upper surface of the substrate and is used for obtaining single cells; the square micro-well penetrates through the substrate in the vertical direction, the bottom of the square micro-well is a square bottom plate provided with a plurality of small micropores, and the square bottom plate and the substrate are connected into a whole. The concrete structure is as follows:

1. header and fluid channel

When cells are identified, light needs to penetrate through the top cover, so that the top cover needs to be made of a transparent material; meanwhile, the top cover needs to be provided with a fluid inlet and outlet interface, so that holes need to be formed; finally, the top cover needs to be sealed with the fluid channel and sealed, so the material selection needs to consider the compatibility with the fluid channel.

The fluid channel plays a role in restricting cell flow, and the characteristic dimension of the fluid channel is in millimeter level, so that materials which are convenient to process, low in cost and good in biocompatibility need to be adopted.

The substrate material may be a material manufactured into a specific specification and shape by evaporation, cutting and thermoforming. Particularly preferred are relatively thin and transparent insulating polymers.

The dimensions of the top cover and the fluid channel are matched to the micro-well array substrate. The width of the fluid channel is also matched to the characteristic dimension (side length or diameter) of the structures on the substrate.

In the preferred embodiment shown in fig. 1, the top cover is made of glass material, and the fluid channel and the bottom plate are made of PDMS (polydimethylsiloxane) material and are hermetically connected in a bonding manner.

In the preferred embodiment shown in fig. 1, the microfluidic channel has a length of 45 mm and a width of 10 mm; this is the optimal condition for application to tumor cells. When other cells need to be processed, the appropriate microfluidic channel size can be selected at the discretion of the skilled person.

2. Substrate with microstructure array

In this embodiment, the microwell array is a key component for containing and processing cells. To accommodate the size of the cell, materials must be selected that are compatible with existing microfabrication techniques. In the preferred embodiment shown in fig. 2, the silicon substrate material is chosen because silicon is well compatible with lithographic etching and etching based micromachining processes, and thus arrays of microwells that conform to the size of the cells are readily available.

In the preferred embodiment shown in fig. 2, the upper layer of the sorting unit has square pores with a side length of 100 microns and a depth of 150 microns; the lower layer of micropores is square, with a side length of 8 microns and a depth of 10 microns (through the chip sorting substrate). 9 small holes are distributed on the bottom of the upper layer of micropores in an array mode. In the larger microwells of the upper layer and the smaller microwells of the lower layer, an extracellular matrix (Matrigel, commercially available) for simulating the environment of tumor cell invasion, 100 μm thick, is laid on the extracellular matrix membrane at the position 13 shown in the figure.

In the preferred embodiment shown in fig. 2, the sorting cells are shown in a staggered array, with each cell being spaced 400 microns apart. The entire array was 10 mm wide and 35 mm long.

3. Base plate

In the embodiment, the bottom plate is provided with micro cavities which are arranged in an array mode, the position of each micro cavity corresponds to the position of the upper-layer micro hole in the sorting substrate, each micro cavity is square, the side length is 200 micrometers, the depth is 1000 micrometers, the bottom of each micro cavity is provided with a connecting hole with the length of 5-10000 micrometers, the connecting hole is connected with the outside in an interactive mode, the opening of the connecting hole at the bottom of each micro cavity is rectangular, and the side length is 5 × 50 micrometers.

In the preferred embodiment shown in fig. 1, the material of the base plate is PDMS.

4. Sealing method

In the preferred embodiment shown in FIG. 1, the materials of the top cover, the microfluidic channels, the array substrate and the bottom plate are glass, PDMS, silicon and PDMS, respectively. The oxygen plasma-assisted bonding method can well realize good sealing between glass and PDMS and between silicon and PDMS; the sealing between the silicon and the glass can be realized by adopting an anodic bonding method.

In other embodiments, suitable sealing methods may be selected depending on the materials of the top cover, microfluidic channel, sorting substrate, and bottom plate.

5. Chip matching system

Also provided in this example are systems for studying tumor single cell invasion and epithelial-mesenchymal transition. Besides the microfluidic chip, a fluorescent Probe (Probe), a gene amplification reagent, a fluorescence microscope, an image processing device, an injection pump, a constant temperature water bath, a PCR amplification instrument and the like are required to form a complete system.

The fluorescent probe is used for researching the condition of the cell expressing protein, in a preferred embodiment shown in figure 2, E-cadherin marked with green fluorescence and Vimentin antibody marked with red fluorescence are used as the fluorescent probe for researching the change of protein expression in the EMT action of tumor cells. In other embodiments, different antibodies or polypeptides may be selected as probes for different proteins.

The fluorescence microscope is used for detecting the binding condition of the antibody with the fluorescent label, thereby indirectly detecting the protein expression condition of the tumor cells.

The syringe pump is used to drive microfluidics and inject relevant reagents into the chip.

Lysis and amplification reagents were used to sort out cells for gene analysis.

6. Chip manufacturing method

The microfluidic chip and the detection system are successfully manufactured by adopting the following manufacturing process. The specific manufacturing method is to help those skilled in the art understand the technical solution of the present invention, and is not to limit the materials, dimensions and manufacturing method of the device of the present invention.

The manufacturing method of the micro-fluidic chip comprises the following steps:

the top cover 2: rectangular pieces were cut from 4 inch Pyrex7740 glass (corning corporation) in 6 cm long by 2 cm wide dimensions and laser drilled at specific locations on each piece to create sample inlet 1 and sample outlet 3 holes.

The fluid passage 4: an N-type 4-inch silicon wafer is adopted, and after the planar shape of the flow channel is photoetched, a groove with the depth of 100 micrometers is formed by using ICP dry etching (induction plasma etching, namely, high-energy plasma of sulfur hexafluoride and carbon tetrafluoride is used for etching silicon) which is commonly used for semiconductors. And pouring the liquid PDMS into a tank, demolding and taking out after the liquid PDMS is solidified, cutting off more PDMS, and cutting the PDMS into rectangular small pieces according to the overall dimensions of 6 cm in length and 2 cm in width to obtain the microfluidic channel layer.

Substrate with sort array 5: an N-type 4-inch silicon wafer is adopted, and then square micropores are etched downwards in a sorting area by utilizing the technologies of photoetching and dry etching. And carrying out alignment photoetching on the back of the silicon wafer to form a channel position at the bottom of the silicon micropore, and carrying out dry etching by adopting reactive ion etching to penetrate through the silicon wafer to finally obtain a complete silicon micropore array. Then, the cut pieces are cut into rectangles according to the external dimensions of 6 cm in length and 2 cm in width.

Bottom plate 7: the PDMS material is adopted, a silicon wafer is used as a mould to carry out the standard photoetching processing, the positions of the cavity 8 of the bottom plate and the communication hole 10 of the cavity are photoetched, a rectangular cavity connecting groove 9 (the connecting groove is a groove on the bottom plate) connected between the cavities 8 of the bottom plate is manufactured by using a secondary photoetching technology to form a cavity group, and a control valve is added to connect the collection cavity with the amplification cavity.

Sealing: bonding a silicon substrate and a base plate PDMS together, cutting the silicon substrate and the base plate PDMS into rectangular small pieces according to the overall dimension of 6 cm in length and 2 cm in width, treating the bottom surface of a top cover, two surfaces of a microfluidic channel and the top surface of the silicon substrate by using oxygen plasma, and sequentially bonding the two surfaces together to finally obtain a complete chip.

Chip system: on the basis of the microfluid chip, a common plastic pipe is used for connecting the pump and a fluid inlet and a fluid outlet on the top cover, and is simultaneously connected with a connecting hole on the bottom plate. When the analysis result is needed, the chip is placed under a fluorescence microscope and connected with an image processing device for analysis. When gene analysis is needed, the needed reagent is injected into the chip to be sealed and put into a constant temperature water bath kettle, and the construction of the whole set of system can be completed.

7. Specific application method

The microfluidic chip and the corresponding detection system of the invention have been successfully applied to the detection of tumor invasion capacity and EMT process at the single cell level by the following methods. The specific method is provided to help those skilled in the art understand the function and application method of the present invention, and is not intended to limit the application scope of the device of the present invention.

Blood or tumor tissue of a tumor patient is subjected to enrichment treatment and the like to prepare a mixed sample, and the mixed sample is introduced into the microfluidic chip. And (4) after standing, washing the cells after the cells fall into the capture microwells, and washing away the cells which do not enter the microwells. After completion, the chip was placed in a cell incubator for culture. The upper layer is filled with a culture medium without fetal calf serum, and the lower layer cavity is filled with a culture medium containing high-concentration fetal calf serum to induce cell invasion. And taking out the chip after a period of time. Injecting E-cadherin with green fluorescence and Vimentin antibody marked with red fluorescence into the bottom plate through a connecting hole of the bottom plate, and analyzing the protein condition by using a fluorescence microscope and an image analysis system. Then cell lysate and gene amplification reagent are introduced into the chamber containing the target cells, and the chip is put into a constant temperature water bath box at 30 ℃ for amplification for 3 hours. Then, the amplified product is taken out for gene analysis such as sequencing.

Example 2

The microfluidic chip provided by the embodiment comprises four layers of structures which are sequentially stacked together and sealed with each other, namely a top cover, a microfluidic channel layer, a substrate with a sorting array and a bottom plate with a cavity from top to bottom, wherein the sorting unit on the substrate and the cavity on the bottom plate are arranged oppositely; the sorting unit is provided with a square micro-well on the upper surface of the substrate and is used for obtaining single cells; the square micro-well penetrates through the substrate in the vertical direction, the bottom of the square micro-well is a square bottom plate provided with a plurality of small micropores, and the square bottom plate and the substrate are connected into a whole.

1. Header and fluid channel

The top cover is provided with a sample inlet and a sample outlet; the microfluidic channel layer is provided with a flow channel, the flow channel is in a strip shape with a wide middle section and gradually narrowed two ends, the middle section of the flow channel is opposite to the region of the substrate provided with the sorting unit array, and the two ends of the flow channel are respectively opposite to the sample inlet and the sample outlet. The microfluidic channel was 45 mm long with a middle section of 10 mm width.

In this embodiment, the cap and the fluid channel are formed in one step using the same polymer material (PDMS), and do not need to be separately manufactured and sealed together.

2. Substrate with microstructure array

In the embodiment, a silicon substrate material is selected, the square micro-wells on the sorting unit are square, the side length is 100 micrometers, and the depth is 180 micrometers; the micro-wells are formed on a square bottom plate, and the side length of each micro-well is 8 micrometers, and the depth of each micro-well is 20 micrometers (penetrating through a substrate). 9 small holes are distributed in the square bottom plate array. An extracellular matrix layer (Matrigel) for simulating the invasion environment of tumor cells is paved on the square bottom plate in the micro-well, and the thickness of the matrix layer is 100 microns.

The sorting cells are in a staggered array with 200 micron spacing between each cell. The entire array was 10 mm wide and 45 mm long.

3. Base plate

The cavity on the bottom plate is square, and the bottom of the cavity is provided with a connecting hole; the side length of the cavity is 150 microns, the depth is 2000 microns, and the position of the cavity corresponds to the micro-well array of the substrate. The height of the cavity connecting hole between the cavities is 8 microns, the width is 50 microns, and the length is 100 microns. The bottom of each micro-cavity is provided with a connecting hole which is mutually connected with the outside. The material of the backplane is PDMS. For the case of multiple cavities on the backplane, PDMS is easier to process.

The other settings were the same as in example 1.

The above embodiments are only for illustrating the invention and are not to be construed as limiting the invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention, therefore, all equivalent technical solutions also belong to the scope of the invention, and the scope of the invention is defined by the claims.

Claims (5)

1. A micro-fluidic chip for researching tumor single-cell invasion and epithelial-mesenchymal transition is characterized by comprising four layers of structures which are sequentially stacked, wherein the four layers of structures comprise a top cover, a micro-fluidic channel layer, a substrate with a sorting unit array and a bottom plate with a cavity group from top to bottom, and the sorting unit on the substrate and the cavity on the bottom plate are oppositely arranged;

the sorting unit is provided with a square micro-well on a substrate, the square micro-well penetrates through the substrate in the vertical direction, the bottom of the square micro-well is a square bottom plate provided with a plurality of small micro-holes, and the square bottom plate and the substrate are connected into a whole;

the sorting units on the substrate are arranged in a staggered array; an extracellular matrix layer simulating the tumor cell invasion environment is paved on the square bottom plate of the square micro-well;

the top cover is provided with a sample inlet and a sample outlet; the microfluidic channel layer is provided with a flow channel, the flow channel is in a strip shape with a wide middle section and two gradually narrowed ends, the middle section of the flow channel is opposite to the region of the substrate provided with the sorting unit array, and the two ends of the flow channel are respectively opposite to the sample inlet and the sample outlet;

the thickness of the spread extracellular matrix layer is 50-200 microns;

the side length of an opening of the sorting unit on the substrate is 10-1000 microns, and the depth of the square micro-well is 10-1000 microns; the side length of each micropore formed in each square bottom plate is 5-100 micrometers, the thickness of each square bottom plate is 10-1000 micrometers, and the number of micropores formed in each square bottom plate is 4-25.

2. The microfluidic chip according to claim 1, wherein the depth of the channel on the microfluidic channel layer is 10 to 200 μm, the width of the middle section of the channel is 1 to 20 mm, and the length of the microfluidic channel is 10 to 100 mm.

3. The microfluidic chip according to claim 1, wherein the cavities on the bottom plate are square, a connecting hole is formed at the bottom of each cavity, the connecting hole has a square opening shape, the side length is 5-1000 micrometers, and the length of the connecting hole is 5-10000 micrometers; the side length of the cavity is 15-5000 microns, and the depth of the cavity is 15-5000 microns.

4. The microfluidic chip according to claim 1, wherein the top cover is made of a transparent material, the microfluidic channel layer is made of an insulating polymer or silicon, and the flow channel is formed by etching; the substrate is made of insulated polymer, glass or silicon, and is made into a sorting unit array by adopting a photoetching and/or etching method; the bottom plate is made of glass, silicon or stainless steel materials, and a square cavity is formed by adopting an acid etching and/or photoetching method.

5. A system for researching tumor single cell invasion and epithelial-mesenchymal transition, which is characterized by comprising the microfluidic chip of any one of claims 1-4, a fluorescent probe, a fluorescent microscope, an image processing device, an injection pump, a constant temperature water bath box and a polymerase chain reaction amplification instrument;

the fluorescent probe is used for monitoring and analyzing protein expression information of a sample, the fluorescent microscope and the image processing equipment are used for observing the microfluidic chip, and the injection pump is connected with the microfluidic chip and used for sample injection; the PCR amplification instrument is used for analyzing the genome information of the separated cells.

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