CN111349541A - Microfluidic chip for capturing and screening single cells and application thereof - Google Patents
Microfluidic chip for capturing and screening single cells and application thereof Download PDFInfo
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Abstract
The invention provides a micro-fluidic chip for capturing and screening single cells and application thereof. The micro-fluidic chip comprises four layers of structures which are sequentially stacked together and sealed with each other, and a micro-valve control layer, a micro-valve thin film layer, a substrate with a flow channel and a micro-pore array and a detection release layer are respectively arranged from top to bottom. The invention develops a multi-layer and multifunctional micro-fluidic chip by introducing a micro-processing technology and a micro-fluidic technology, realizes the capture, identification, culture and release of cells, provides a new method for cell screening, and can obtain high-secretion cells more quickly, accurately and conveniently.
Description
Technical Field
The invention relates to a microfluidic chip for capturing and screening single cells and application thereof.
Background
The monoclonal antibody has high specificity and high targeting property, and becomes an important means for tumor targeted therapy and probe detection. At present, CHO cell, NS0 cell and hybridoma cell are mammalian expression systems for producing monoclonal antibody. During long-term culture, the genes expressing the heavy and light chains of the antibody on the chromosome are deleted or rearranged, so that the capacity of the cells for secreting specific antibodies is lost, and the specific mechanism is still unclear. The high-yield cells secreting target protein molecules tend to have lower proliferation efficiency, but unstable heterogeneous hybridoma cells proliferate faster, and the occupied ratio is continuously enlarged to influence the secretion efficiency of antibodies, so that the high-yield cells secreting target protein molecules become barriers for improving the yield of the antibodies. To ensure the expression efficiency and stability of the secreted antibody, it is important to optimize the cell population and establish a stable cell line.
The species for preparing the antibody includes mice, rabbits, camels, people infected with foreign substances, and the like, and the species with immune response. The commonly used preparation methods are hybridoma technology, single B cell technology, antibody library technology and immune library technology. Among them, (1) in the most classical hybridoma technique, B lymphocytes, such as bone marrow, peripheral blood, lymph nodes, spleen and other B cell-rich tissues and organs, are isolated from a species such as a mouse, rabbit or camel, which is inoculated with an antigen, a cell or other substances. Fusing with immortal myeloma cell in polyethylene glycol (PEG) to obtain hybridoma, or inducing virus such as Sendai virus, and promoting cell fusion by electroporation. HAT medium is used for primary screening of fused cells of B lymphocytes and myeloma cells, and fused cells of the same kind or unfused cells are excluded. (2) the single B cell technology is to separate B cells from the above various species, screen out the B cells of specific antibodies by high-throughput methods such as flow cytometry, and then amplify the variable region or full-length sequence of the antibodies for random pairing; or naturally paired antibody genes amplified from the same cell. (3) The antibody library technology is to insert antibody sequences separated from B cells into carriers such as bacteriophage to form a bacteriophage antibody library, and then to screen specific antibodies with high affinity from the antibody library. The immune bank technology is based on Next-generation Sequencing (NGS) technology to realize the Sequencing analysis of the whole transcriptome of the B cell population. B cells are separated from immunized species, mRNA is extracted to carry out reverse transcription and amplification on antibody genes, all the antibody genes are obtained, and the pairing antibody is presumed by analyzing the frequency of heavy chains and light chains through an NGS technology, so that the diversity of the antibody is improved.
Despite the different methods of preparation, a large number of cell populations are produced, which are heterogeneous, comprising cells secreting specific antibodies, non-specific cells; the antibodies secreted by different cells may also differ in their functional activity or non-functional activity, or in their affinity. Therefore, screening of functionally active, high affinity and high yield cells from a large heterogeneous population is a difficult point in antibody development.
The following methods are available for antibody screening: enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunofluorescence technology, immunohistochemistry and the like, microfluidic technology and other methods based on antigen-antibody combination, or combination with biotin-avidin system, or magnetic beads as reaction carriers and the like. In addition, there is a need for further drug-pressure screening of engineered cells expressing antibodies, such as Geneticin (G418) and other reagents.
First, the traditional ELISA method: using a limiting dilution method, each microwell contains at most 1 or several cells, and after the cells are propagated from a single cell to a cell clone, culture supernatants are collected, and expression efficiency of hybridoma cells and specificity of antibodies are identified by ELISA. The stable cells can be obtained after 3-5 days of culture and repeated circulating screening every time, and the process can be used for screening the cells for stably expressing the antibody for several months. The method has limited cell number of 103Individual cells, and therefore no antibody information is available for all cells, thereby discarding cells that may have functional antibodies. Therefore, the method is time-consuming, laborious and inaccurate in evaluating the antibody. Most of ELISA detection antigens are expressed by genetic engineering such as mammalian cells, escherichia coli, yeast cells, bacillus subtilis and the like, and have certain difference with natural antigens in structure. The natural antigen is expressed on the cell surface or in the cell, and the natural conformation can be obtained, so that the binding specificity of the antibody is really reflected. For more complete and accurate evaluation of antibodies, cell and tissue sample level tests are usedTo confirm the specificity of the antibody. Often identified with flow cytometry, immunofluorescence, immunohistochemistry.
The flow cytometry is based on a fluorescent probe, and realizes the sorting of target cells by combining an external electric field under the level of single cells and flowing liquid; within a few minutes, the analysis is complete 106Individual cells, a high throughput assay and screening method that increases the efficiency of cell line establishment, but requires a cell density of at least 105The proportion of target cells is greater than 0.1% per ml, and therefore, for rare cell or small target cell samples, sorting by flow cytometry is not appropriate. Moreover, the ability to secrete antibodies is not always positively correlated with the cell surface fluorescence intensity.
In recent years, the advent of microfluidic technology has provided a new approach to antibody screening.fabrication of geometric chambers of micro-nanoliter volume, uniform or different size physical barrier structures, etc. consistent with cell size by microfabrication techniques are used to capture single cells.theoretically, the antibody secretion rate of a single cell is 3.66 × 105fM/min, in nanoliter or picoliter volume, can reach ng level concentration level within 1 minute; but the antibody can reach the detection level within 0-4 h under the influence of manual operation and culture environment, so that the detection time is shortened. In the case of physical barrier structures such as microwells, the detection antigen is immobilized within the structure and expressed, and the detection device is in direct contact with or immobilized on a cell, and the antibody secreted by the cell is bound to distinguish specific cells with a fluorescently labeled secondary antibody or other probe. The other method is a droplet-based method, and comprises the steps of wrapping cells, antibody capture magnetic beads and a fluorescence detection reagent into a nano-liter or pico-liter droplet chamber, combining secreted antibodies with the magnetic beads, enriching fluorescent antibodies, and screening cells with strong fluorescence. In addition, the microfluidic-based amplification of single cells also finds application in the field of antibody discovery. The cells, the mRNA capture magnetic beads and the amplification reagent components are simultaneously wrapped in a nano-liter picoliter droplet chamber, heavy chain and light chain genes of the antibody of each cell are obtained, and an antibody sequence is obtained by combining Sanger sequencing or NGS.
These methods cannot continuously detect and culture single or small amount of cells, and establish stable cell strains on the chip; in addition, the data volume generated by the immune repertoire is large, and the length of the fragment read by the NGS is limited, so that the later screening and antibody pairing are not facilitated. The screening and amplification based on the microfluidic chip still needs to be combined with flow cytometry, input a large number of cells, and complete the establishment of stable cell strains by off-chip operation.
At present, no relevant technical report capable of integrating cell capture, multiple identification, culture and release on the same chip has been found.
Disclosure of Invention
The invention aims to provide a microfluidic chip for capturing and screening single cells and application thereof.
The invention also aims to provide a microfluidic chip system which can realize the capture, multiple identification, culture and release of single cells and achieve the screening of high-secretion cells.
The invention designs a brand-new microfluidic chip aiming at the screening of cells, and aims to screen cells with high secretion from a large number of cells on the level of single cells; the realization method is to adopt the micro-fluidic technology and the micro-processing method, integrate the functions of cell capture, multiple identification, culture and release on the same chip, and provide a corresponding operation method to achieve the purpose of rapidly screening cells.
In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a microfluidic chip for capturing and screening single cells, the microfluidic chip comprises four layers of structures stacked in sequence and sealed with each other, and a microvalve control layer, a microvalve 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 (a plurality of holes for gas circulation are formed on the surface of the micro valve control layer and are used as gas inlets and outlets), 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, the micro valve is used for controlling the opening and the closing of the flow channel on the substrate, and meanwhile, the micro valve can also control the opening and the closing of the micro-pore unit and the opening and the closing of the interception unit.
Preferably, the microvalve control layer has two or more independently controlled gas channels, and only one 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 sample and reagents.
The micro valve film layer can be bent and deformed in the film layer area corresponding to the gas channel under the control of the gas pressure. 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. Preferably, the microvalve film layer is a single film with uniform thickness, and is provided with four holes penetrating through the film, and the four holes are respectively communicated with the four holes of the microvalve control layer correspondingly.
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.
Preferably, the sample inlet of the first identification area is divided into two flow channels for sample distribution, and more preferably, a plurality of parallel flow channels are distributed in the functional area; and a micropore array is arranged in the flow passage and is communicated up and down.
The micropore array is used for capturing single cells. The enrichment region has a microvalve structure. To enrich the cells, air bubbles that may be present in the structure are excluded.
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 (a plurality of holes for liquid circulation are formed on the lower surface of the detection release layer and are used as liquid inlets and outlets) for backflushing the inflow of cell liquid, and the other end of the release flow channel is in a closed state. The release flow channel is also used for fixing detection protein.
Specifically, the detection release layer is provided with a plurality of liquid flow channels and corresponding injection ports, the flow channels are vertically overlapped with the flow channels of the first identification area and the second identification area of the substrate, and correspond to micropores of the identification areas. In a preferred embodiment of the invention, each release flow path may act on the same row of 6 microwells in the first identification region or the same row of 4 microwells in the second identification region.
The thickness of the micro valve control layer is 1-5 mm.
The thickness of the micro valve 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 which is arranged in a bent or zigzag manner, or a plurality of channels which are arranged in parallel. Preferably a plurality of channels arranged in parallel. Each flow channel is controlled by a separate 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 micropore units on the substrate are squares or rectangles with the side length of 20-500 micrometers, or circles with the diameter of 10-500 micrometers, or other geometrical structures.
Preferably, the depth of the release flow channel on the detection release layer is 1-50 microns, and the width is 20-2000 microns. Preferably, the release flow channels are arranged in parallel.
Preferably, the width of the gas channel on the micro valve control layer is 20-2000 microns, and the depth is 20-2000 microns.
In the present invention, the material of the micro valve control layer and the detection release layer is selected from transparent materials with good biocompatibility and good protein adsorption, and Polydimethylsiloxane (PDMS) is preferable. The micro valve control layer and the detection release layer are made of solidified rigid materials or are made of rigid materials.
The material of the micro valve film layer is selected from transparent flexible materials with good ductility and strong elasticity, and PDMS and the like are preferred.
The substrate is made of materials (such as silicon wafers and the like) which are easy to process and can be accurately controlled in size, and can be made of transparent materials or opaque materials.
The structure of the microfluidic chip for capturing and screening single cells is schematically shown in figure 1. The cross-sectional view along AA 'and BB' shows the substrate, sample flow channel and capture microwell structure of the chip as shown in FIG. 2. Fig. 3 is a top perspective view of the four-layer structure of fig. 1 after encapsulation. The physical diagram of the microfluidic chip for single cell capture and screening of the present invention is shown in FIG. 5.
In a second aspect, the invention provides any one of the following applications of the microfluidic chip:
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.
In a third aspect, the present invention provides a microfluidic chip system for single cell capture, identification, culture and release, comprising: the device comprises a microfluidic chip, a fluorescent probe, a fluorescent microscope, image processing software, a pump and an air pressure control device controlled by a single chip microcomputer.
In a fourth aspect, the present invention provides a method for screening a hybridoma cell secreting a specific antibody using the microfluidic chip, 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 into the interception unit of 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 into the second identification area, carrying out second identification, releasing the screened hybridoma cells capable of secreting the specific antibody into the interception unit of 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.
The schematic diagram of the process of single cell capture, identification, culture and release by using the microfluidic chip is shown in figure 4.
The cell suspension may be derived from living tissue, blood or a cell suspension cultured in vitro.
The molecular probe with fluorescence is an antibody, polypeptide, biotin-avidin and derivatives thereof marked with fluorescent molecules, and can be used for identifying a single protein by using one fluorescent probe independently and also can be used for identifying multiple proteins by using multiple fluorescent probes simultaneously.
In one 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 fig. 3 and 4, first, the first assay region was pretreated with 75% ethanol, sterile Phosphate Buffered Saline (PBS), while the microvalves of the other regions were closed, prohibiting fluid entry. Then pumping in the detection protein, incubating excessively at 4 ℃ or incubating at 37 ℃ for 2 hours, and adsorbing and fixing the detection protein on the detection release layer. Pumping the cell sample into the first identification area, capturing single cells, and incubating for 0.5-4 hours; then washing the free target molecules with sterile PBS, pumping the fluorescent probe molecules to form a compound with the target molecules, and reacting for 0.5-4 hours. And finally, washing the unbound fluorescent probe molecules by sterile PBS, automatically scanning the micropores from the front by using a fluorescence microscope, and continuously pumping into a culture medium for continuous culture after obtaining a fluorescence image. After culturing for 24-72 hours, opening an inlet micro valve, a waste liquid outlet and a semi-open micro valve of the enrichment area, and closing other micro valves at the same time; and from the detection release area, flushing the selected cells to the enrichment area, opening the inlet micro valve of the second identification area, completely opening the outlet micro valve of the enrichment area, closing other micro valves, and releasing the cells to the second identification area. The second identification area pre-treatment method and identification steps are the same as the first identification area, and after the identification is finished, the cells are released to obtain ideal cells after the second identification area is cultured for 24-72 hours again. According to the experiment requirement, a plurality of screening modules can be connected in parallel and in series.
The sample is a cell population that exhibits heterogeneity. The reagent is a commercial product, or is prepared by self or processed and produced by biological companies.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention develops a multi-layer and multifunctional micro-fluidic chip by introducing a micro-processing technology and a micro-fluidic technology, realizes the capture, identification, culture and release of cells, provides a new method for cell screening, and can acquire high-secretion cells more quickly, accurately and conveniently.
The invention realizes the capture, identification, culture and release of single cells, and has the following remarkable advantages in the field of cell screening for the single cells of heterogeneous cell populations: firstly, compared with the traditional technology, the invention captures single cells by the functions of designing micropores and size, obtains higher capture efficiency according to the concentration and flow rate of the cells, and is beneficial to analyzing the single cells; meanwhile, when the sample is in the micropore, the secreted target molecules can quickly reach the detection concentration and are identified by the detected protein, and the time is shortened. Secondly, PDMS and silicon are biocompatible materials, have no toxicity to cells, and are beneficial to the culture of the cells; PDMS has good air permeability and is beneficial to the exchange of oxygen. In addition, the operation process adopts fluid to release cells, damages the cells to external mechanical force and ensures the activity of the cells. Therefore, the microfluidic chip has diversified functions.
The invention realizes the integration of cell screening: the invention adopts a mode of combining functional areas, integrates 2 times of identification areas, 2 times of enrichment areas and a micro valve control area, integrates the whole operation process on the same chip, reduces the operation outside the chip, avoids the loss and damage of cells in the process and ensures the activity of the cells.
The micro-fluidic chip and the application method designed by the invention have wide application range, and the sizes of the micro-pore array and the flow channel can be adjusted according to different cells; the number of modules can also be increased according to the different cell amounts, which is easy to realize and hardly increases the cost for the skilled person.
Drawings
FIG. 1 is a schematic structural diagram of a microfluidic chip for single cell capture and screening according to the present invention.
FIG. 2 is a cross-sectional view along AA 'and BB' showing the specific structure of the substrate, sample flow channel and capture microwell in the chip.
Fig. 3 is a top perspective view of the four-layer structure of fig. 1 after encapsulation.
FIG. 4 is a schematic diagram of the process of single cell capture, identification, culture and release by using the microfluidic chip.
FIG. 5 is a physical diagram of the microfluidic chip for single cell capture and screening according to the present invention.
FIGS. 1-4 are schematic views, not to scale.
In the figure, 1-microvalve control layer; 2-a microvalve membrane layer; 3-a substrate with 4 functional regions; 4-detection release layer; 5-sample inlet (a well), 6-sample outlet (d well), 7-sample inlet (b well), 8-sample inlet (c well), 12-liquid inlet (c well); 9. 10-gas inlet and outlet; 11-a release flow channel; 13-microwell array structure; 14-a gas channel; 15-entrapment unit controlled by micro-valve; 16-cells; 17-micro valve structures on the substrate flow channels; 18-a sample tube for holding cells; 19-a first identified region; 20-a first enrichment zone; 21-a second identification zone; 22-a second enrichment zone; 23-a microvalve control region; 24-microvalve in open state; 25-microvalve in closed state; 26-detection of proteins. Dashed arrow-gas flow channel; solid arrows: a liquid flow passage.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the 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 commercially available products.
Example 1 microfluidic chip for Single cell Capture and screening and microfluidic chip System for capturing, multiple identification, culture and Release of Single cells
1. Microvalve control layer and microvalve film layer
When cells are identified, light is required to penetrate through the micro valve control layer and the micro valve thin film layer, so that the micro valve control layer and the micro valve thin film layer are required to be made of transparent materials; meanwhile, the micro valve control layer and the micro valve film layer need to be provided with interfaces for fluid to enter and flow out, so that holes need to be formed; in addition, the aim of opening and closing the micro valve is achieved by extruding the membrane to bend and deform through gas, so that the material of the membrane layer of the micro valve is required to be a soft material with good elasticity; in addition, since cells need to be cultured on a chip, all relevant materials need good biocompatibility; finally, the microvalve control layer needs to be sealed with the microvalve thin film layer, and sealing is achieved, so compatibility with the microvalve thin film layer needs to be considered in material selection. Preferred materials are those that can be formed into specific specifications and shapes by evaporation, cutting and thermoforming. Particularly preferred are relatively thin and transparent polymers.
In the preferred embodiment shown in fig. 1 and 4, the microvalve control layer, the microvalve thin film layer and the detection release layer can all be made of PDMS materials, and the required gas channel and film thickness of the microvalve control layer are obtained by soft lithography, and the two layers are hermetically connected in a bonding manner. In other implementation methods, the micro valve control layer may also be made of a glass material, and a gas channel is obtained by wet etching or laser etching, and the two are sealed together by a bonding method.
In the preferred embodiment shown in fig. 1 and 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 membrane layer is 40 microns, which is a preferred dimension for effective control of the microvalve switch. When the length, width and depth of the flow channel of the micro valve control switch are required to be changed, the proper size of the gas channel and the micro valve film can be selected by the skilled person according to the requirement.
In the preferred embodiment shown in fig. 1 and 4, the detection release layer serves two functions: firstly, fixing a detection antigen; second, the cells are released. Thus, in addition to being biocompatible, the test release layer should also have polypeptide or protein adsorption or surface modification. In the preferred embodiment, PDMS is selected as the preferred material, and the surface plasma treatment is performed and then the PDMS is incubated with the protein together, so as to achieve the purpose of protein immobilization. In addition to the preferred embodiment, other transparent materials, such as polystyrene, etc., which have high adsorbability or can be activated to introduce functional groups, can also be selected.
2. Substrate with micropore array and flow channel
In the present invention, the microwell array is a key functional region for capturing and culturing cells. To accommodate the size of the cell, materials must be selected that are compatible with microfabrication techniques. In the preferred embodiment shown in fig. 1 and 2, silicon is chosen as the material for the substrate microstructure array because silicon is well compatible with lithographic and etch based micromachining processes and the machining accuracy is easily controlled so that a capture array conforming to the cell size can be easily obtained. The skilled person can also select a processable material such as glass, as desired. It is noted that the substrate integrates 4 functional areas: the identification area of the cell needs to be etched twice on a silicon chip to obtain a flow channel and a micropore array structure, and the cell-rich collection area can be finished by only once etching.
In the preferred embodiment shown in fig. 2, the first identification region of the substrate is provided with 6 flow channels, 6 flow channels share 1 sample inlet port, and the second identification region is provided with 4 flow channels. For the cell identification micropore array, each micropore is a chamber with the side length of 50 micrometers and the height of 160 micrometers, the whole micropore array has 260 micropores, wherein the first identification area has 180 micropores (30 micropores are uniformly distributed on each flow channel on average), and the second identification area has 80 micropores (20 micropores are uniformly distributed on each flow channel on average); for the sample flow channel, to accommodate flow rate, the cells were kept as monolayer as possible, with a height of 30 microns and a width of 200 microns. The above are the preferred conditions for analysis of hybridoma cells. When other cells need to be processed, the proper size and number of micropores can be selected by those skilled in the art according to the size specification of the silicon wafer and the requirements of cell experiments.
The physical diagram of the microfluidic chip for single cell capture and screening of the present invention is shown in FIG. 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 release layer are PDMS, silicon and PDMS, respectively. By adopting the oxygen plasma-assisted bonding method, good sealing between PDMS and between silicon and PDMS can be well realized.
In other embodiments, the suitable sealing method may 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 matching system
Besides the microfluidic chip, the invention also needs a fluorescent Probe (Probe), a fluorescence microscope, ImageJ image analysis software and a pump to jointly form a complete system to complete the capture, identification, culture and release of cells.
Fluorescent probes are used for identification of antibody molecules, and 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 recognizing antibodies secreted by cells. In other embodiments, different antibodies, polypeptides, or biotin-avidin systems and derivatives thereof may be selected as probes for different detection methods.
The fluorescence microscope is used for detecting whether the cells captured in the micropore array have fluorescence or not, and scanning and imaging the micropore array to obtain a fluorescence picture.
ImageJ image analysis software was used to analyze the images obtained by the fluorescence microscope and obtain the number of microwells corresponding to the fluorescent cells. The software screens the micropores meeting the requirements by calculating the intensity of fluorescence in the image.
Pumps are used to drive liquid samples, liquid media, gases and related reagents.
5. Specific manufacturing method of chip
The chip and system of the present invention have been successfully fabricated by two different fabrication processes as follows. The specific fabrication methods are given to help those skilled in the art understand the fabrication method and gist of the present invention, and are not intended to limit the materials, dimensions, and fabrication methods of the devices of the present invention.
The preparation method A comprises the following steps:
a microvalve control layer: a4-inch Pyrex7740 glass sheet (Corning corporation) is adopted, the plane positions of micropores and gas channels are photoetched on the front surface of a silicon wafer, hydrofluoric acid is adopted to corrode the glass, the gas channels and holes with the depth of 200 micrometers are formed, and the positions of the micropores are punched through by laser. Finally, rectangular small pieces are cut according to the external dimensions of 6 cm in length and 3 cm in width.
Micro-valve film layer: an N-type 4-inch silicon wafer is adopted, a layer of PDMS liquid with the thickness of 30 micrometers is coated on the surface of the N-type 4-inch silicon wafer by using a spin coating method, the PDMS liquid is taken out after solidification, and a rectangular film is cut according to the overall dimensions of 6 cm in length and 3 cm in width.
Substrate with microwell array and flow channels: an N-type 4-inch silicon wafer is adopted, a wet oxidation is adopted, an oxidation layer with the thickness of 0.5 micrometer is obtained on the surface of the silicon wafer, the plane positions of a flow channel and a liquid injection port are photoetched on the front surface of the silicon wafer, and a flow channel with the depth of 40 micrometers is manufactured by a potassium hydroxide wet etching method. And photoetching the back of the silicon wafer to form the position corresponding to the bottom of the micropore, and etching through the silicon wafer by adopting an ICP dry method to finally obtain a complete silicon micropore array and a micro channel. A4-inch silicon wafer was cut into rectangular pieces in the overall dimensions of 6 cm in length and 3 cm in width.
Detection of the release layer: an N-type 4-inch silicon wafer was used, and after the planar shape of the flow channel (release flow channel) was photoetched, a projection having a height of 5 μm was etched by an ICP dry method. Pouring liquid PDMS into a tank, demoulding after the PDMS is solidified, taking out, cutting the solidified liquid PDMS into rectangular small pieces according to the overall dimensions of 6 cm in length and 3 cm in width, and punching holes (serving as liquid inlets and outlets) at required positions by using a puncher, thereby obtaining the detection release layer.
Sealing: firstly, oxygen plasma treatment is carried out on the back surface of the micro valve control layer, the two surfaces of the silicon substrate, the two surfaces of the micro valve film and the front surface of the detection release layer, and the micro valve control layer, the silicon substrate, the micro valve film and the detection release layer are bonded together in sequence to finally obtain a complete chip.
Chip system: on the basis of a microfluid chip, a polytetrafluoroethylene tube is used for connecting a pump and a microvalve control layer, and a fluid inlet and a fluid outlet of a detection release layer, the polytetrafluoroethylene tube is used for connecting a gas channel inlet of the pump and the microvalve control layer, the chip is placed under an upright fluorescence microscope capable of automatically carrying out fluorescence imaging, and ImageJ image processing software is used for automatically analyzing and processing a fluorescence image, so that the construction of the whole set of system can be completed.
The preparation method B comprises the following steps:
a microvalve control layer: an N-type 4-inch silicon wafer is adopted, and after the planar shape of the gas channel is photoetched, a convex body with the height of 30 micrometers is etched by using an ICP dry method. And pouring the liquid PDMS into a tank, demolding and taking out after the PDMS is solidified, cutting the PDMS into rectangular small pieces according to the overall dimensions of 6 cm in length and 3 cm in width, and punching holes at required positions by using a puncher, thereby obtaining the microvalve control layer.
Micro-valve film layer: an N-type 4-inch silicon wafer is adopted, a layer of PDMS liquid with the thickness of 30 micrometers is coated on the surface of the N-type 4-inch silicon wafer by using a spin coating method, the PDMS liquid is taken out after solidification, and a rectangular film is cut according to the overall dimensions of 6 cm in length and 3 cm in width.
Substrate with microwell array and flow channels: an N-type 4-inch silicon wafer is adopted, and the planar shape of the micro-channel structure is photoetched on one surface of a double-polished silicon wafer of 200 microns; and the other side is photoetched to form the plane shape of the micropore structure. And respectively adopting an ICP dry etching method to obtain a flow channel with the depth of 40 micrometers and a micropore with the depth of 160 micrometers, namely etching and punching through the micropore part. A4-inch silicon wafer was cut into rectangular pieces in the overall dimensions of 6 cm in length and 3 cm in width.
Detection of the release layer: an N-type 4-inch silicon wafer was used, and after the planar shape of the flow channel (release flow channel) was photoetched, a projection having a height of 5 μm was etched by an ICP dry method. Pouring liquid PDMS into a tank, demoulding after the PDMS is solidified, taking out, cutting the solidified liquid PDMS into rectangular small pieces according to the overall dimensions of 6 cm in length and 3 cm in width, and punching holes (serving as liquid inlets and outlets) at required positions by using a puncher, thereby obtaining the detection release layer.
Sealing: the back surface of the micro valve control layer, the two surfaces of the silicon substrate, the two surfaces of the micro valve film and the front surface of the detection release layer are firstly subjected to oxygen plasma treatment, and then are bonded together in sequence to finally obtain a complete chip.
Chip system: on the basis of a microfluid chip, a polytetrafluoroethylene tube is used for connecting a pump and a microvalve control layer, and a fluid inlet and a fluid outlet of a detection release layer, the polytetrafluoroethylene tube is used for connecting a gas channel inlet of the pump and the microvalve control layer, the chip is placed under an upright fluorescence microscope capable of automatically carrying out fluorescence imaging, and ImageJ image processing software is used for automatically analyzing and processing a fluorescence image, so that the construction of the whole set of system can be completed.
6. Specific application
The following method successfully applies the microfluidic chip and the corresponding system of the invention to the screening of hybridoma cells. The specific method is given 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.
The specific application method is illustrated by taking hybridoma cells aiming at the CD45 protein as an example as follows:
all experimental procedures were performed intracellularly in sterile environment, consumables and instruments were sterilized in advance, and reagents were sterile.
And (3) pretreating the microfluidic chip, namely injecting 75% ethanol into the microfluidic chip, continuously injecting for 5 minutes, and sterilizing and infiltrating the surface of the flow channel to increase the hydrophilicity. Sterile Phosphate Buffered Saline (PBS) was injected and the flow channel was purged with ethanol. The assay region was then pumped with the CD45 test protein (leukocyte common antigen) and incubated overnight at 4 ℃. The next day, 5% sterile BSA was injected, incubated for 1 hour, and the sites that did not bind antigen were blocked.
First identification: and collecting heterogeneous hybridoma cells, re-suspending the hybridoma cells in a complete culture medium, adjusting the cell concentration, injecting the hybridoma cells into a first identification area of the microfluidic chip through a micropump, and closing other micro valves such as the enrichment area and the like. When the cell suspension flows through the first identified region, the liquid fills the flow channel and flows out of the release channel along the direction of the micropores. Due to the size effect, the size of the cells is larger than 5 microns and larger than the height of the release flow channel, and the cells are captured in the micropores. The cells were incubated in a carbon dioxide incubator for 2 hours. Injecting a fluorescent secondary antibody, injecting sterile PBS, washing unbound antibody, injecting a fluorescent labeled secondary antibody, and placing in a carbon dioxide incubator for incubation for 1 hour. Fluorescence was assayed by injecting sterile PBS and washing unbound secondary antibody. And (3) automatically scanning the micropores from the front by using a fluorescence microscope, and continuously pumping the obtained fluorescence image into a complete culture medium for culture.
Single cell enrichment: after culturing for 48 hours, selecting micropores with high fluorescence intensity, injecting culture solution from the detection release layer, closing the microvalve at the inlet of the second identification area, and opening the microvalve at the outlet of the enrichment area. The cells are back-washed from the microwells into the enrichment zone.
And (3) second identification: the microvalve of the second identification zone is opened and cells enter the second identification zone from the enrichment zone. Other operations are the same as the first authentication.
Finally, the cells were harvested and transferred to 24-well plates for expanded culture.
The structure of the microfluidic chip for capturing and screening single cells is schematically shown in figure 1. The cross-sectional view along AA' shows the substrate, sample flow channel and capture microwell structure of the chip as shown in FIG. 2. Fig. 3 is a top perspective view of the four-layer structure of fig. 1 after encapsulation. The schematic diagram of the process of single cell capture, identification, culture and release by using the microfluidic chip is shown in figure 4.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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.
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