CN112920951A - Cell screening chip and manufacturing and cell screening and collecting method thereof - Google Patents
Cell screening chip and manufacturing and cell screening and collecting method thereof Download PDFInfo
- Publication number
- CN112920951A CN112920951A CN202110250079.3A CN202110250079A CN112920951A CN 112920951 A CN112920951 A CN 112920951A CN 202110250079 A CN202110250079 A CN 202110250079A CN 112920951 A CN112920951 A CN 112920951A
- Authority
- CN
- China
- Prior art keywords
- screening
- cell
- chip
- pdms
- cavity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000012216 screening Methods 0.000 title claims abstract description 130
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 12
- 239000004205 dimethyl polysiloxane Substances 0.000 claims abstract description 104
- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims abstract description 104
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims abstract description 104
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims abstract description 103
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims abstract description 103
- 238000003860 storage Methods 0.000 claims abstract description 43
- 239000000758 substrate Substances 0.000 claims abstract description 42
- 238000011534 incubation Methods 0.000 claims abstract description 40
- 239000011521 glass Substances 0.000 claims abstract description 35
- 239000007788 liquid Substances 0.000 claims abstract description 26
- 239000002699 waste material Substances 0.000 claims abstract description 20
- 238000003491 array Methods 0.000 claims abstract description 15
- 238000001914 filtration Methods 0.000 claims abstract description 8
- 238000002347 injection Methods 0.000 claims abstract description 7
- 239000007924 injection Substances 0.000 claims abstract description 7
- 230000000149 penetrating effect Effects 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 27
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 21
- 229910052710 silicon Inorganic materials 0.000 claims description 21
- 239000010703 silicon Substances 0.000 claims description 21
- 238000004140 cleaning Methods 0.000 claims description 15
- 238000005406 washing Methods 0.000 claims description 13
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 230000001678 irradiating effect Effects 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 238000001259 photo etching Methods 0.000 claims description 6
- 229920002120 photoresistant polymer Polymers 0.000 claims description 6
- 238000004080 punching Methods 0.000 claims description 6
- 238000004113 cell culture Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 4
- 108010019160 Pancreatin Proteins 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 3
- XPBBUZJBQWWFFJ-UHFFFAOYSA-N fluorosilane Chemical compound [SiH3]F XPBBUZJBQWWFFJ-UHFFFAOYSA-N 0.000 claims description 3
- 239000003292 glue Substances 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 229910021421 monocrystalline silicon Inorganic materials 0.000 claims description 3
- 229940055695 pancreatin Drugs 0.000 claims description 3
- 238000004528 spin coating Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000001413 cellular effect Effects 0.000 claims 1
- 238000012545 processing Methods 0.000 description 4
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- -1 Polydimethylsiloxane Polymers 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- KPUWHANPEXNPJT-UHFFFAOYSA-N disiloxane Chemical class [SiH3]O[SiH3] KPUWHANPEXNPJT-UHFFFAOYSA-N 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 238000000018 DNA microarray Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 125000001183 hydrocarbyl group Chemical group 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000005459 micromachining Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 229920002545 silicone oil Polymers 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
- B01L2300/0845—Filaments, strings, fibres, i.e. not hollow
Abstract
Providing a cell screening chip, which comprises a glass substrate (1) and a PDMS sheet (2) which are mutually attached, wherein an incubation cavity (5), a screening cavity (6), a storage cavity (7), a sample injection flow channel (8), a first flow channel (9), a second flow channel (10) and a sample outlet flow channel (11) are formed on the attachment surface of the PDMS sheet (2) from a sample inlet (3) to a sample outlet (4); a valve is arranged at the first flow passage (9); the screening cavity (6) is provided with 2 groups of screening micro-column (12) arrays in parallel, and the distance between two adjacent screening micro-columns (12) is larger than a single cell and smaller than a combined cell; a main channel (13) and an auxiliary channel (14) are formed, and waste liquid penetrating ports (15) are respectively formed at 2 corners of the screening cavity (6); the edge of the storage cavity (7) is provided with a filtering microcolumn (16) array; the chip integrates multiple functions; the invention also provides a manufacturing method of the chip and a cell screening and collecting method.
Description
Technical Field
The invention belongs to the technical field of microfluidic biochips, and particularly relates to a cell screening chip and a manufacturing and cell screening and collecting method thereof.
Background
The screening and collection of target cells in a mixed cell solution sample containing various cells is always a common subject in cell biology, and different cell screening and collection methods exist based on different cell properties, for example, screening can be performed by using a filter screen according to the size of the cells; adherent cells and suspension cells can be separated according to the adherent characteristics of the cells; depending on the requirements for a particular growth factor or inhibitory factor, selection media may be used for screening; for some specific antibody cells, they can also be screened by flow cytometry after fluorescent labeling.
Highly differentiated cells will generally have some specificity compared to other cells, and depending on the different specific antigens or antibodies of different cells, the target highly differentiated cells can be labeled with labeled magnetic beads, specific antibodies or antigens, and cells producing specific antibodies or antigens. Thereby purifying and screening the target cells. However, the conventional cell screening methods all have the same disadvantages: the operation is complex, the breeding period is long, and the pollution is easy.
With the continuous development of microfluidic technology and micromachining technology, the application advantages of Micro total analysis systems (μ -TAS) in the field of cell biology are becoming more and more significant. In the microfluidic chip, the size of the unit for performing various functions can be close to the size of the cell, so that the method can be used for accurately analyzing the single cell markers and the whole cells in the target sample, and has the advantage of high precision. The method has obvious advantages in cell screening. The technology can integrate all the existing cell screening steps and processes (cell culture, cell capture, cell screening and cell enrichment) on one microchip, realize the integration of screening operation, can reduce the damage and pollution to cell samples in the operation process, and is very suitable for the rapid screening and collection of a small amount of target cells.
At present, a series of novel microfluidic chips and devices are developed for cell screening, and the microfluidic chips and the devices can be mainly classified into the following types: (1) the method is characterized in that an antibody is simply spotted on a substrate by using a spotting instrument, and a target living cell is grabbed by using the antibody. Such as patent document CN 112098642A. (2) And (4) screening target cells by using a flow channel for fixing the size. Such as document CN 112175792A.
These chip devices for cell screening all have some obvious disadvantages: (1) the method is very limited because of the dependence on external sample treatment, (2) the need for many external operating or control instruments, and (3) the screening can be performed only by physical or biological characteristics.
Polydimethylsiloxane, abbreviated as PDMS. Is a high molecular organic polymer, has optical transparency and internal fine structure porosity, and has good biocompatibility. Applications of PDMS include micro-fluidic channel systems in bio-microelectromechanical systems, caulks, lubricants, contact lenses. The liquid dimethyl siloxane is a viscous liquid called silicone oil, and is an organic siloxane mixture with chain structures with different polymerization degrees, and the end group and the side group of the organic siloxane mixture are all hydrocarbon groups (such as methyl, ethyl, phenyl and the like).
Disclosure of Invention
The invention aims to provide a cell screening chip and a manufacturing and cell screening and collecting method thereof. That is, the function of the cell screening chip is realized based on the chip.
In order to solve the technical problems, the cell screening chip adopts the following technical scheme:
a cell screening chip comprises a glass substrate and a PDMS sheet which are mutually attached, and is characterized in that a sample inlet and a sample outlet which penetrate through the PDMS sheet are formed in the PDMS sheet at intervals, an attachment surface of the PDMS sheet is provided with an incubation cavity, a screening cavity and a storage cavity which are sequentially connected from the sample inlet to the sample outlet and are used for cell solution to flow through to complete cell screening, a sample inlet flow channel is formed between the sample inlet and the incubation cavity, a first flow channel is formed between the incubation cavity and the screening cavity, a second flow channel is formed between the screening cavity and the storage cavity, and a sample outlet flow channel is formed between the storage cavity and the sample outlet; after the PDMS sheet is attached to the glass substrate, a communicated closed cavity is formed from the sample inlet to the sample outlet; the PDMS sheet is provided with a valve which can open or block the first flow channel at the first flow channel; the screening cavity is provided with 2 groups of screening micro-column arrays in parallel, and the distance between two adjacent screening micro-columns in the screening micro-column arrays is larger than a single cell and smaller than a binding cell; a main channel directly connecting the incubation cavity and the storage cavity is formed between the 2 groups of screening micro-column arrays, auxiliary channels are formed on two sides of the 2 groups of screening micro-column arrays, and 2 corners of the screening cavity close to the side of the storage cavity are respectively provided with a waste liquid port penetrating through the PDMS sheet; the edge of the storage cavity is provided with a filtering micro-column array, and the distance between two adjacent filtering micro-columns in the filtering micro-column array is larger than a single cell and smaller than a combined cell.
The following are further schemes for the cell screening chip of the present invention:
the sample injection flow channel is arranged as follows: the initial runner section that draws from the introduction port divide into 2 relatively thin branch runner sections, and every branch runner section subdivides into 2 more thin subdividing runner sections, and 4 subdividing runner sections are each parallel connection the incubation chamber.
The valve is arranged as follows: in the area between the incubation cavity and the screening cavity of the PDMS sheet, vent grooves are respectively formed in the upper side and the lower side of the first flow channel, thin layers which deform under pressure to enable the thin layers to block the first flow channel are respectively reserved on the upper side and the lower side of the first flow channel, vent holes penetrating the PDMS sheet are respectively formed in the terminals of the vent grooves on the two sides, and after the PDMS sheet is attached to the glass substrate, the vent grooves on the two sides respectively form a closed vent cavity.
The incubation cavity and the screening cavity are rectangular, the side of the screening cavity close to the storage cavity respectively extends towards the upper right direction and the lower right direction to serve as the auxiliary channel extension section, the auxiliary channel extension section is horn-shaped, and the opening of the auxiliary channel extension section is far larger than the width of the second flow channel; and the waste liquid ports are respectively arranged at the terminal of the auxiliary channel extension section.
The cavity department of hatching chamber, storage chamber is provided with the support column array that prevents the cavity and cave in, the screening microcolumn array in screening chamber plays the supporting role simultaneously.
In order to solve the technical problems, the manufacturing method of the cell screening chip adopts the following technical scheme:
a method for preparing cell screening chip includes preparing PDMS sheet and glass substrate separately and bonding them, making PDMS sheet including making silicon substrate and PDMS sheet as mould; the method is characterized in that:
the silicon substrate manufacturing method specifically comprises the following steps: adopting four-inch monocrystalline silicon as a substrate, spin-coating 30-micron thick photoresist on a silicon wafer by using a rotary glue coating machine, and pre-drying: baking at 65 ℃ for 30 minutes; then, photoetching is carried out according to the designed layout; and (3) after photoetching is finished, post-drying: postbaking for 30 minutes at 95 ℃; developing by using a developing solution, removing the photoresist and cleaning to obtain a microstructure with a designed height; then hard-baking for 30 minutes at 95 ℃; and obtaining the silicon wafer molds with microstructures of different heights.
The preparation process of the PDMS sheet is as follows: putting the prepared silicon chip mold into a fluorosilane atmosphere for incubation for 4 hours so as to facilitate the subsequent stripping between the PDMS chip and the mold; according to the weight ratio of 15: 1, weighing PDMS prepolymer and curing agent, placing the PDMS prepolymer and the curing agent in a container, uniformly stirring the PDMS prepolymer and the curing agent by using a glass rod, placing the container in a vacuum drier, vacuumizing until the vacuum degree is 13psi, and standing for 30min to remove bubbles; taking out the PDMS mixture, placing the silicon wafer mold on a horizontal table, pouring PDMS, and standing for 30min to fill the PDMS in the mold; putting the mould into an oven, heating for 1h at the temperature of 80 ℃, and carefully stripping the PDMS from the silicon wafer after the PDMS is completely cured; punching holes at the positions of a sample inlet, a waste liquid outlet and a sample outlet on the PDMS chip by using a needle head with a specific aperture, and punching holes at the air inlet of a valve; placing the PDMS chip into a plasma cleaning machine, keeping the PDMS chip in vacuum for 1h, and irradiating for glow for 2 min; taking out the chip, dropwise adding a PEG (6-9) -siloxane and acetone mixed solution (v: v =1: 1) at a sample inlet, filling the mixed solution in the inner cavity and the pipeline of the whole chip by using negative pressure in the PDMS chip as a driving force, and incubating for 1h at room temperature; and then, washing with ultrapure water to ensure that the surfaces of all the inner cavities and pipelines in the PDMS chip are hydrophilic so as to reduce the adhesion of all the inner cavities and pipelines to cells.
The manufacturing of the glass substrate comprises the selection and the cutting of the material of the glass substrate.
The bonding process of the PDMS sheet and the glass substrate comprises the steps of putting the bonding surfaces of the PDMS sheet and the glass substrate which face upwards together into a plasma cleaning machine for cleaning, irradiating for 2min, taking out, rapidly bonding the PDMS sheet and the glass substrate together, and heating on a hot plate at 90 ℃ for 20 min to 30min to enhance the bonding degree of the PDMS sheet and the glass substrate.
In order to solve the technical problems, the cell screening and collecting method adopts the following technical scheme:
a cell screening and collecting method, realize screening and collecting from mixing cell solution target cell, wherein use the above-mentioned cell screening chip, choose the appropriate capture cell that can attract each other and combine with target cell and form and combine the cell, open the said valve, make the sample inlet ventilate to the outlet port, inject the capture cell solution from the sample inlet under pressure, observe its flow condition at the same time, wait to capture cell solution and will be covered with and incubate the cavity, close the valve, stop the pressurization of the sample port, put the chip into cell incubator to incubate, wait to capture cell all adhere to the wall, withdraw the chip; opening a valve, injecting a mixed cell solution sample to be screened from a sample inlet under pressure, closing the valve when the sample to be tested is fully distributed in an incubation cavity, stopping the pressurization of the sample inlet, placing the chip in a cell culture box again for incubation, and enabling the captured cells and the target cells in the mixed cell solution to be fully contacted and combined with each other to form combined cells; opening a valve, injecting washing liquid under pressure from the sample inlet to wash the incubation area, and enabling other cells which are not combined with the captured cells to flow into the screening area and flow out from the waste liquid port; then washing with pancreatin-containing cleaning solution from the sample inlet, so that all the captured cells are detached from the wall, all the cells enter the screening area, the captured cells which are not combined with the target cells pass through 2 groups of screening micro-column arrays and auxiliary channels, and flow out from 2 waste liquid ports; the combined cells can not pass through the 2 groups of screening micro-column arrays and can only enter the storage area along the main channel; a small amount of single cells entering the storage area can also flow out from the sample outlet through the filtering micro-column array at the edge of the storage area; after the screening collection is completed, the target cells are subjected to subsequent treatment.
The subsequent treatment comprises the following steps: and directly carrying out cell analysis on the target cells in the combined cells in the storage cavity.
The subsequent treatment comprises the following steps: and injecting a cleaning solution into the sample outlet of the chip under pressure to back flush the bound cells, so that the bound cells can be flushed out through the sample inlet and collected.
The subsequent treatment comprises the following steps: washing the chip with separating reagent capable of separating the captured cell from the target cell, and collecting the target cell after the captured cell is separated from the target cell.
The cell screening chip is manufactured by a micro-nano manufacturing process, the chip comprises an incubation area, a screening area and a storage area, and the traditional cell screening steps of cell culture, cell capture, cell screening, cell enrichment and the like are integrated on one microchip, so that the integration of cell screening process operation is realized, the complicated operation steps in each process can be saved, the possible damage and pollution to a cell sample in the operation process can be greatly reduced, in addition, the high-precision characteristic of the micro-fluidic chip is combined, and the micro-fluidic chip is very suitable for the rapid screening and the high-efficiency enrichment of a small amount of target cells in a complex sample. The screened target cells are collected in a storage area, and can be directly used for carrying out the next experiment in the storage area or taken out from the storage area.
Drawings
FIG. 1 is a perspective view showing the assembled state of the basic components of the cell screening chip of the present invention.
FIG. 2 is a schematic perspective view of the cell screening chip PDMS sheet and the glass substrate in a separated state.
Fig. 3 is a schematic perspective view of a bonding surface of a PDMS sheet.
Fig. 4 is a schematic view of the valve in the open state.
Fig. 5 is a schematic view of the valve closed state.
The parts indicated by the reference numerals in the figures are: 1. a glass substrate; 2. a PDMS sheet; 3. a sample inlet; 4. a sample outlet; 5. an incubation cavity; 6. a screening chamber; 7. a storage chamber; 8. a sample injection flow channel; 9. a first flow passage; 10. a second flow passage; 11. a sample outlet flow channel; 12. screening the microcolumns; 13. a main channel; 14. an auxiliary channel; 15. a waste liquid port; 16. filtering the microcolumn; 17. a vent channel; 18. a thin layer; 19. and (4) a vent.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples. The cells involved in the present invention are unicellular except the binding cells.
As shown in FIG. 1, the cell screening chip of the present invention includes a glass substrate 1 and a PDMS sheet 2 attached to each other. As shown in fig. 2 and 3, a sample inlet 3 and a sample outlet 4 penetrating through a PDMS sheet 2 are formed in the PDMS sheet 2 at intervals, an incubation cavity 5, a screening cavity 6 and a storage cavity 7 which are sequentially connected and used for cell solution to flow through to complete cell screening are formed in a joint surface of the PDMS sheet 2 from the sample inlet 3 to the sample outlet 4, a sample inlet channel 8 is formed between the sample inlet 3 and the incubation cavity 5, a first channel 9 is formed between the incubation cavity 5 and the screening cavity 6, a second channel 10 is formed between the screening cavity 6 and the storage cavity 7, and a sample outlet channel 11 is formed between the storage cavity 7 and the sample outlet 4; after the PDMS sheet 2 is attached to the glass substrate 1, a communicated closed cavity is formed from the sample inlet 3 to the sample outlet 4; the PDMS sheet 2 is provided with a valve which can open or block the first flow channel 9 at the first flow channel 9; the screening cavity 6 is provided with 2 groups of screening micro-column 12 arrays in parallel, and the distance between two adjacent screening micro-columns 12 in the screening micro-column 12 arrays is larger than a single cell and smaller than a combined cell; a main channel 13 directly connecting the incubation cavity 5 and the storage cavity 7 is formed between the 2 groups of screening micro-column 12 arrays, auxiliary channels 14 are formed at two sides of the 2 groups of screening micro-column 12 arrays, and 2 corners of the screening cavity 6 close to the side of the storage cavity 7 are respectively provided with a waste liquid port 15 penetrating through the PDMS sheet 2; the edge of the storage cavity 7 is provided with an array of filter micro-pillars 16, and the distance between two adjacent filter micro-pillars 16 in the array of filter micro-pillars 16 is larger than a single cell and smaller than a combined cell.
As shown in fig. 2 and 3, the sample injection flow channel 8 is configured as follows: the initial runner section 8a led out from the sample inlet 3 is divided into 2 relatively thin branch runner sections 8b, each branch runner section 8b is divided into 2 thinner branch runner sections 8c, and the 4 branch runner sections 8c are respectively connected with the incubation cavity 5 in parallel.
As shown in fig. 2 and 3, the valve is arranged as follows: in the area between the incubation cavity 5 and the screening cavity 6 of the PDMS sheet 2, vent grooves 17 are respectively formed in the upper side and the lower side of the first flow channel 9, thin layers 18 which are deformed to block the first flow channel 9 when meeting pressure are respectively reserved on the upper side and the lower side of the first flow channel 9, vent holes 19 penetrating the PDMS sheet 2 are respectively formed in the terminals of the vent grooves 17 on the two sides, and after the PDMS sheet 2 is attached to the glass substrate 1, the vent grooves 17 on the two sides respectively form a closed vent cavity. In normal, or original, condition, the valve is in an open condition as shown in fig. 4. When it is desired to close the valve, pressurized gas is simultaneously supplied to the 2 vent channels 17 through the 2 vent openings 19, causing the oppositely directed bulges of the lamellae 18 to deform, as shown in fig. 5, blocking the first flow channel 9. That is, the valve is in an open state when no pressure gas is introduced, and the valve is in a closed state when the pressure gas is continuously introduced.
As shown in fig. 2 and 3, the incubation cavity 5 and the screening cavity 6 are both rectangular, and the side of the screening cavity 6 close to the storage cavity 7 respectively extends to the upper right direction and the lower right direction as an extension section of the auxiliary channel 14, the extension section of the auxiliary channel 14 is trumpet-shaped, and the opening of the extension section is much larger than the width of the second flow channel 10; waste liquid ports 15 are respectively opened at the terminal ends of the extension sections of the auxiliary channels 14.
Because the cavities of the incubation cavity 5 and the storage cavity 7 are large, in order to prevent the cavities from collapsing, an array of support pillars for preventing the cavities from collapsing can be arranged at the cavities of the incubation cavity 5 and the storage cavity 7. Since the array of screening micro-pillars 12 of the screening chamber 6 can simultaneously serve as a support, the screening chamber 6 does not need to be provided with an array of support pillars for preventing the collapse of the cavity.
A method for preparing cell screening chip includes preparing PDMS sheet 2 and glass substrate 1 separately, and bonding them, making PDMS sheet 2 including making silicon substrate as mould and making PDMS sheet 2; the method is characterized in that:
the silicon substrate manufacturing method specifically comprises the following steps: adopting four-inch monocrystalline silicon as a substrate, spin-coating 30-micron thick photoresist on a silicon wafer by using a rotary glue coating machine, and pre-drying: baking at 65 ℃ for 30 minutes; then, photoetching is carried out according to the designed layout; and (3) after photoetching is finished, post-drying: postbaking for 30 minutes at 95 ℃; developing by using a developing solution, removing the photoresist and cleaning to obtain a microstructure with a designed height; then hard-baking for 30 minutes at 95 ℃; obtaining silicon chip molds with microstructures of different heights;
the PDMS slab 2 was made as follows: putting the prepared silicon chip mold into a fluorosilane atmosphere for incubation for 4 hours so as to facilitate the subsequent stripping between the PDMS chip and the mold; according to the weight ratio of 15: 1, weighing PDMS prepolymer and curing agent, placing the PDMS prepolymer and the curing agent in a container, uniformly stirring the PDMS prepolymer and the curing agent by using a glass rod, placing the container in a vacuum drier, vacuumizing until the vacuum degree is 13psi, and standing for 30min to remove bubbles; taking out the PDMS mixture, placing the silicon wafer mold on a horizontal table, pouring PDMS, and standing for 30min to fill the PDMS in the mold; putting the mould into an oven, heating for 1h at the temperature of 80 ℃, and carefully stripping the PDMS from the silicon wafer after the PDMS is completely cured; punching holes at the positions of a sample inlet 3, a waste liquid inlet 15 and a sample outlet 4 on the PDMS chip by using a needle head with a specific aperture, and punching holes at the air inlet of a valve; placing the PDMS chip into a plasma cleaning machine, keeping the PDMS chip in vacuum for 1h, and irradiating for glow for 2 min; taking out the chip, dripping a mixed solution of PEG 6-9-siloxane and acetone (v: v =1: 1) at the injection port 3, filling the whole inner cavity and pipeline of the chip with the mixed solution by using the negative pressure in the PDMS chip as a driving force, and incubating for 1h at room temperature; and then, washing with ultrapure water to ensure that the surfaces of all the inner cavities and pipelines in the PDMS chip are hydrophilic so as to reduce the adhesion of all the inner cavities and pipelines to cells.
The glass substrate 1 is produced by selecting and cutting the material of the glass substrate 1.
The bonding process of the PDMS sheet 2 and the glass substrate 1 comprises the steps of putting the bonding surfaces of the PDMS sheet 2 and the glass substrate 1 together in a plasma cleaning machine to clean, irradiating for 2min, taking out and rapidly bonding together, and heating on a hot plate at 90 ℃ for 20 min to 30min to enhance the bonding degree of the PDMS sheet 2 and the glass substrate.
The cell screening chip of the present invention can realize the screening and collection of target cells from a mixed cell solution. The specific operation can be performed as follows.
Selecting proper capture cells which can mutually attract and combine with target cells to form combined cells, opening a valve to ventilate a sample inlet 3 to a sample outlet 4, injecting capture cell solution from the sample inlet 3 under pressure, and observing the flow condition of the PDMS sheet 2 due to the transparency of the PDMS sheet; opening a valve, injecting a mixed cell solution sample to be screened from a sample inlet 3 under pressure, closing the valve when the sample to be tested is fully distributed in an incubation cavity 5, stopping the pressurization of the sample inlet 3, placing the chip in a cell culture box again for incubation, and enabling the captured cells and the target cells in the mixed cell solution to be fully contacted and combined with each other to form combined cells; opening a valve, injecting washing liquid under pressure from the sample inlet 3 to wash the incubation area, so that other cells which are not combined with the captured cells flow into the screening area and flow out from the waste liquid outlet 15; then, the sample inlet 3 is washed by a washing solution containing pancreatin, so that all the captured cells are detached from the wall, all the cells enter the screening area, the captured cells which are not combined with the target cells pass through the 2 groups of screening microcolumn 12 arrays and the auxiliary channel 14, and flow out from the 2 waste liquid outlets 15. The bound cells cannot pass through the 2 groups of screening microcolumn 12 arrays, and can only enter the storage region along the main channel 13; a small number of single cells entering the storage region will also flow out of the sample outlet 4 through the array of filter micropillars 16 at the edge of the storage region. After the screening collection is completed, the target cells are subjected to subsequent treatment. The subsequent processing can be performed in the following manner.
The first subsequent processing mode: the cell analysis of the target cells among the bound cells is performed directly in the storage chamber 7.
And a second subsequent processing mode: the washing liquid is injected into the sample outlet 4 of the chip under pressure to recoil the combined cells, so that the combined cells can be flushed out through the sample inlet 3 for collection.
And a third subsequent processing mode: washing the chip with separating reagent capable of separating the captured cell from the target cell, and collecting the target cell after the captured cell is separated from the target cell.
Claims (10)
1. A cell screening chip comprises a glass substrate (1) and a PDMS sheet (2) which are mutually attached, and is characterized in that the PDMS sheet (2) is provided with a sample inlet (3) and a sample outlet (4) which penetrate through the PDMS sheet (2) at intervals, the attachment surface of the PDMS sheet (2) is provided with an incubation cavity (5), a screening cavity (6) and a storage cavity (7) which are sequentially connected and used for cell solution to flow through to complete cell screening from the sample inlet (3) to the sample outlet (4), a sample inlet flow channel (8) is arranged between the sample inlet (3) and the incubation cavity (5), a first flow channel (9) is arranged between the incubation cavity (5) and the screening cavity (6), a second flow channel (10) is arranged between the screening cavity (6) and the storage cavity (7), and a sample outlet flow channel (11) is arranged between the storage cavity (7) and the sample outlet (4); the device comprises a PDMS sheet (2), a glass substrate (1), a sample inlet (3), a sample outlet (4), a valve, a screening cavity (6), 2 groups of screening micro-columns (12) arrays, a main channel (13), an auxiliary channel (14), a waste liquid port (15), a filtering micro-column (16) array, a storage cavity (7), a waste liquid outlet (15), a waste liquid outlet and a waste liquid outlet, wherein the PDMS sheet (2) is attached to the glass substrate (1) and is communicated with the storage cavity (7), the valve is arranged at the first flow channel (9) of the PDMS sheet (2) and can enable the first flow channel (9) to be opened or blocked, the screening cavity (6) is parallelly provided with 2 groups of screening micro-columns (12), the distance between two adjacent screening micro-columns (12) in the screening micro-column (12) array is larger than a single cell and smaller than a combined cell, the main channel (13) is formed between 2 groups of screening, the spacing between two adjacent filter micro-pillars (16) in the array of filter micro-pillars (16) is larger than a single cell and smaller than a bound cell.
2. The cell screening chip of claim 1, wherein the sample injection flow channel (8) is configured to: the initial runner section (8a) that draws from introduction port (3) divide into 2 branch runner sections (8b) that are relatively thinner, and every branch runner section (8b) subdivides into 2 subdivided runner sections (8c) that are thinner, and 4 subdivided runner sections (8c) are each parallelly connected incubation chamber (5).
3. The cell screening chip of claim 1, wherein the valve is configured such that: in the area between the incubation cavity (5) and the screening cavity (6) of the PDMS sheet (2), vent grooves (17) are respectively formed in the upper side and the lower side of the first flow channel (9), thin layers (18) which are deformed to block the first flow channel (9) under pressure are respectively reserved in the upper side and the lower side of the first flow channel (9), vent holes (19) penetrating the PDMS sheet (2) are respectively formed in the terminals of the vent grooves (17) in the two sides, and after the PDMS sheet (2) is attached to the glass substrate (1), the vent grooves (17) in the two sides form closed vent cavities respectively.
4. The cell screening chip of claim 1, wherein the incubation cavity (5) and the screening cavity (6) are rectangular, and the side of the screening cavity (6) close to the storage cavity (7) extends in the upper right direction and the lower right direction respectively as the extension of the auxiliary channel (14), and the extension of the auxiliary channel (14) is flared, and the opening of the extension is far larger than the width of the second flow channel (10); and the waste liquid ports (15) are respectively arranged at the terminal of the extension section of the auxiliary channel (14).
5. The cell screening chip of claim 3, wherein the cavities of the incubation cavity (5) and the storage cavity (7) are provided with an array of support pillars for preventing the cavities from collapsing, and the array of screening micropillars (12) of the screening cavity (6) simultaneously plays a role of support.
6. A method for preparing cell screening chip includes preparing PDMS sheet (2) and glass substrate (1) separately and bonding them, preparing PDMS sheet (2) including preparing silicon substrate as mould and preparing PDMS sheet (2); the method is characterized in that:
the silicon substrate manufacturing method specifically comprises the following steps: adopting four-inch monocrystalline silicon as a substrate, spin-coating 30-micron thick photoresist on a silicon wafer by using a rotary glue coating machine, and pre-drying: baking at 65 ℃ for 30 minutes; then, photoetching is carried out according to the designed layout; and (3) after photoetching is finished, post-drying: postbaking for 30 minutes at 95 ℃; developing by using a developing solution, removing the photoresist and cleaning to obtain a microstructure with a designed height; then hard-baking for 30 minutes at 95 ℃; obtaining silicon chip molds with microstructures of different heights;
the preparation process of the PDMS sheet (2) is as follows: putting the prepared silicon chip mold into a fluorosilane atmosphere for incubation for 4 hours so as to facilitate the subsequent stripping between the PDMS chip and the mold; according to the weight ratio of 15: 1, weighing PDMS prepolymer and curing agent, placing the PDMS prepolymer and the curing agent in a container, uniformly stirring the PDMS prepolymer and the curing agent by using a glass rod, placing the container in a vacuum drier, vacuumizing until the vacuum degree is 13psi, and standing for 30min to remove bubbles; taking out the PDMS mixture, placing the silicon wafer mold on a horizontal table, pouring PDMS, and standing for 30min to fill the PDMS in the mold; putting the mould into an oven, heating for 1h at the temperature of 80 ℃, and carefully stripping the PDMS from the silicon wafer after the PDMS is completely cured; punching holes at the positions of a sample inlet (3), a waste liquid inlet (15) and a sample outlet (4) on a PDMS chip by using a needle head with a specific aperture, and punching holes at a gas inlet of a valve; placing the PDMS chip into a plasma cleaning machine, keeping the PDMS chip in vacuum for 1h, and irradiating for glow for 2 min; taking out the chip, dropwise adding a mixed solution of PEG (6-9) -siloxane and acetone (v: v =1: 1) at the injection port (3), filling the whole inner cavity and pipeline of the chip with the mixed solution by using the negative pressure in the PDMS chip as a driving force, and incubating for 1h at room temperature; then, washing with ultrapure water to ensure that the surfaces of all inner cavities and pipelines in the PDMS chip are hydrophilic so as to reduce the adhesion of all the inner cavities and pipelines to cells;
the manufacturing of the glass substrate (1) comprises the selection and cutting of the material of the glass substrate (1);
the bonding process of the PDMS sheet (2) and the glass substrate (1) comprises the steps of putting the bonding surfaces of the PDMS sheet (2) and the glass substrate (1) upwards together into a plasma cleaning machine for cleaning, irradiating for glow for 2min, taking out and rapidly bonding together, and heating on a hot plate at 90 ℃ for 20 min to 30min to enhance the bonding degree of the PDMS sheet and the glass substrate.
7. A cell screening and collecting method for screening and collecting target cells from a mixed cell solution, which is characterized in that the cell screening chip of any one of claims 1 to 5 is used, proper capture cells capable of mutually attracting and combining with the target cells to form combined cells are selected, the valve is opened, the gas is introduced from the sample inlet (3) to the sample outlet (4), the captured cell solution is injected from the sample inlet (3) under pressure, the flowing condition of the captured cell solution is observed, when the cell solution to be captured is fully distributed in the incubation cavity (5), the valve is closed, the pressurization of the sample inlet is stopped, the chip is placed in a cell incubator for incubation, and the chip is taken back after all the captured cells are attached to the wall; opening a valve, injecting a mixed cell solution sample to be screened from the sample inlet (3) under pressure, closing the valve when the sample to be tested is fully distributed in the incubation cavity (5), stopping the pressurization of the sample inlet (3), placing the chip in a cell culture box again for incubation, and enabling the captured cells and the target cells in the mixed cell solution to be fully contacted and combined with each other to form combined cells; opening a valve, injecting washing liquid under pressure from the sample inlet (3) to wash the incubation area, and enabling other cells which are not combined with the captured cells to flow into the screening area and flow out from the waste liquid port (15); then, washing with a pancreatin-containing cleaning solution through a sample inlet (3) to ensure that all captured cells are detached from the wall, all the cells enter a screening area, the captured cells which are not combined with target cells pass through 2 groups of screening microcolumn (12) arrays and auxiliary channels (14), and flow out from 2 waste liquid ports (15); the bound cells cannot pass through the array of 2 groups of screening microcolumns (12) and can only enter the storage region along the main channel (13); a small amount of single cells entering the storage area can also flow out from the sample outlet (4) through the filtering microcolumn (16) array at the edge of the storage area; after the screening collection is completed, the target cells are subjected to subsequent treatment.
8. The method for screening and collecting cells according to claim 7, wherein the subsequent treatment comprises: the target cells in the bound cells are subjected to cellular analysis directly in the storage chamber (7).
9. The method for screening and collecting cells according to claim 7, wherein the subsequent treatment comprises: and injecting a cleaning solution into the sample outlet (4) of the chip under pressure to perform backflushing on the combined cells, so that the combined cells can be flushed out through the sample inlet (3) and collected.
10. The method for screening and collecting cells according to claim 7, wherein the subsequent treatment comprises: washing the chip with separating reagent capable of separating the captured cell from the target cell, and collecting the target cell after the captured cell is separated from the target cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110250079.3A CN112920951B (en) | 2021-03-08 | 2021-03-08 | Cell screening chip and manufacturing and cell screening and collecting method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110250079.3A CN112920951B (en) | 2021-03-08 | 2021-03-08 | Cell screening chip and manufacturing and cell screening and collecting method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112920951A true CN112920951A (en) | 2021-06-08 |
| CN112920951B CN112920951B (en) | 2022-07-22 |
Family
ID=76171852
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110250079.3A Active CN112920951B (en) | 2021-03-08 | 2021-03-08 | Cell screening chip and manufacturing and cell screening and collecting method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112920951B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113576541A (en) * | 2021-08-04 | 2021-11-02 | 张希武 | Capture method of vesicles in tears and contact lens capture chip |
| CN115970775A (en) * | 2022-12-08 | 2023-04-18 | 中南大学 | Centrifugal driving micro-fluidic chip, preparation method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834558A (en) * | 2012-11-21 | 2014-06-04 | 中国科学院深圳先进技术研究院 | Blood cell rapid sorting device and manufacturing method thereof |
| CN106076441A (en) * | 2016-06-07 | 2016-11-09 | 中国科学院上海微系统与信息技术研究所 | A kind of micro fluidic device based on size detection circulating tumor cell and method |
| WO2019085388A1 (en) * | 2017-11-01 | 2019-05-09 | 深圳市瑞格生物科技有限公司 | Chip for isolating and capturing cells and application thereof for sorting tumor cells |
-
2021
- 2021-03-08 CN CN202110250079.3A patent/CN112920951B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834558A (en) * | 2012-11-21 | 2014-06-04 | 中国科学院深圳先进技术研究院 | Blood cell rapid sorting device and manufacturing method thereof |
| CN106076441A (en) * | 2016-06-07 | 2016-11-09 | 中国科学院上海微系统与信息技术研究所 | A kind of micro fluidic device based on size detection circulating tumor cell and method |
| WO2019085388A1 (en) * | 2017-11-01 | 2019-05-09 | 深圳市瑞格生物科技有限公司 | Chip for isolating and capturing cells and application thereof for sorting tumor cells |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113576541A (en) * | 2021-08-04 | 2021-11-02 | 张希武 | Capture method of vesicles in tears and contact lens capture chip |
| CN115970775A (en) * | 2022-12-08 | 2023-04-18 | 中南大学 | Centrifugal driving micro-fluidic chip, preparation method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112920951B (en) | 2022-07-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20230016193A1 (en) | Cell capture system and method of use | |
| US20230234061A1 (en) | Manipulation of fluids, fluid components and reactions in microfluidic systems | |
| US8573259B2 (en) | Modular microfluidic assembly block and system including the same | |
| EP1654347B1 (en) | Improved materials for constructing cell-chips, cell-chip covers, cell-chip coats, processed cell-chips and uses thereof | |
| WO2017177839A1 (en) | Super-hydrophobic micro-pit array chip, preparation method therefor and applications thereof | |
| CN112920951B (en) | Cell screening chip and manufacturing and cell screening and collecting method thereof | |
| KR100733914B1 (en) | Microfluidic 3-dimensional cell culture system | |
| US20080257735A1 (en) | Microfluidic Device for Enabling the Controlled Growth of Cells and Methods Relating to Same | |
| CN106065391A (en) | For unicellular sorting and the micro-fluidic chip of unicellular whole genome amplification | |
| WO2008130623A1 (en) | Manipulation of fluids, fluid components and reactions in microfluidic systems | |
| CN108499619A (en) | A kind of integrated micro-fluidic filtrating chip of film and its preparation method and application | |
| KR20120012793A (en) | Microorganism culture device and method of operation thereof | |
| CN110305788A (en) | The array chip and its preparation and operating method of cell capture and the culture of tumour ball | |
| CN205127987U (en) | Micro -fluidic chip for multi -index detection | |
| CN102719359A (en) | Cell culture device and its application | |
| WO2011135339A2 (en) | Reactor | |
| WO2017031017A1 (en) | A microfluidic hydrodynamic shuttling chip device for highthroughput multiple single cells capture | |
| CN206033771U (en) | A micro -fluidic chip that is used for single cell sorting and unicellular full genome to amplify | |
| TWI588256B (en) | Device and method for single cell isolation and cultivation | |
| CN112063510A (en) | Structure of high-flux cell culture chip and manufacturing and using method thereof | |
| KR101106022B1 (en) | Cell-based chemotaxis kit and fabricating method thereof | |
| CN100504389C (en) | Transmission through applied microflow liquid, and method of analyzing system | |
| CN111316957A (en) | Microfluidic chip, preparation method thereof, nematode culture device and application | |
| US20210260576A1 (en) | Single Cell Isolation and Processing System With Reversible Well Shape | |
| CN114686372A (en) | Hybridoma cell screening chip and screening and collecting method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230620 Address after: Room 4-1, No.1, Qidi Science and Technology Park (Ningbo), No. 1277 Zhongguanxi Road, Zhuangshi Street, Zhenhai District, Ningbo City, Zhejiang Province, 315000 (Building A, 404-1) Patentee after: Ningbo Weiliu Biotechnology Co.,Ltd. Address before: 315211, Fenghua Road, Jiangbei District, Zhejiang, Ningbo 818 Patentee before: Ningbo University |
|
| TR01 | Transfer of patent right |