CN109991423B - Efficient single cell capture and rapid single cell secretory protein detection platform and detection method - Google Patents
Efficient single cell capture and rapid single cell secretory protein detection platform and detection method Download PDFInfo
- Publication number
- CN109991423B CN109991423B CN201910083613.9A CN201910083613A CN109991423B CN 109991423 B CN109991423 B CN 109991423B CN 201910083613 A CN201910083613 A CN 201910083613A CN 109991423 B CN109991423 B CN 109991423B
- Authority
- CN
- China
- Prior art keywords
- cell
- channel
- capture
- single cell
- detection
- 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.)
- Active
Links
Images
Classifications
-
- 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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
-
- 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
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
-
- 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
- B01L2300/0887—Laminated structure
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/06—Valves, specific forms thereof
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Clinical Laboratory Science (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Sustainable Development (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Fluid Mechanics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Computational Biology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a high-efficiency single cell capture and rapid single cell secretory protein detection platform and a detection method; the detection platform consists of a single cell capture and droplet incubation microfluidic chip, a secreted protein capture glass plate and a chip clamp; the micro-fluidic chip for cell capture and droplet incubation has two layers of structures, namely a single cell capture and droplet incubation layer and an isolation valve layer. The platform can efficiently and quickly capture single cells, micro liquid drops containing single cells can be quickly and stably generated by adding the isolation phase, long-term liquid drop incubation can be carried out, the high activity of the single cells is ensured, and the chip is combined with an antibody array glass plate for detecting the secretory protein, so that the high-flux, quick and high-sensitivity detection of the secretory protein of the single cells can be realized.
Description
Technical Field
The invention relates to a high-efficiency single cell capture and rapid single cell secretory protein detection platform, and belongs to the technical field of microfluidic single cell analysis methods.
Background
Traditional cell research methods are based on population studies of a large number of cells, and the results obtained are often averaged over all cells. However, there are studies that show some differences in gene expression and phenotype among cells of the same type and among different individuals (Zhang, Y et al (2014) Journal of the American Chemical Society136:15257), which is called cellular heterogeneity. The experimental results obtained from the population experiments can cover the heterogeneity of cells and influence the treatment, prognosis and recurrence probability of cancer. The single cell research means that cells are dispersed in independent experimental units and researched to obtain independent experimental results without mutual influence, so that the heterogeneity of the cells can be fully researched to obtain more accurate and instructive results, but the single cell research has good statistical significance only if the single cell research has higher flux. The secretory protein has the capacity of promoting the proliferation and metastasis of cancer cells (Konty, T at al (2011) Biosensors & bioelectronics 26:2707), and the research on the single-cell secretory protein of the cancer cells is helpful for understanding the metastatic capacity of the cancer cells and the relationship between the cancer cells and an immune system.
At present, the detection of single cell secretory protein mainly combines a microfluidic chip with a secretory protein capture tool to realize the detection of cell secretory protein in single cells, multiple cells or blood plasma, and outputs through a fluorescent signal. The micro-fluidic chip for detecting the secreted protein comprises two major types of micropore array chips (Lu, Y at al (2013) anal. chem.85:2548) and fishbone-shaped liquid drop generating chips; the principle is that the single cell is captured through Poisson distribution, so that the problems of low cell utilization rate, low single cell capture efficiency, serious reagent waste, large detection cavity volume, low detection sensitivity and the like exist, and the secretory protein can be fully captured only by incubating for more than 12 hours due to the large detection cavity volume. A detection platform which has a small detection volume, can realize rapid single-cell secretory protein capture in a short incubation time, can allow micro-droplets of cells to incubate for a long time, can ensure the high activity of single cells and realizes high flux, rapidness and high sensitivity of single-cell secretory protein is lacked.
Disclosure of Invention
Aiming at the problems of low single cell capture efficiency, large secreted protein detection cavity volume, low single cell secreted protein detection sensitivity, serious reagent waste and the like of the conventional single cell secreted protein detection platform, the invention provides the efficient single cell capture and rapid single cell secreted protein detection platform which can realize high single cell capture efficiency, small secreted protein detection cavity volume, high secreted protein detection sensitivity, rapid single cell secreted protein capture and reagent saving.
The technical scheme of the invention is as follows:
the high-efficiency single cell capturing and rapid single cell secretory protein detection platform comprises a cell capturing and droplet incubation micro-fluidic chip and a secretory protein capturing glass plate; wherein, the single cell capture and droplet incubation microfluidic chip comprises: an isolation valve layer (23) positioned at the upper layer and a single cell capturing and droplet incubation layer (24) positioned at the lower layer;
the single cell capturing and liquid drop incubating layer comprises a plurality of units, each unit comprises straight cell flow channels (7) positioned at the left end and the right end of each cell flow channel, the two straight cell flow channels (7) are connected by using a U-shaped or arc-shaped cell flow channel (11), the single cell capturing and liquid drop incubating layer further comprises a cell capturing cavity (6), the cell capturing cavity (6) is connected with 3 channels, the first channel is a cell flow channel and is connected with one arm end of the U-shaped or arc-shaped cell flow channel (11), and the width of the channel is larger than the diameter of a cell to be captured; the second channel is a connecting channel (8), the width of which is less than the diameter of the cell to be captured, and is used for connecting the cell capturing cavity (6) with the other arm end of the U-shaped or arc-shaped cell flow channel (11); the third channel is a detection channel (9) and leads to a solution channel (10); the two ends of the solution channel (10) are respectively provided with a solution inlet (2) and a solution outlet (4);
the isolation valve layer comprises two isolation valve channels (16) with opposite flow directions, and the two isolation valve channels are respectively crossed with the detection channel (9); a diaphragm (29) is arranged between the isolating valve channel (16) and the detection channel (9); the cell solution inlet (1) and the cell solution outlet (3) are arranged at two ends of the cell flow channel, and the two ends of the cell flow channel are respectively provided with a solution inlet (2) and a solution outlet (4); the inlet (5) of the isolated phase channel is connected with the tail ends of the cell flow channels (7, 11);
the secreted protein capture glass plate comprises: a glass plate (26) with the surface modified with polylysine, and a secretory protein capture antibody array strip (20) is modified on the polylysine glass plate (26); the secretory protein capture antibody array strip (20) is positioned in the detection channel (9) and between two intersections formed by the two isolation valve channels (16) and the detection channel, and the secretory protein capture antibody array strip (20) faces the detection channel (9) and is in direct contact with a solution in the detection channel (9).
In the invention, the plurality of units can be connected in series, in parallel or in a mixed series-parallel manner.
Preferably, the width of the cell channel (7, 11), the solution channel (10) and the isolation phase channel (5) is 5-1000 microns, and the depth is 5-1000 microns; the detection channel has a width of 5-1000 microns, a length of 5-1000 microns, and a height of 5-200 microns.
Preferably, a cell capture chamber (6) and a detection channel (9) form an incubation unit, and a cell secreted protein detection unit is formed by adding a group of secreted protein capture antibody bands (20); the secreted protein capture antibody strip (20) is contacted with the solution in the detection channel (9).
Preferably, the number of secreted protein detection units in each secreted protein detection platform is 1-50000.
Preferably, the isolation valve channel (16) has a width of 5-1000 microns, a height of 5-1000 microns, and the two isolation channels are separated (14) by a distance of 5-1000 microns.
Preferably, the high-efficiency single cell capturing and rapid single cell secreted protein detection platform further comprises a chip clamp, the chip clamp comprises a clamp upper plate and a clamp lower plate, and the isolation valve layer (23) and the single cell capturing and droplet incubation layer (24) are clamped between the clamp upper plate and the clamp lower plate
Preferably, the chip holder upper plate opening is 0.1-2 cm wide and 0.1-2 cm long.
Preferably, the chip holder upper plate is provided with screw fixing openings (30), and 7 chip channel openings, which are cell channel openings (31, 33), solution channel openings (32, 34), isolation valve channel openings (35, 36), and isolation phase inlet openings (37), respectively.
The invention also provides application of the high-efficiency single-cell capture and rapid single-cell secretory protein detection platform, which is applied to detection and/or analysis of single-cell secretory protein/intracellular protein.
The invention also provides a single cell secretory protein detection method, which comprises the following steps:
1) preparing a cell capture and droplet incubation microfluidic chip and a secreted protein capture glass plate according to the high-efficiency single-cell capture and rapid single-cell secreted protein detection platform structure;
2) introducing a capture antibody into a modified chip cavity thermally bonded with a polylysine capture glass plate to modify a secreted protein capture antibody array; after modification, sealing by using a buffer solution, washing, and finally spin-drying the secretory protein capture glass plate;
3) aligning, jointing and fixing the single cell capturing and droplet incubation microfluidic chip with the working area of the single cell secretory protein capturing glass plate; soaking the chip in ultrapure water; capturing the single cells;
A. injecting ultrapure water into the isolating valve channel (16) to increase the pressure of the ultrapure water, causing the diaphragm (29) to deform downwards, and isolating the detection channel (9) from the cell capture cavity (6) and the solution channel (10);
B. the isolated phase is reversely led in from the isolated phase inlet (5) and the solution channel outlet (4) to generate liquid drops with single cells in the cell capturing cavity (6),
C. after the liquid drop array is generated, an air pump switch is closed, the liquid drop array is placed still, then an air pipe is pulled out, water pipes at inlets (12 and 13) of isolating valve channels are pulled out, the pressure of the isolating valve channel (16) is released, the appearance of a diaphragm (29) is restored, an isolating effect is not generated any more, a detection channel (9) is communicated with a cell capturing cavity (6) again to form a single cell incubation unit, and the inlet and the outlet of a chip are sealed by using an adhesive tape; .
4) Then putting the platform into a cell culture box, incubating cell droplets, wherein single cells are in a cell capture cavity in the whole incubation process, and secreted protein is transferred to a cell detection channel through diffusion and captured by a secreted protein capture antibody array; and disassembling the incubated single-cell secretory protein capture glass plate from the chip, detecting signals on the single-cell secretory protein capture glass plate, and analyzing the secretion condition of the single-cell secretory protein.
Preferably, step (3) captures single cells, and the cell concentration is controlled at 5 x 105-5*107The flow rate of the cell phase and the solution phase is 0.005-0.05mL/h, the flow rate of the isolated phase is 0.5-2.0mL/h when the isolated phase is reversely introduced, an air pump switch is closed after the liquid drop array is generated, the air pump switch is kept stand for 1-10min, then the air pipe is pulled out, and the inlet and the outlet of the chip are sealed by using an adhesive tape.
Preferably, the single-cell secreted protein capture glass plate after incubation is disassembled from the chip, immediately sealed by buffer solution, and then incubated with the detection antibody of 4 secreted proteins with biotin groups for 45 minutes, and the detection antibody is diluted by 200 times; then washed with 1% BSA in PBS and incubated with 100-fold diluted SA-APC with secreted protein capture glass plates for 20 min; and finally, performing gradient washing and spin-drying by using PBS, 50% PBS, ultrapure water and ultrapure water, detecting a fluorescence signal on the single-cell secretory protein capture glass plate by using a scanner, and analyzing the secretion condition of the single-cell secretory protein.
The invention adopts the principle of fluid mechanics to make the single cell captured by the capturing cavity, the capturing cavity of the single cell and the detection channel connected with the capturing cavity of the single cell form an incubation unit, and different incubation units are not interfered with each other; the high-flux detection of the single-cell secretory protein can be realized by increasing the number of the incubation units. The single cell capture device has the advantages of high single cell capture efficiency, small secretory protein detection cavity volume, high secretory protein detection sensitivity, rapid single cell secretory protein capture and reagent saving.
Drawings
FIG. 1 is a top view of a single cell trapping and droplet incubation layer structure
FIG. 2 is a top view of a unit structure of a single cell capture and droplet incubation layer
FIG. 3 is a top view of the isolation valve layer structure
FIG. 4 is a top view of the isolation valve unit structure
FIG. 5 is a top view of a chip structure modified by a secreted protein capture glass plate
FIG. 6 is a top view of a structure of a secretory protein capturing glass plate modified chip unit
FIG. 7 is a top view of a structure of a single cell capture and droplet incubation microfluidic chip
FIG. 8 is a top view of the structure of single cell capture and droplet incubation unit
FIG. 9 is a top view of a chip structure for efficient single-cell capture and rapid single-cell secretory protein detection
FIG. 10 is a top view of a chip unit structure for efficient single-cell capture and rapid single-cell secretory protein detection
FIG. 11 is a side view of a platform for efficient single cell capture and rapid single cell secreted protein detection
FIG. 12 shows the upper plate of the chip fixture for efficient single-cell capture and rapid single-cell secretory protein detection
FIG. 13 is a lower plate of a chip clamp for efficient single cell capture and rapid single cell secretory protein detection
Fig. 14 is a diagram generated by a fluorescent droplet incubation array of a high-efficiency single-cell capture and droplet incubation microfluidic chip.
FIG. 15 shows the single cell capture on chip. The upper is the single cell capture efficiency histogram with average single cell capture efficiency of 80%, and the lower is the chip single cell capture graph.
FIG. 16 shows the survival of the cells after the chip was incubated with the droplets for 7 hours. A is a picture of a bright field before the chip is placed in the incubator; panel B is a graph showing cell survival after 7 hours incubation using calcein (CA-AM) staining; panel C shows cell death after 7 hours incubation using Propidium Iodide (PI) staining.
FIG. 17 shows the survival/death of cells after 7 hours incubation. The survival rate of the medicine reaches 94.8 percent.
FIG. 18 is a heat map of secretion of 4 secreted proteins after 4 hours incubation of macrophages.
Number mark in the figure
1 cell solution inlet 2 solution inlet 3 cell solution outlet 4 solution outlet 5 isolation phase inlet 6 cell capture cavity 7 straight cell flow channel 8 connecting channel 9 detection channel 10 solution channel 11U-shaped or arc cell flow channel 12 isolation valve channel inlet 13 isolation valve channel inlet 14 isolation valve channel spacer 15 out-diffusion region 16 isolation valve channel 17 modification chip channel outlet 18 modification chip channel inlet 19 secreted protein capture antibody array spacer 20 secreted protein capture antibody array strip 21 clamp upper plate 22 clamp lower plate
23 isolation valve layer 24 single cell capture and drop incubation layer 25 bent iron sheet 26 polylysine glass plate 27 drop incubation layer channel 28 screw 29 single cell capture and drop incubation layer, diaphragm 30 screw fixation opening between isolation valve layers
31 cell channel opening 32 solution channel opening 33 cell channel opening 34 solution channel opening 35 and 36 isolation valve channel opening 37 isolation phase inlet opening.
Detailed Description
Example 1
Referring to fig. 1 to 14, the platform for detecting high-efficiency single-cell capture and rapid single-cell secreted protein comprises a single-cell capture and droplet incubation microfluidic chip, a secreted protein capture glass plate and a chip clamp; wherein the content of the first and second substances,
1. referring to fig. 11, the single cell capture and droplet incubation microfluidic chip comprises: an isolation valve layer 23 at the upper layer and a single cell capture and droplet incubation layer 24 at the lower layer; the single cell capturing and liquid drop incubation layer is formed by connecting a plurality of experiment units end to end, each experiment unit comprises straight cell flow channels 7 positioned at the left end and the right end of each cell flow channel, the two straight flow channels are connected by using a U-shaped or arc-shaped cell flow channel 11, the left end of a cell capturing cavity 6 is connected with the U-shaped or arc-shaped cell flow channel 11, the right end of the cell capturing cavity 6 is connected with the right end of the U-shaped or arc-shaped cell flow channel 11 through a connecting channel 8, a detection channel 9 is connected below the cell capturing cavity 6, and the lower part of the detection channel is connected with a solution channel 10;
wherein, referring to fig. 10, the cell capturing chamber 6 is connected with 3 channels, the first channel is a cell flow channel, which is connected with the left end of one end of the U-shaped or arc-shaped channel 11, the width of the channel is larger than the diameter of the cell for capturing the cell; the second channel is a connecting channel 8, the width of which is less than the diameter of the cell and is used for connecting the right end of the U-shaped or arc-shaped channel 11; the third channel is a detection channel 9 leading to a solution channel 10; the two ends of the solution channel 10 are respectively provided with a solution inlet 2 and a solution outlet 4;
referring to fig. 3 and 4, the isolation valve layer is composed of a plurality of identical structural units, each structural unit is composed of two opposite identical isolation valve channels 16 which are separated by a certain distance 14, the middle part of the structural unit is provided with an outward expansion area 15, and each isolation valve channel is provided with only one inlet (12 or 13); the isolation valve channel 16 is arranged above the detection channel 9, a layer of PDMS membrane 29 is arranged in the middle of the detection channel 9, and is positioned in the range of the detection channel 9, and the isolation valve outward expansion area 15 is overlapped with the detection channel 9; the cell solution inlet 1 and the cell solution outlet 3 are arranged at two ends of the cell flow channel; the inlet 5 of the isolated phase channel is connected to the end of the cell flow channel.
2. The secreted protein capture glass plate comprises: a glass plate 26 modified with polylysine on the surface, and a secretory protein capture antibody band 20 is modified on the polylysine glass plate 26.
3. The clamp includes: the clamp upper plate 21 and the clamp lower plate 22, the glass plate 26 is located between the chip clamp upper plate 21 and the clamp lower plate 22, and a clip-shaped iron sheet 25 is placed on the clamp upper plate 21 and fixed by a screw 28 to form a high-efficiency single cell capture and rapid single cell secretory protein detection platform, which is shown in fig. 12 and 13.
4. Assembling a high-efficiency single cell capture and rapid single cell secretory protein detection platform: calibrating a polylysine glass plate 26 modified with a secretory protein capture antibody array 20 with a single cell capture and droplet incubation microfluidic chip, wherein the secretory protein capture antibody array 20 is positioned in the detection channel 9 and between the two isolation valve channels 16, and the antibody array 20 faces the detection channel 9 and is in direct contact with the solution in the detection channel 9. Fixing the calibrated chip by using a clamp, wherein the outlet and the inlet of the chip channel correspond to the openings of the clamp corresponding to the outlet and the inlet of the chip channel; the platform is a clip iron sheet 25, a clamp upper plate 21, an isolation valve layer 23, a single cell capture and droplet incubation layer 24, a secreted protein capture antibody glass plate 26, and a clamp lower plate 22 from top to bottom, respectively, see fig. 13.
Preferably, the width of the cell channel (7, 11), the solution channel 10 and the isolation phase channel 5 is 5-1000 microns, and the depth is 5-1000 microns; the detection channel has a width of 5-1000 microns, a length of 5-1000 microns, and a height of 5-200 microns.
Preferably, a cell capture chamber 6 and a detection channel 9 constitute an incubation unit, and a cell secreted protein detection unit is formed by the incubation unit and a group of secreted protein capture antibody strips 20; the secreted protein capture antibody strip 20 is contacted with the solution in the detection channel 9.
Preferably, the number of secreted protein detection units in each secreted protein detection platform is 1-50000.
Preferably, the isolation valve channel 16 is 5-1000 microns wide and 5-1000 microns high, with the two isolation channels being separated 14 from each other by a distance of 5-1000 microns.
Preferably, the membrane thickness between the isolation valve channel 16 and the detection channel 9 therebelow is 3-50 microns
Preferably, the chip holder upper plate opening is 0.1-2 cm wide and 0.1-2 cm long. Preferably, the chip holder upper plate is provided with screw fixing openings 30, and 7 chip channel openings, which are cell channel openings (31, 33), solution channel openings (32, 34), isolation valve channel openings (35, 36), and isolation phase inlet openings 37, respectively.
The efficient single cell capturing and fast single cell secreted protein detecting process includes the following steps:
(1) manufacturing a single cell capturing and droplet incubation layer and an isolation valve layer by using PDMS (polydimethylsiloxane), pasting the two layers together for calibration, and thermally bonding the single cell capturing and droplet incubation layer and the isolation valve layer together in a thermal bonding manner;
(2) carrying out hydrophobic modification and water boiling treatment on the manufactured chip, and reserving the processed chip for later use;
(3) manufacturing a modified channel PDMS chip by using a modified chip silicon chip template, thermally bonding the modified chip and a polylysine glass plate, and modifying the secreted protein capture antibody on the polylysine glass plate;
(4) designing and manufacturing a clamp of the single cell capturing and droplet incubation microfluidic chip, and combining the single cell capturing and droplet incubation microfluidic chip and a polylysine glass plate modified with a secretory protein capturing antibody together by the clamp to manufacture a single cell capturing and rapid single cell secretory protein detection platform;
(5) capturing single cells by using a platform, and reversely introducing an isolation phase to generate a liquid drop array with cells after capturing; putting the generated liquid drop array platform with the cells into a cell incubator for incubation for a period of time;
(6) and (3) performing the immune enzyme chain reaction treatment on the polylysine glass plate which captures the single-cell secretory protein, outputting a fluorescent signal, and detecting and analyzing the fluorescent signal.
In the preferred embodiment of the invention, in step (4), a BSA-FITC fluorescent strip is modified on the secretory protein capture glass plate to serve as a reference strip for calibrating the single-cell capture and incubation microfluidic chip and the single-cell secretory protein capture glass plate; and then fixing the single cell capture and droplet incubation microfluidic chip and the single cell secretory protein capture glass plate together by using a clamp, and balancing the pressure of each screw site by using a clip-shaped stainless steel plate to carry out an experiment.
In a preferred embodiment of the present invention, in the step (5), the operation time of the single cell capture and the droplet incubation microfluidic chip should be controlled within 1.5 hours; when the isolated phase is reversely led in to generate liquid drops, the flow rate of the isolated phase is controlled to be 0.8 mL/h; the chip should be soaked in ultrapure water during operation.
Example 2
(1) Thermal bonding of single cell capture and droplet incubation layer PDMS chip and isolation valve layer PDMS chip
Aligning and attaching the single cell capturing and liquid drop incubation layer and the isolation valve layer together by using an upright microscope, then putting the chip into a 60-degree oven to heat for 20 minutes, then peeling the chip from a silicon chip template, punching holes at the inlet and outlet of a chip channel, and finishing the manufacture of the single cell capturing and liquid drop incubation microfluidic chip.
(2) Modification of single cell capture and droplet incubation microfluidic chip
Modifying a chip channel by using the electronic fluoridizing liquid 1720, heating the modified chip by using an oven for 1 hour to ensure that the solvent is completely volatilized, then putting the chip into PBS for water boiling, soaking the chip into the PBS after the water boiling, and taking out the chip when in experiment.
(3) Modification of single cell secreted protein capture glass plate
Manufacturing a micro-fluidic chip of a decoration channel, and punching holes at an inlet and an outlet of the micro-fluidic chip; putting the chip into absolute ethyl alcohol for 10 minutes of ultrasound, then putting the chip into ultrapure water for 10 minutes of ultrasound, taking out the chip, drying the surface moisture by using nitrogen, putting the chip into a clean dish, and heating the chip in an 80-DEG oven for 30 minutes for drying; removing impurities remained on the surface of the modified chip channel by using an adhesive tape, naturally attaching the chip to a polylysine glass plate, and heating the chip in an 80-DEG oven for 2 hours for thermal bonding; after cooling, 1.5 microliters of secreted protein capture antibody was introduced for modification for 4 hours. After modification, blocking is carried out for 1 hour by using PBS (3% BSA), then gradient washing is carried out by sequentially using pure 1X PBS, 50% PBS, ultrapure water and ultrapure water, and finally, the secretory protein capture glass plate is spin-dried and is placed in a clean dish to be protected from light for storage.
(4) Single cell capture and droplet incubation microfluidic chip single cell capture and droplet array generation and incubation
During the single cell capture process of the platform, the platform is soaked in ultrapure water; when single cell is captured, ultrapure water is injected into the isolation valve channel, the diaphragm between the isolation valve channel and the detection channel below the isolation valve channel deforms downwards, the detection channel is isolated from the cell capture cavity and the flow channel, and the cells are prevented from flowing into the detection channel to influence the capture of the single cells; the flow rate of the cells was 0.01mL/h, and the cells were captured by and stayed in the cell-capturing chamber for 5 minutes. Standing for 5 minutes after the capture is finished, releasing the pressure of the mobile phase to facilitate the reverse introduction of the isolated phase; reversely leading the isolated phase from the isolated phase inlet and the solution channel outlet, wherein the flow rate of the isolated phase is 0.8mL/h, generating capture cavity liquid drops with single cells one by one, and closing the air valve after the array is generated; standing for 5 minutes after the liquid drops are generated, exhausting the air pressure in the channel, then pulling out the air pipe, pulling out the water pipe in the channel of the isolation valve, releasing the water pressure, recovering the appearance of the diaphragm, and connecting the detection channel and the cell capturing cavity together to form an incubation unit; then, sealing the inlet and the outlet by using an adhesive tape, immersing the microfluidic chip in ultrapure water, and putting the microfluidic chip into a cell culture box for incubation; during the cell incubation process, the single cell is in the cell capture cavity, the secretory protein produced by the cell can diffuse to the detection channel under the diffusion effect, and the secretory protein in the detection channel is captured by the secretory protein detection antibody array.
Example 3 modification of Single-cell secreted protein Capture glass plate
Manufacturing a microfluidic PDMS chip of the modification channel as shown in FIG. 5, and punching holes at the inlet and outlet of the chip; putting the chip into absolute ethyl alcohol for 10 minutes of ultrasound, then putting the chip into ultrapure water for 10 minutes of ultrasound, taking out the chip, drying the surface moisture by using nitrogen, putting the chip into a clean dish, and heating the chip in an 80-DEG oven for 30 minutes for drying; removing impurities remained on the surface of the modified chip channel by using an adhesive tape, naturally attaching the chip to a polylysine glass plate, and heating the chip in an 80-DEG oven for 2 hours for thermal bonding; after the antibody is cooled, introducing BSA-FITC, an IL-8 capture antibody, an MCP-1 capture antibody, a TNF-a capture antibody and an MIP-1b capture antibody into 5 modification channels respectively, wherein each modification channel is 1.5 microliters, carrying out modification and modification on the secretory protein capture antibody, diluting the used capture antibody by one time, and carrying out modification for 8 hours. After modification, blocking is carried out for 1 hour by using PBS (3% BSA), then gradient washing is carried out by sequentially using PBS (1X pure), PBS (50% 1X pure), ultrapure water and ultrapure water, and finally, the secretory protein capture glass plate is spin-dried and is placed in a clean dish to be protected from light for storage.
Example 4 Capture of Single cells and verification of viability of cells
Microfluidic chip for capturing single cells and incubating liquid drops and method for preparing the sameAligning and attaching the single cell secretory protein capture glass plate working areas and fixing the single cell secretory protein capture glass plate working areas by using a clamp so as to carry out an experiment; during the experiment, the chip should be soaked in ultrapure water. Injecting ultrapure water into the isolating valve layer channel to cause the diaphragm to deform downwards, and isolating the detection channel from the cell capture cavity and the solution channel; introducing cells (cells A549) at cell channel inlet for capture, introducing cell culture solution in accordance with cell suspension in solution channel, and controlling cell concentration at 5 x 106About one/mL, the flow rate of the cell phase and the solution phase is 0.01mL/h, the single cell capture rate can reach 80%, as shown in FIG. 15, standing for 5 minutes after capture is completed, then pulling out the tubes of the cell inlet and the solution inlet, reversely introducing the isolation phase from the isolation phase inlet and the solution phase outlet, and the flow rate of the isolation phase is 0.8mL/h, generating a liquid drop array with cells, then closing an air pump switch, standing for 5 minutes, then pulling out the air tube, sealing the inlet and the outlet of the chip by using an adhesive tape, putting the chip into ultrapure water, then putting the chip into a cell incubator, and incubating after incubating for a period of time, the cell liquid drops.
And after incubation, the closed entrance and exit of the chip are uncovered, mixed dye of CA-AM and PI is introduced for cell staining, the viability of the cells is judged, and after 20 minutes, fluorescent signals of the cells are photographed and observed as shown in figures 16 and 17, and the survival rate of the incubated single cells in 7 hours reaches 94.8%.
Example 5 detection of Single cell secreted proteins
Disassembling the incubated single-cell secretory protein capture glass plate from the chip, immediately blocking the glass plate by 3% BSA PBS for 30 minutes, then incubating the glass plate with a detection antibody of 4 secretory proteins with biotin groups for 45 minutes, and diluting the detection antibody by 200 times; then washed with 1% BSA in PBS and incubated with 100-fold diluted SA-APC with secreted protein capture glass plates for 20 min; and finally, performing gradient washing and spin-drying by using PBS, 50% PBS, ultrapure water and ultrapure water, detecting a fluorescence signal on the single-cell secretory protein capture glass plate by using a scanner, and analyzing the secretion condition of the single-cell secretory protein, wherein FIG. 18 is a secretion heat map of four kinds of secretory proteins of macrophages.
Claims (10)
1. Single cell capture and single cell secretion protein testing platform, its characterized in that: the detection platform comprises a cell capture and droplet incubation microfluidic chip and a secreted protein capture glass plate; wherein, the single cell capture and droplet incubation microfluidic chip comprises: the isolation valve layer is positioned at the upper layer, and the single cell capturing and droplet incubation layer is positioned at the lower layer;
the single cell capturing and liquid drop incubating layer comprises a plurality of units, each unit comprises straight cell flow channels (7) positioned at the left end and the right end of each cell flow channel, the two straight cell flow channels (7) are connected by using a U-shaped or arc-shaped cell flow channel, the single cell capturing and liquid drop incubating layer also comprises a cell capturing cavity (6), the cell capturing cavity (6) is connected with 3 channels, the first channel is a cell flow channel and is connected with one arm end of the U-shaped or arc-shaped cell flow channel, and the width of the channel is larger than the diameter of a cell to be captured; the second channel is a connecting channel (8), the width of which is less than the diameter of the cell to be captured, and the connecting channel is used for connecting the cell capturing cavity (6) with the other arm end of the U-shaped or arc-shaped cell flow channel; the third channel is a detection channel (9), and the detection channel (9) leads to a solution channel (10); the two ends of the solution channel (10) are respectively provided with a solution inlet (2) and a solution outlet (4);
the isolation valve layer comprises two isolation valve channels (16) with opposite flow directions, and the two isolation valve channels are respectively crossed with the detection channel (9); a diaphragm (29) is arranged between the isolating valve channel (16) and the detection channel (9); the cell solution inlet (1) and the cell solution outlet (3) are arranged at two ends of the cell flow channel, and the two ends of the cell flow channel are respectively provided with a solution inlet (2) and a solution outlet (4); the inlet (5) of the isolated phase channel is connected with the tail end of the cell flow channel;
the secreted protein capture glass plate comprises: a glass plate (26) with the surface modified with polylysine, and a secretory protein capture antibody array strip (20) is modified on the glass plate (26); the secretory protein capture antibody array strip (20) is positioned in the detection channel (9) and between two intersections formed by the two isolation valve channels (16) and the detection channel, and the secretory protein capture antibody array strip (20) faces the detection channel (9) and is in direct contact with a solution in the detection channel (9).
2. The single cell capture and single cell secreted protein detection platform of claim 1, wherein: the width of the cell flow channel, the solution channel (10) and the isolation phase channel is 5-1000 microns, and the depth is 5-1000 microns; the detection channel has a width of 5-1000 microns, a length of 5-1000 microns, and a height of 5-200 microns.
3. The single cell capture and single cell secreted protein detection platform of claim 1, wherein: a cell capture cavity (6) and a detection channel (9) form an incubation unit, and the incubation unit and a group of secretory protein capture antibody array strips (20) form a cell secretory protein detection unit; the secretory protein capture antibody array strip (20) is contacted with the solution in the detection channel (9).
4. The single cell capture and single cell secreted protein detection platform of claim 1, wherein: the isolation valve channel (16) has a width of 5-1000 microns, a height of 5-1000 microns, and a spacing (14) between two isolation valve channels of 5-1000 microns.
5. The single cell capture and single cell secreted protein detection platform of claim 1, wherein: the device is characterized by further comprising a chip clamp, wherein the chip clamp comprises an upper clamp plate and a lower clamp plate, and the isolation valve layer, the single cell capturing and droplet incubation layer and the glass plate are clamped between the upper clamp plate and the lower clamp plate.
6. The single cell capture and single cell secreted protein detection platform of claim 5, wherein: the width of the opening of the upper plate of the clamp is 0.1-2 cm, and the length of the opening is 0.1-2 cm.
7. The single cell capture and single cell secreted protein detection platform of claim 5, wherein: the upper plate of the clamp is provided with a screw fixing opening (30) and 7 chip channel openings which are respectively two cell channel openings, two solution channel openings, two isolating valve channel openings and an inlet (5) opening of an isolating phase channel.
8. The use of the single cell capture and single cell secreted protein assay platform of any one of claims 1 to 7 for the detection and/or analysis of single cell secreted proteins and/or intracellular proteins.
9. A single cell secretory protein detection method comprises the following steps:
1) the structure of the single-cell capture and single-cell secreted protein detection platform according to any one of claims 1 to 7, wherein a cell capture and droplet incubation microfluidic chip and a secreted protein capture glass plate are prepared;
2) introducing a capture antibody into a modified chip cavity thermally bonded with a polylysine capture glass plate to modify a secreted protein capture antibody array; after modification, sealing by using a buffer solution, washing, and finally spin-drying the secretory protein capture glass plate;
3) aligning, jointing and fixing the single cell capturing and droplet incubation microfluidic chip with the working area of the single cell secretory protein capturing glass plate; soaking the chip in ultrapure water; capturing single cells:
A. injecting ultrapure water into the isolating valve channel (16) to increase the pressure of the ultrapure water, causing the diaphragm (29) to deform downwards, and separating the detection channel (9) from the cell capture cavity (6) and the solution channel (10);
B. reversely leading the isolated phase from the inlet (5) of the isolated phase channel and the outlet (4) of the solution channel, and generating liquid drops with single cells in the cell capturing cavity (6);
C. after the liquid drop array is generated, an air pump switch is closed, the liquid drop array is placed still, then an air pipe is pulled out, a water pipe at the inlet of an isolation valve channel is pulled out, the pressure of the isolation valve channel (16) is released, the appearance of a diaphragm (29) is restored, the isolation effect is not generated, the detection channel (9) is communicated with the cell capturing cavity (6) again to form a single cell incubation unit, and then the inlet and the outlet of the chip are sealed by using an adhesive tape;
4) placing the platform into a cell culture box, incubating cell droplets, wherein single cells are in a cell capture cavity in the whole incubation process, and secreted proteins are transferred to a cell detection channel through diffusion and captured by a secreted protein capture antibody array; and disassembling the incubated single-cell secretory protein capture glass plate from the chip, detecting signals on the single-cell secretory protein capture glass plate, and analyzing the secretion condition of the single-cell secretory protein.
10. The method for detecting the secreted protein from a single cell according to claim 9, wherein: step (3) capturing single cells, and controlling the cell concentration to be 5 x 105-5*107The flow rate of the cell phase and the solution phase is 0.005-0.05mL/h, the flow rate of the isolated phase is 0.5-2.0mL/h when the isolated phase is reversely introduced, an air pump switch is closed after the liquid drop array is generated, the air pump switch is kept stand for 1-10min, then the air pipe is pulled out, and the inlet and the outlet of the chip are sealed by using an adhesive tape.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910083613.9A CN109991423B (en) | 2019-01-29 | 2019-01-29 | Efficient single cell capture and rapid single cell secretory protein detection platform and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910083613.9A CN109991423B (en) | 2019-01-29 | 2019-01-29 | Efficient single cell capture and rapid single cell secretory protein detection platform and detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109991423A CN109991423A (en) | 2019-07-09 |
| CN109991423B true CN109991423B (en) | 2020-06-26 |
Family
ID=67130112
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910083613.9A Active CN109991423B (en) | 2019-01-29 | 2019-01-29 | Efficient single cell capture and rapid single cell secretory protein detection platform and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109991423B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112834754B (en) * | 2019-11-22 | 2022-06-03 | 中国科学院大连化学物理研究所 | Portable high-throughput single-cell outer vesicle detection device and visual detection method |
| CN112827517B (en) * | 2019-11-25 | 2023-05-02 | 杭州微著生物科技有限公司 | Use method and device of micro-fluidic chip |
| CN113588963B (en) * | 2020-04-30 | 2024-02-23 | 厦门德运芯准科技有限公司 | High-throughput single-cell proteome analysis and transcriptome combined analysis method |
| EP4150336A1 (en) * | 2020-05-14 | 2023-03-22 | Bifrost Biosystems | A microfluidic device having specifically designed detection chambers |
| CN113008764B (en) * | 2020-06-17 | 2022-12-02 | 山东大学 | Single cell function evaluation kit and evaluation method |
| CN112538428B (en) * | 2020-12-01 | 2023-01-13 | 中国科学院上海微系统与信息技术研究所 | Microfluidic chip based on droplet microfluidic technology and detection method thereof |
| CN113070107B (en) * | 2021-02-23 | 2022-03-29 | 厦门大学 | Micro-fluidic chip for accurately assembling single particles and single particle assembling method |
| CN114062679B (en) * | 2021-11-16 | 2023-09-08 | 中国科学院上海微系统与信息技术研究所 | Single-cell secretion high-flux detection method and system based on droplet microfluidic |
| CN114752476A (en) * | 2022-04-08 | 2022-07-15 | 厦门大学 | Device for single cell capture and centrifugal droplet generation and application thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923816A (en) * | 2014-03-28 | 2014-07-16 | 大连理工大学 | Cell capturing array based on microfluidic technology |
| CN104388301A (en) * | 2014-11-28 | 2015-03-04 | 清华大学 | Single-cell high-efficiency capture device and system based on fluid mechanics |
| WO2016112344A1 (en) * | 2015-01-08 | 2016-07-14 | The Regents Of The University Of California | Position-defined cell culture and characterization platform |
| CN107012067A (en) * | 2017-04-10 | 2017-08-04 | 厦门大学 | A kind of high flux pairing captures micro-fluidic chip and its application of unicellular/individual particle |
| CN107012220A (en) * | 2017-04-10 | 2017-08-04 | 厦门大学 | It is a kind of to utilize the method for matching the unicellular inclusion of micro-fluidic chip high throughput analysis |
| CN108267581A (en) * | 2012-08-24 | 2018-07-10 | 耶鲁大学 | For the systems, devices and methods of high-throughput multi detection |
| KR101897250B1 (en) * | 2017-07-11 | 2018-09-10 | 광주과학기술원 | A single cell analysis chip for drug or drug combination |
| CN108693144A (en) * | 2018-04-28 | 2018-10-23 | 天津大学 | Durability single cell protein group Hygienic monitoring on hands of childhood method is carried out based on SPRM technologies |
| CN108940387A (en) * | 2017-05-18 | 2018-12-07 | 中国科学院大连化学物理研究所 | It is a kind of for unicellular isolated drop micro-fluidic chip and preparation method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8465706B2 (en) * | 2007-06-20 | 2013-06-18 | Trustees Of Columbia University In The City Of New York | On-demand microfluidic droplet or bubble generation |
-
2019
- 2019-01-29 CN CN201910083613.9A patent/CN109991423B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108267581A (en) * | 2012-08-24 | 2018-07-10 | 耶鲁大学 | For the systems, devices and methods of high-throughput multi detection |
| CN103923816A (en) * | 2014-03-28 | 2014-07-16 | 大连理工大学 | Cell capturing array based on microfluidic technology |
| CN104388301A (en) * | 2014-11-28 | 2015-03-04 | 清华大学 | Single-cell high-efficiency capture device and system based on fluid mechanics |
| WO2016112344A1 (en) * | 2015-01-08 | 2016-07-14 | The Regents Of The University Of California | Position-defined cell culture and characterization platform |
| CN107012067A (en) * | 2017-04-10 | 2017-08-04 | 厦门大学 | A kind of high flux pairing captures micro-fluidic chip and its application of unicellular/individual particle |
| CN107012220A (en) * | 2017-04-10 | 2017-08-04 | 厦门大学 | It is a kind of to utilize the method for matching the unicellular inclusion of micro-fluidic chip high throughput analysis |
| CN108940387A (en) * | 2017-05-18 | 2018-12-07 | 中国科学院大连化学物理研究所 | It is a kind of for unicellular isolated drop micro-fluidic chip and preparation method thereof |
| KR101897250B1 (en) * | 2017-07-11 | 2018-09-10 | 광주과학기술원 | A single cell analysis chip for drug or drug combination |
| CN108693144A (en) * | 2018-04-28 | 2018-10-23 | 天津大学 | Durability single cell protein group Hygienic monitoring on hands of childhood method is carried out based on SPRM technologies |
Non-Patent Citations (1)
| Title |
|---|
| High-Throughput Secretomic Analysis of Single Cells to Assess Functional Cellular Heterogeneity;Yao Lu,et al;《Anal. Chem.》;20130122;第85卷;2548-2556 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109991423A (en) | 2019-07-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109991423B (en) | Efficient single cell capture and rapid single cell secretory protein detection platform and detection method | |
| Tavakoli et al. | Recent advances in microfluidic platforms for single-cell analysis in cancer biology, diagnosis and therapy | |
| US9895690B2 (en) | Microfluidic chip and application thereof | |
| CN107118938B (en) | Fluid enhances the unicellular arrangement of dielectrophoresis and control chip and preparation method thereof | |
| CN109852530A (en) | A kind of micro-fluidic chip and its device and method integrating circulating tumor cell capture, cracking and detection of nucleic acids | |
| CN109682962B (en) | Immunofluorescence detection system and detection method based on microfluidic chip | |
| CN107497503B (en) | Microfluidic chip for researching tumor single cell invasion and epithelial mesenchymal transition | |
| CN210916029U (en) | Simple micro-fluidic chip for separating and detecting circulating tumor cells | |
| CN101948741A (en) | Microfluidic gene chip for nucleic acid sequencing | |
| US11358147B2 (en) | System and method for isolating and analyzing cells | |
| CN108117968A (en) | A kind of single celled method of high-throughput automatic capture based on drop micro-fluidic chip | |
| WO2021088936A1 (en) | Microfluidic chip for isolating and capturing single cell, manufacturing method for same, and applications thereof | |
| CN112630282A (en) | Preparation of array electrode chip combined with micro-fluidic clamp and construction method of electrochemical immunosensor | |
| CN108728327A (en) | A kind of full-automatic sample preparation microfluidic system and preparation method thereof and application method | |
| CN207130273U (en) | A kind of magnetic force micro-fluidic chip for capturing bacterium | |
| Chen et al. | Reconfigurable microfluidics combined with antibody microarrays for enhanced detection of T-cell secreted cytokines | |
| CN213203076U (en) | Multichannel unicellular magnetic bead capture test device | |
| CN212845415U (en) | Micro-fluidic chip for detecting tumor marker | |
| CN112980677A (en) | Micro-fluidic chip for analyzing and sorting tumor cell migration capacity and preparation process | |
| CN213423201U (en) | Micro-fluidic chip for cancer marker detection | |
| CN211847950U (en) | Cell capturing and screening device | |
| CN209784162U (en) | fixing device for red blood cell super-resolution imaging | |
| CN113820487A (en) | Micro-fluidic chip for cancer marker detection and preparation method thereof | |
| WO2022205399A1 (en) | Integrated arrayed micro-fluidic chip for capturing and stretching cells | |
| CN113009134A (en) | Micro-fluidic chip for detecting tumor marker and detection method |
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 | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220426 Address after: 361000 rooms a, B, C, D, e and F, 14th floor, building 2, No. 1888, Hongxiang West Road, Jinhai street, Xiang'an District, Xiamen City, Fujian Province Patentee after: Xiamen Deyun xinzhun Technology Co.,Ltd. Address before: 361000 Siming South Road, Xiamen, Fujian Province, No. 422 Patentee before: XIAMEN University |