CN111349541A - Microfluidic chip for single cell capture and screening and its application - Google Patents

Abstract

The present invention provides a microfluidic chip for capturing and screening single cells and its application. The microfluidic chip includes a four-layer structure that is stacked together and sealed with each other, and from top to bottom are a microvalve control layer, a microvalve film layer, a substrate with a flow channel and a micropore array, and a detection release layer. . The invention develops a multi-layer and multi-functional microfluidic chip by introducing micro-processing technology and micro-fluidic technology, which realizes the capture, identification, culture and release of cells, and provides a new method for cell screening. It can obtain high secretory cells more quickly, accurately and conveniently.

Description

Microfluidic chip for single cell capture and screening and its application

technical field

The present invention relates to a microfluidic chip for single cell capture and screening and its application.

Background technique

Monoclonal antibodies have high specificity and high targeting, and have become an important means of tumor targeted therapy and probe detection. At present, the mammalian expression systems for the production of monoclonal antibodies include CHO cells, NS0 cells, and hybridoma cells. During long-term culture, the genes expressing antibody heavy and light chains on chromosomes are deleted or rearranged, resulting in the loss of the ability of cells to secrete specific antibodies. The specific mechanism is still unclear. High-producing cells with secreted target protein molecules tend to have lower proliferation efficiency, while unstable heterozygous hybridoma cells proliferate faster, and their proportion continues to increase, which affects the secretion efficiency of antibodies and becomes a method for improving antibody production. obstacle. To ensure the expression efficiency and stability of secreted antibodies, it is critical to optimize cell populations and establish stable cell lines.

The species that prepare antibodies include mice, rabbits, camels, and people infected with foreign substances, as well as species with immune responses. Commonly used preparation methods include hybridoma technology, single B cell technology, antibody library technology and immune library technology. Among them, (1) Hybridoma technology is the most classic, and B lymphocytes are isolated from species such as mice, rabbits or camels inoculated with antigens, cells or other substances, such as bone marrow, peripheral blood, lymph nodes and spleen enriched in B cells. tissue organs. It is fused with immortalized myeloma cells in polyethylene glycol (PEG) to form hybridoma cells, or the method of virus induction such as Sendai virus, electroporation and other methods are used to promote cell fusion. The cells fused with B lymphocytes and myeloma cells were preliminarily screened with HAT medium, and fused congener cells or unfused cells were excluded. (2) Single B cell technology is to separate B cells from the above-mentioned species, use high-throughput methods such as flow cytometry to screen out B cells with specific antibodies, and then amplify the variable region or whole antibody of the antibody. Random pairing of long sequences; or amplification of naturally paired antibody genes from the same cell. (3) Antibody library technology is to insert antibody sequences isolated from B cells into vectors such as phage to form a phage antibody library, and then screen high-affinity specific antibodies from the antibody library. Immune library technology, based on Next-generation Sequencing (NGS), realizes whole transcriptome sequencing analysis of B cell population. B cells were isolated from the immunized species, mRNA was extracted and reverse-transcribed to amplify antibody genes, and all antibody genes were obtained. Through NGS technology, the frequencies of heavy and light chains were analyzed to infer paired antibodies to improve the diversity of antibodies.

Although there are different preparation methods, they all produce a large number of cell populations, which are heterogeneous, including cells that secrete specific antibodies and non-specific cells; the antibodies secreted by different cells are also different, with functional activity or no. Functional activity, or affinity varies. Therefore, it is difficult to screen out cells with functional activity, high affinity and high yield from a large number of heterogeneous populations in antibody development.

Antibody screening methods are as follows: enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunofluorescence technology, immunohistochemistry, etc., microfluidic technology and other methods based on antigen-antibody binding, or with biotin -Avidin system combination, or magnetic beads as reaction carrier and other methods. In addition, for the engineered cells expressing antibodies, additional drug pressure screening is required, such as geneticin (Geneticin, G418) and other reagents.

First, the traditional ELISA method: using the method of limiting dilution, each microwell contains at most one or several cells, and after the single cell is propagated into a cell clone, the culture supernatant is collected, and the expression efficiency of the hybridoma cells is identified by ELISA. specificity of the antibody. It takes 3 to 5 days of culture and multiple rounds of screening to obtain stable cells each time. This process takes several months to screen cells that stably express antibodies. The number of cells analyzed by this method is limited, only up to 10 ³ cells, so the antibody information of all cells cannot be obtained, and cells that may have functional antibodies are discarded. Therefore, this method is time-consuming, laborious, and inaccurate in evaluating antibodies. Most of the antigens detected by ELISA are expressed by genetic engineering such as mammalian cells, Escherichia coli, yeast cells and Bacillus subtilis, which are structurally different from natural antigens. Natural antigens are expressed by cells on the cell surface or in cells, and their natural conformation can be obtained, which truly reflects the specificity of antibody binding. For a more comprehensive evaluation and a more accurate evaluation of antibodies, the specificity of antibodies should be verified at the level of cells and tissue samples. Often identified with flow cytometry, immunofluorescence, and immunohistochemistry.

Flow cytometry is based on fluorescent probes, and at the level of single cells and flowing liquid, combined with an external electric field, to achieve the sorting of target cells; in a few minutes, the analysis of 10 ⁶ cells is completed, which is a high-throughput method. Analysis and screening method to improve the efficiency of establishing cell lines, but this method requires a cell density of at least 10 ⁵ /ml and a proportion of target cells greater than 0.1%. Therefore, flow cytometry is not suitable for rare cells or a small amount of target cell samples. Instrument sorting. Furthermore, the ability to secrete antibodies does not always correlate positively with the intensity of cell surface fluorescence.

In recent years, the emergence of microfluidic technology has provided new ideas for antibody screening. Micro-nanoliter volume geometry chambers, uniform or different size physical barrier structures, etc. are fabricated by microfabrication technology to capture individual cells. Theoretically, the rate of antibody secretion by a single cell is 3.66×10 ⁵ fM/min, and in nanoliter or picoliter volume, it can reach ng-level concentration level in 1 minute; The detection level is reached, thereby shortening the detection time. If it is a physical barrier structure such as a micropore, the detection antigen is fixed in the internal expression of the structure, and the detection device is directly contacted or fixed with the cells, combined with the antibody secreted by the cell, and the fluorescently labeled secondary antibody or other probes are used to distinguish specific cells. The other is a droplet-based method, in which cells, antibody-capturing magnetic beads, and fluorescent detection reagents are packaged into nanoliter or picoliter droplet chambers, and the secreted antibodies are bound to the magnetic beads to enrich fluorescent antibodies and screen for fluorescence comparison. strong cells. In addition, single-cell expansion based on microfluidic technology is also used in the field of antibody discovery. Cells, mRNA capture magnetic beads and amplification reagent components are simultaneously packaged in a nanoliter picoliter droplet chamber to obtain the heavy chain and light chain genes of each cell's antibody, and combine with Sanger sequencing or NGS to obtain the antibody sequence.

These methods cannot continuously detect and culture a single or a small number of cells, and establish stable cell lines on the chip; in addition, the large amount of data generated by the immune repertoire and the limited length of fragments read by NGS are not conducive to the later stage. Screening and antibody pairing. Among them, the screening and amplification based on microfluidic chips still need to combine flow cytometry, input a large number of cells, and off-chip operations to complete the establishment of stable cell lines.

At present, there are no related technical reports that can integrate cell capture, multiple identification, culture and release on the same chip.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a microfluidic chip for single cell capture and screening and its application.

Another object of the present invention is to provide a microfluidic chip system capable of capturing, identifying, culturing and releasing single cells for multiple times to screen high secretory cells.

The present invention designs a brand-new microfluidic chip for cell screening, the purpose of which is to screen out cells with high secretion from a large number of cells at the single cell level; The functions of cell capture, multiple identification, culture and release are integrated on the same chip, and corresponding operation methods are provided to achieve the purpose of rapid cell screening.

In order to achieve the object of the present invention, in the first aspect, the present invention provides a microfluidic chip for single cell capture and screening, the microfluidic chip includes a four-layer structure stacked together in sequence and sealed with each other, The bottom is the microvalve control layer, the microvalve thin film layer, the substrate with the flow channel and the microwell array, and the detection and release layer.

The microvalve control layer is provided with at least 4 holes, respectively a, b, c and d, wherein a is used as a sample inlet, b and c are used as a sample inlet and outlet, and d is a sample outlet; the 4 holes are independently It penetrates through the control layer, the micro-valve film layer and the substrate; the side of the micro-valve control layer in contact with the micro-valve film layer is provided with several gas channels, and one end of each gas channel is opened on the top of the micro-valve control layer The surface (a number of holes for gas circulation are formed on the surface of the microvalve control layer as gas inlets and outlets), and the other end is in a closed state; there are several gas branch channels on the gas channel, which are branched by the gas The channel constitutes a gas micro-valve structure, and the gas micro-valve structure and the flow channel on the substrate are vertically arranged in a crisscross pattern; the gas micro-valve structure corresponds to the micro-valve film layer and the position of the micro-valve on the substrate; The valve is used as the interception structure of the flow channel on the substrate, its width and depth are consistent with the flow channel, and the thickness is 20-1000 microns. of opening and closing.

Preferably, the microvalve control layer has two or more independently controlled gas channels, and only one end of the two ends is open for gas (air) circulation to control the opening and closing of the microvalve. There are four holes through the microvalve control layer for the inflow and outflow of samples and reagents.

The microvalve thin film layer, in the thin film layer region corresponding to the gas channel, can be bent and deformed under the control of air pressure. The microvalve film layer is provided with at least 4 holes, corresponding to a, b, c and d on the microvalve control layer respectively. Preferably, the microvalve thin film layer is a single thin film with uniform thickness, with four holes penetrating the thin film, respectively communicating with the four holes of the microvalve control layer.

The substrate with flow channels and microwell arrays includes at least four functional areas, which are two identification areas and two enrichment areas, wherein the first identification area, the first enrichment area, the second identification area and the first identification area. The two enrichment regions are connected through a flow channel in sequence; the identification region includes a flow channel and a micropore array composed of each micropore unit communicated with the flow channel for capturing cells; the enrichment region refers to The interception unit controlled by the microvalve is used to intercept the cells; the micropore unit is a micropore structure arranged on the swimming channel of the flow channel and penetrates through the substrate, and communicates with the release channel on the detection release layer; One end of the flow channel of the identification area is provided with a sample inlet port, corresponding to a on the microvalve control layer; at least one sample collection port is provided between the first identification area and the first enrichment area, corresponding to the b on the microvalve control layer; at least one sample collection port is set between the first enrichment area and the second identification area, corresponding to c on the microvalve control layer; the second enrichment The zone is provided with at least one sample collection port, corresponding to d on the microvalve control layer.

Preferably, in the first identification area, the sample inlet is divided into two flow channels, which are used for sample splitting. More preferably, a plurality of parallel flow channels are arranged in the functional area; a micropore array is arranged in the flow channel, and the upper and lower passages are connected. .

The microwell array is used to capture single cells. The enrichment zone has a microvalve structure. In order to enrich the cells, air bubbles that may be present in the structure are excluded.

The detection release layer is provided with a release channel, the release channel and the channel on the substrate are vertically arranged in a crisscross pattern and correspond to the microporous unit on the substrate, and the depth of the release channel is smaller than that of a single channel. The diameter of the cell; one end of the release channel is opened on the lower surface of the detection release layer (a number of holes for liquid circulation are formed on the lower surface of the detection release layer, as the liquid inlet and outlet), used for backflushing cells Inflow of liquid, the other end is closed. The release channel is also used to immobilize the detection protein.

Specifically, the detection and release layer has a plurality of liquid flow channels and corresponding injection ports. The flow channels are vertically superimposed with the flow channels of the first identification area and the second identification area of the substrate, and are vertically overlapped with the micropores of the identification area. Corresponding. In a preferred embodiment of the present invention, each release channel can act on 6 micropores in the same row of the first identification area or 4 micropores in the same row in the second identification area.

The thickness of the microvalve control layer is 1-5 mm.

The thickness of the microvalve thin film layer is 5-500 microns.

The thickness of the substrate is 150-1000 microns.

The thickness of the detection release layer is 1-5 mm.

Preferably, the flow channel of the identification area on the substrate is a single channel arranged in a curved or folded line, or a plurality of channels arranged in parallel. Preference is given to multiple channels arranged in parallel. Each flow channel is controlled by an individual microvalve structure.

Preferably, the width of the flow channel on the substrate is 20-2000 microns, and the depth is 20-500 microns.

Preferably, the microporous unit on the substrate is a square or rectangle with a side length of 20-500 microns, or a circle with a diameter of 10-500 microns, or other geometric structures.

Preferably, the depth of the release channel on the detection release layer is 1-50 microns, and the width is 20-2000 microns. Preferably, the release channels are arranged in parallel.

Preferably, the width of the gas channel on the microvalve control layer is 20-2000 microns, and the depth is 20-2000 microns.

In the present invention, the materials of the microvalve control layer and the detection and release layer are selected from transparent materials with good biocompatibility and good protein adsorption, preferably polydimethylsiloxane (PDMS) and the like. The microvalve control layer and the detection release layer are hardened hard materials or hard materials themselves.

The material of the microvalve film layer is selected from transparent flexible materials with good ductility and strong stretchability, preferably PDMS and the like.

The substrate is made of a material (such as a silicon wafer, etc.) that is easy to process and whose dimensions can be precisely controlled, and can be a transparent material or an opaque material.

The schematic diagram of the structure of the microfluidic chip used for single cell capture and screening of the present invention is shown in Figure 1 . The cross-sectional views along AA' and BB' show the specific structure of the substrate in the chip, the sample flow channel and the capture micropores, as shown in Figure 2. A top-view perspective view of the four-layer structure in FIG. 1 after encapsulation is shown in FIG. 3 . The physical diagram of the microfluidic chip used for single cell capture and screening of the present invention is shown in Figure 5 .

In a second aspect, the present invention provides any of the following applications of the microfluidic chip:

1) for single cell capture, identification, culture and release;

2) Application in the preparation of a microfluidic chip system for single cell capture, identification, culture and release;

3) for screening hybridoma cells that can secrete specific antibodies;

4) For screening cells that can secrete specific cytokines.

In a third aspect, the present invention provides a microfluidic chip system for capturing, identifying, culturing and releasing single cells, comprising: the microfluidic chip, a fluorescent probe, a fluorescent microscope, image processing software, a pump, and a Air pressure control device controlled by single chip microcomputer.

In a fourth aspect, the present invention provides a method for screening hybridoma cells that can secrete specific antibodies using the microfluidic chip, comprising the following steps:

S1. Pour alcohol and phosphate buffer into the microfluidic chip for pretreatment, and then add a detection protein solution to the detection release layer, so that the detection protein is coated on the release channel of the detection release layer;

S2. Pass the cell suspension to be selected into the microfluidic chip, so that the cells fall into the micropore unit of the substrate, and then pass fluorescent molecular probes into the microfluidic chip, and use a fluorescence microscope Observe the microwell array from the front of the chip, and identify and screen the cells according to whether the cells in the microwell unit have fluorescence and the intensity of fluorescence;

S3. For the cells in the selected microporous unit, pass the liquid medium from the detection release area, flush the cells in the microporous unit into the retention unit of the first enrichment area, and then open the entrance of the second identification area Microvalve, fully open the outlet microvalve of the first enrichment area, close other microvalves, release the cells to the second identification area, and carry out the second identification. After the identification, the selected hybridoma cells that can secrete specific antibodies will be screened. It is released into the retention unit of the second enrichment zone, and finally the screened cells are taken out from the sample outlet of the microvalve control layer.

Figure 4 shows a schematic diagram of the process of capturing, identifying, culturing and releasing single cells using the microfluidic chip.

The cell suspension can be derived from living tissue, blood or cell suspension cultured in vitro.

The fluorescent molecular probes are antibodies, polypeptides, biotin-avidin and their derivatives labeled with fluorescent molecules. One fluorescent probe can be used alone to complete the identification of a single protein, or multiple fluorescent probes can be used simultaneously. A variety of fluorescent probes are used to complete the multiple identification of multiple proteins.

In a specific embodiment of the present invention, the application method of the microfluidic chip system for single cell capture, identification, culture and release is as follows:

As shown in Figures 3 and 4, first, the first identification area was pre-treated with 75% ethanol, sterile phosphate buffered solution (PBS), and the microvalves of other areas were closed at the same time to prevent fluid from entering. Then, the detection protein was pumped in, and incubated at 4°C for over 2 hours or at 37°C for 2 hours, and the detection protein was adsorbed and immobilized on the detection release layer. The next day, the cell samples were pumped into the first identification area, and single cells were captured and incubated for 0.5-4 hours; then the free target molecules were washed with sterile PBS, and the fluorescent probe molecules were pumped to form complexes with the target molecules, reacting for 0.5- 4 hours. Finally, sterile PBS was used to wash the unbound fluorescent probe molecules, and a fluorescence microscope was used to automatically scan the microwells from the front. After acquiring the fluorescent images, the medium was continuously pumped into the culture medium. After culturing for 24-72 hours, open the inlet microvalve, waste liquid outlet and half-open microvalve of the enrichment area, and close other microvalves at the same time; from the detection and release area, flush the selected cells to the enrichment area and open the The inlet microvalve of the second identification area fully opens the outlet microvalve of the enrichment area, closes other microvalves, and releases the cells to the second identification area. The pretreatment method and identification steps of the second identification area are the same as those of the first identification area. After the identification, the cells are released after culturing again for 24-72 hours to obtain ideal cells. According to experimental requirements, multiple screening modules can be connected in parallel or in series.

The sample is a heterogeneous population of cells. The reagents used are commercial products, either self-prepared or processed by biological companies.

By the above-mentioned technical scheme, the present invention at least has the following advantages and beneficial effects:

(1) The present invention develops a multi-layer, multi-functional microfluidic chip by introducing microfabrication technology and microfluidic technology, which realizes the capture, identification, culture and release of cells, and provides a kind of cell screening. The new method can obtain high secretory cells more quickly, accurately and conveniently.

(2) The present invention realizes the capture, identification, culture and release of single cells. In the field of cell screening, the single cell screening of heterogeneous cell populations has the following significant advantages: First, compared with traditional technologies, the present invention has the following advantages: By designing the micropores, the size of the cells can capture a single cell. According to the concentration of the cells, the flow rate can obtain a higher capture efficiency, which is conducive to the analysis of single cells. At the same time, when the sample is in the micropore, the secreted target molecules can quickly reach the detection concentration. , is recognized by the detected protein and shortens the time. Secondly, both PDMS and silicon are biocompatible materials, which are non-toxic to cells, which is conducive to cell culture; PDMS has good gas permeability, which is conducive to the exchange of oxygen. In addition, fluids are used to release cells during the operation, which can damage the external mechanical force of cells and ensure the activity of cells. Therefore, microfluidic chips have functional diversification.

(3) The present invention realizes the integration of cell screening: the present invention adopts the method of functional area combination, integrates two identification areas, two enrichment areas, and micro-valve control areas, and integrates the entire operation process in the same block It is completed on the chip, reducing the operation outside the chip, avoiding the loss and damage of cells in this process, thus ensuring the activity of cells.

(4) The microfluidic chip and application method designed by the present invention have a wide range of applications. The size of the micropore array and flow channel involved in the present invention can be adjusted according to different cells; the module can also be increased according to different cell amounts. , which is easily achieved by those skilled in the art and adds little cost.

Description of drawings

FIG. 1 is a schematic exploded view of the structure of the microfluidic chip used for single cell capture and screening of the present invention.

Figure 2 is a cross-sectional view along AA', BB', showing the specific structure of the substrate in the chip, the sample flow channel and the capture micropore.

FIG. 3 is a top perspective perspective view of the four-layer structure in FIG. 1 after encapsulation.

FIG. 4 is a schematic diagram of the process of capturing, identifying, culturing and releasing single cells using the microfluidic chip.

Fig. 5 is a physical diagram of the microfluidic chip used for single cell capture and screening of the present invention.

Figures 1-4 are schematic diagrams and are not drawn to scale.

In the figure, 1-microvalve control layer; 2-microvalve film layer; 3-substrate with 4 functional areas; 4-detection release layer; 5-sample inlet (a hole), 6-sample outlet (d hole) ), 7-sample inlet and outlet (b hole), 8-sample inlet and outlet (c hole), 12-liquid inlet and outlet; 9,10-gas inlet and outlet; 11-release flow channel; 13-micropore array structure; 14-gas channel; 15- interception unit controlled by microvalve; 16-cell; 17-microvalve structure on substrate flow channel; 18-sample tube for holding cells; 19-first identification area; 20-first enrichment area; 21-second identification area; 22-second enrichment area; 23-microvalve control area; 24-microvalve in open state; 25-microvalve in closed state; 26-detection protein. Dotted arrows - gas flow path; solid arrows: liquid flow path.

Detailed ways

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available commodities.

Example 1 Microfluidic chip for single cell capture and screening and microfluidic chip system capable of capturing, multiple identification, culturing and releasing single cells

1. Microvalve control layer and microvalve film layer

When identifying cells, light needs to pass through the microvalve control layer and the microvalve film layer. Therefore, the microvalve control layer and the microvalve film layer need to be made of transparent materials; at the same time, the microvalve control layer and the microvalve film layer need to be made of fluid. The interface for entering and exiting, so it needs to be able to open holes; in addition, because the gas squeezes the film to produce bending deformation to achieve the purpose of micro-valve switching, the material of the micro-valve film layer must be a soft material with good elasticity; Cells need to be cultured on the chip, and all related materials need to have good biocompatibility; finally, the microvalve control layer needs to be sealed together with the microvalve film layer and sealed, so the choice of material should be considered. Microvalve membrane layer compatibility. Preferred materials are those that can be fabricated into specific gauges and shapes by evaporation, cutting and thermoforming. Particularly preferred are thinner and transparent polymers.

In the preferred embodiment shown in FIG. 1 and FIG. 4 , the microvalve control layer, the microvalve film layer and the detection and release layer can all be made of PDMS material, and the required gas channel and film thickness of the microvalve control layer can be obtained by soft lithography. , the two are sealed and connected by bonding. In other implementation methods, the microvalve control layer can also be made of glass material, and gas channels are obtained by wet etching or laser etching, and then the two are sealed together by bonding.

In the preferred embodiment shown in FIG. 1 and FIG. 4 , the thickness of the microvalve control layer is 2000 microns, the depth of the gas channel is 200 microns, and the thickness of the microvalve film layer is 40 microns, which are effective for controlling the opening and closing of the microvalve. Preferred size. When the length, width and depth of the flow channel of the microvalve control switch are required to be changed, those skilled in the art can choose the appropriate gas channel and the size of the microvalve membrane according to their own needs.

In the preferred embodiment shown in Figures 1 and 4, the detection release layer has two functions: one is to fix the detection antigen; the other is to release cells. Therefore, in addition to biocompatibility, the detection release layer should also have adsorption properties or surface modifiability of polypeptides or proteins. In a preferred embodiment, PDMS is selected as the preferred material and incubated with the protein after surface plasma treatment to achieve the purpose of immobilizing the protein. In addition to the preferred embodiment, other transparent materials, such as polystyrene and other materials with high adsorption properties, or materials that can be activated and introduced into functional groups can also be selected.

2. Substrate with microwell array and flow channel

In the present invention, the microwell array is a key functional area for capturing and culturing cells. To accommodate the size of the cells, materials that are compatible with microfabrication techniques must be selected. In the preferred embodiment shown in FIG. 1 and FIG. 2 , silicon is selected as the material of the substrate microstructure array, because silicon can be well compatible with the micromachining process based on photolithography and etching, and the processing accuracy is easy to control, so that it can be Capture arrays that match the cell size are easily obtained. Those skilled in the art can also select machinable materials such as glass according to requirements. It is worth noting that the substrate integrates 4 functional areas: 2 cell identification areas and 2 cell enrichment areas, and these four functional areas and the microstructure processing methods they contain are different, and the cell identification area needs to be Two etchings are performed on a silicon wafer to obtain the flow channel and microwell array structure, and the cell-rich area can be completed with only one etching.

In the preferred embodiment shown in FIG. 2 , the first identification area of the substrate is provided with 6 flow channels, the 6 flow channels share one sample inlet, and the second identification area is provided with 4 flow channels. In the microwell array for cell identification, each microwell is a chamber with a side length of 50 microns and a height of 160 microns. There are 30 micropores evenly arranged on each flow channel), and 80 micropores in the second identification area (an average of 20 micropores are evenly arranged on each flow channel); for the flow channel of the sample, in order to adapt to the flow rate, the cells should be kept as monolayer as possible distribution with a height of 30 microns and a width of 200 microns. The above are the preferred conditions applied to the analysis of hybridoma cells. When other cells need to be processed, those skilled in the art can choose the appropriate size and number of micropores by themselves according to the size specification of the silicon wafer and the requirements of cell experiments.

The physical diagram of the microfluidic chip used for single cell capture and screening of the present invention is shown in Figure 5 .

3. Sealing method

In the preferred embodiment shown in FIG. 1 , the materials of the microvalve control layer, the microvalve thin film layer, the substrate layer and the detection and release layer are PDMS, PDMS, silicon and PDMS, respectively. By adopting the oxygen plasma-assisted bonding method, good sealing and sealing between PDMS and PDMS, silicon and PDMS can be well achieved.

In other implementation methods, a suitable sealing method can be selected according to the materials of the microvalve control layer, the microvalve thin film layer, the substrate and the detection release layer.

4. Chip supporting system

In addition to the microfluidic chip, the present invention also requires a fluorescent probe (Probe), a fluorescent microscope, ImageJ image analysis software and a pump to form a complete system to complete the capture, identification, culture and release of cells.

Fluorescent probes are used for the identification of antibody molecules. In the preferred embodiment shown in FIG. 4 , a goat anti-mouse Ig secondary antibody labeled with green fluorescence is used as a fluorescent probe for identifying antibodies secreted by cells. In other embodiments, different antibodies, polypeptides, or biotin-avidin reaction systems and derivatives thereof can also be selected as probes for different detection methods.

Fluorescence microscopy is used to detect whether the cells captured in the microwell array are fluorescent, and the microwell array is scanned and imaged to obtain fluorescence pictures.

ImageJ image analysis software was used to analyze the images acquired by the fluorescence microscope and obtain the corresponding microwell numbers of fluorescent cells. The software selects the required microwells by calculating the intensity of the fluorescence in the image.

Pumps are used to drive liquid samples, liquid media, gases and related reagents.

5. The specific production method of the chip

The chip and system of the present invention have been successfully fabricated by the following two different fabrication processes. The specific fabrication method is given to help those skilled in the art to understand the fabrication method and gist of the present invention, rather than to limit the material, size and fabrication method of the device of the present invention.

Production method A:

Micro-valve control layer: Using a 4-inch Pyrex7740 glass sheet (Corning), the plane positions of micro-holes and gas channels are etched on the front side of the silicon wafer, and the glass is etched with hydrofluoric acid to form gas channels and holes with a depth of 200 microns. The laser penetrates the location of the micro-holes. Finally, cut into small rectangular pieces according to the dimensions of 6 cm long and 3 cm wide.

Microvalve film layer: N-type 4-inch silicon wafer is used, and a layer of PDMS liquid with a thickness of 30 microns is coated on its surface by spin coating. After it is solidified, it is demolded and taken out. Cut into rectangular films.

Substrate with micro-hole array and flow channel: N-type 4-inch silicon wafer is used, and wet oxidation is used to obtain a 0.5-micron-thick oxide layer on the surface of the silicon wafer. For the plane position of the liquid injection port, a flow channel with a depth of 40 microns was made by the method of potassium hydroxide wet etching. Photolithography is performed on the back of the silicon wafer, and the position corresponding to the bottom of the microhole is lithography, and the silicon wafer is etched through the ICP dry method, and finally a complete silicon microwell array and microchannel are obtained. Cut a 4-inch silicon wafer into small rectangular pieces according to the dimensions of 6 cm in length and 3 cm in width.

Detect release layer: N-type 4-inch silicon wafer is used, and after the plane shape of the flow channel (release flow channel) is photo-etched, a 5-micron-high convex body is dry-etched by ICP. Pour the liquid PDMS into the tank, take it out of the mold after it solidifies, cut it into small rectangular pieces according to the dimensions of 6 cm in length and 3 cm in width, and use a hole puncher to punch holes at the desired positions (as the liquid inlet and outlet), so that A detection release layer was obtained.

Sealing: First, the reverse side of the microvalve control layer, both sides of the silicon substrate, both sides of the microvalve film and the front side of the detection release layer are subjected to oxygen plasma treatment, and they are bonded together in sequence to obtain a complete chip.

Chip system: On the basis of the microfluidic chip, a PTFE tube is used to connect the pump and the microvalve control layer, the fluid inlet and outlet of the detection release layer, and the PTFE tube is used to connect the pump and the gas channel inlet of the microvalve control layer. Place the chip under an upright fluorescence microscope that can automatically perform fluorescence imaging, and use ImageJ image processing software for automatic analysis and processing of fluorescence images to complete the construction of the entire system.

Production method B:

Microvalve control layer: N-type 4-inch silicon wafer is used. After the plane shape of the gas channel is etched, a 30-micron-high convex body is etched by ICP dry method. Pour the liquid PDMS into the tank, take it out of the mold after it solidifies, cut it into small rectangular pieces according to the dimensions of 6 cm in length and 3 cm in width, and use a hole puncher to punch holes at the desired positions, so that the micro-valve control is obtained. Floor.

Microvalve film layer: N-type 4-inch silicon wafer is used, and a layer of PDMS liquid with a thickness of 30 microns is coated on its surface by spin coating. After it is solidified, it is demolded and taken out. Cut into rectangular films.

Substrate with micro-hole array and flow channel: N-type 4-inch silicon wafer is used, and the plane shape of the micro-channel structure is photo-etched on one side of the 200-micron double-polished silicon wafer; the plane of the micro-pore structure is photo-etched on the other side. shape. The ICP dry etching method was used to obtain a flow channel with a depth of 40 microns and a micro-hole with a depth of 160 microns, that is, the micro-holes were partially etched and penetrated. The 4-inch silicon wafer was cut into small rectangular pieces according to the dimensions of 6 cm long and 3 cm wide.

Detect release layer: N-type 4-inch silicon wafer is used, and after the plane shape of the flow channel (release flow channel) is photo-etched, a 5-micron-high convex body is dry-etched by ICP. Pour the liquid PDMS into the tank, take it out of the mold after it solidifies, cut it into small rectangular pieces according to the dimensions of 6 cm in length and 3 cm in width, and use a hole puncher to punch holes at the desired positions (as the liquid inlet and outlet), so that A detection release layer was obtained.

Sealing: First, oxygen plasma treatment is performed on the reverse side of the microvalve control layer, the two sides of the silicon substrate, the two sides of the microvalve film and the front side of the detection and release layer, and they are bonded together in sequence, and finally a complete chip is obtained.

Chip system: On the basis of the microfluidic chip, a PTFE tube is used to connect the pump and the microvalve control layer, the fluid inlet and outlet of the detection release layer, and the PTFE tube is used to connect the pump and the gas channel inlet of the microvalve control layer. Place the chip under an upright fluorescence microscope that can automatically perform fluorescence imaging, and use ImageJ image processing software for automatic analysis and processing of fluorescence images to complete the construction of the entire system.

6. Specific applications

The microfluidic chip and the corresponding system of the present invention are successfully applied to the screening of hybridoma cells as follows. The specific methods are given to help those skilled in the art to understand the functions and application methods of the present invention, but not to limit the scope of application of the device of the present invention.

Taking hybridoma cells targeting CD45 protein as an example, the specific application method is described as follows:

All experimental steps are carried out in a sterile environment between cells, consumables and instruments are sterilized in advance, and reagents are sterile.

For the pretreatment of the microfluidic chip, 75% ethanol was injected into the microfluidic chip for 5 minutes, and the surface of the flow channel was sterilized and infiltrated to increase the hydrophilicity. Sterile phosphate buffered solution (PBS) was injected to wash the ethanol in the flow channel. Then, CD45 detection protein (leukocyte common antigen) was pumped into the identification area, and then placed at 4°C for overnight incubation. The next day, 5% sterile BSA was injected and incubated for 1 hour to block unbound antigenic sites.

The first identification: collect heterogeneous hybridoma cells, resuspend them in complete medium, adjust the cell concentration, inject them into the first identification area of the microfluidic chip through a micropump, and close other areas such as the enrichment area at the same time Microvalve. When the cell suspension flows through the first identification zone, the liquid fills the flow channel and flows out from the release channel in the direction of the micropores. Due to the effect of size, the size of the cells is greater than 5 microns, which is greater than the height of the release channel, and the cells are trapped into the micropores. Place in a carbon dioxide incubator for 2 hours. Inject fluorescent secondary antibody, inject sterile PBS, wash unbound antibody, inject fluorescently-labeled secondary antibody, and incubate in a carbon dioxide incubator for 1 hour. For fluorescence identification, inject sterile PBS to wash unbound secondary antibodies. The microwells were automatically scanned from the front with a fluorescence microscope, and after the fluorescence images were acquired, the complete medium was continuously pumped for cultivation.

Single cell enrichment: After 48 hours of culture, select micropores with high fluorescence intensity, inject culture medium from the detection release layer, close the microvalve at the entrance of the second identification area, and open the outlet microvalve of the enrichment area. The cells are backwashed from the microwells and into the enrichment zone.

The second identification: open the micro valve of the second identification area, and the cells enter the second identification area from the enrichment area. Other operations are the same as the first identification.

Finally, cells were collected and transferred to 24-well plates for expansion.

The schematic diagram of the structure of the microfluidic chip used for single cell capture and screening of the present invention is shown in Figure 1 . The cross-sectional view along AA' shows the specific structure of the substrate in the chip, the sample flow channel and the capture micropores, as shown in Figure 2. A top-view perspective view of the four-layer structure in FIG. 1 after encapsulation is shown in FIG. 3 . Figure 4 shows a schematic diagram of the process of capturing, identifying, culturing and releasing single cells using the microfluidic chip.

Although the present invention has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (10)

1. The microfluidic chip is used for capturing and screening single cells and is characterized by comprising four layers of structures which are sequentially stacked together and sealed with each other, namely a micro valve control layer, a micro valve thin film layer, a substrate with a flow channel and a micropore array and a detection release layer from top to bottom;

the micro valve control layer is at least provided with 4 holes, namely a, b, c and d, wherein a is used as a sample inlet, b and c are used as sample inlets and outlets, and d is used as a sample outlet; the 4 holes respectively and independently penetrate through the control layer, the micro valve film layer and the substrate; a plurality of gas channels are arranged on one surface of the micro valve control layer, which is in contact with the micro valve film layer, one end of each gas channel is opened on the upper surface of the micro valve control layer, and the other end of each gas channel is in a closed state; the gas channel is provided with a plurality of gas branch channels, the gas branch channels form a gas micro valve structure, and the gas micro valve structure and the flow channel on the substrate are vertically arranged in a cross shape; the gas micro-valve structure corresponds to the positions of the micro-valve film layer and the micro-valve on the substrate; the micro valve is used as an interception structure of a flow channel on the substrate, the width and the depth of the micro valve are consistent with those of the flow channel, the thickness of the micro valve is 20-1000 microns, and the micro valve is used for controlling the opening and the closing of the flow channel on the substrate;

the micro valve film layer is provided with at least 4 holes which respectively correspond to a, b, c and d on the micro valve control layer;

the substrate with the flow channel and the micropore array at least comprises four functional areas which are respectively two identification areas and two enrichment areas, wherein the first identification area, the first enrichment area, the second identification area and the second enrichment area are sequentially connected and communicated through the flow channel; the identification area comprises a flow channel and a micropore array which is communicated with the flow channel and is composed of micropore units for capturing cells; the enrichment area is an interception unit controlled by a micro valve and used for intercepting cells; the micropore unit is a micropore structure which is arranged on a lane of the flow channel and runs through the substrate, and is communicated with the release flow channel on the detection release layer; wherein, one end of the first identification area flow channel is provided with a sample inlet corresponding to a on the micro valve control layer; at least one sample collection port is disposed between the first identified region and the first enrichment region, corresponding to b on the microvalve control layer; at least one sample collection port is provided between the first enrichment zone and the second identification zone, corresponding to c on the microvalve control layer; the second enrichment region is provided with at least one sample collection port corresponding to d on the microvalve control layer;

the detection release layer is provided with release flow channels, the release flow channels and the flow channels on the substrate are vertically arranged in a cross manner and correspond to the micropore units on the substrate, and the depth of the release flow channels is smaller than the diameter of a single cell; one end of the release flow channel is opened on the lower surface of the detection release layer and used for backflushing inflow of cell liquid, and the other end of the release flow channel is in a closed state.

2. The microfluidic chip according to claim 1, wherein the thickness of the microvalve control layer is 1-5 mm; and/or

The thickness of the micro valve film layer is 5-500 microns; and/or

The thickness of the substrate is 150-1000 microns; and/or

The thickness of the detection release layer is 1-5 mm.

3. The microfluidic chip according to claim 1, wherein the flow channel of the identification region on the substrate is a single channel arranged in a curved or broken line manner, or a plurality of channels arranged in parallel.

4. The microfluidic chip according to claim 1, wherein the width of the flow channel on the substrate is 20-2000 microns, and the depth is 20-500 microns; and/or

The micropore unit on the substrate is a square or rectangle with the side length of 20-500 micrometers, or a circle with the diameter of 10-500 micrometers.

5. The microfluidic chip according to claim 1, wherein the depth of the release flow channel on the detection release layer is 1-50 microns, and the width thereof is 20-2000 microns.

6. The microfluidic chip according to claim 1, wherein the width of the gas channel on the microvalve control layer is 20-2000 μm and the depth is 20-2000 μm.

7. The microfluidic chip according to any of claims 1 to 6, wherein the material of the microvalve control layer and the detection release layer is selected from transparent materials with good biocompatibility and good protein adsorption, preferably PDMS; and/or

The material of the micro-valve film layer is selected from transparent flexible materials with good ductility and strong elasticity, and PDMS is preferred; and/or

The substrate is made of silicon wafers.

8. The microfluidic chip of any one of claims 1 to 7, for any one of the following applications:

1) for single cell capture, identification, culture and release;

2) the application in preparing a microfluidic chip system for capturing, identifying, culturing and releasing single cells;

3) screening hybridoma cells capable of secreting specific antibodies;

4) used for screening cells capable of secreting specific cytokines.

9. A microfluidic chip system for single cell capture, identification, culture and release, comprising: the microfluidic chip of any one of claims 1 to 7, a fluorescent probe, a fluorescence microscope, image processing software, a pump, and a pressure control device controlled by a single-chip microcomputer.

10. The method for screening hybridoma capable of secreting a specific antibody using the microfluidic chip of any one of claims 1 to 7, comprising the steps of:

s1, introducing alcohol and phosphate buffer solution into the microfluidic chip for pretreatment, and then adding a detection protein solution into the detection release layer to coat the detection protein on a release flow channel of the detection release layer;

s2, introducing a cell suspension to be selected into the microfluidic chip to enable the cells to fall into the micropore units of the substrate, introducing a fluorescent molecular probe into the microfluidic chip, observing the micropore array from the front of the chip by using a fluorescence microscope, and identifying and screening the cells according to the existence of fluorescence in the micropore units and the strength of the fluorescence;

s3, introducing a liquid culture medium into the selected microporous unit from the detection release area, flushing the cells in the microporous unit to the first enrichment area, then opening the inlet micro valve of the second identification area, fully opening the outlet micro valve of the first enrichment area, closing other micro valves, releasing the cells to the second identification area, carrying out second identification, releasing the screened hybridoma cells capable of secreting the specific antibody to the second enrichment area after the identification is finished, and finally taking out the screened cells from the sample outlet of the micro valve control layer.

Images