CN110499251A - The extracellular vesica analysis chip of the unicellular source property of high flux multiparameter and application - Google Patents
The extracellular vesica analysis chip of the unicellular source property of high flux multiparameter and application Download PDFInfo
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- 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
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
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- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
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- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
- C12M3/08—Apparatus for tissue disaggregation
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Abstract
The present invention provides the extracellular vesica analysis chip of unicellular source property and its application of a kind of high flux multiparameter, the chip includes double-layer structure, one layer is bioanalysis substrate for the capture of extracellular vesica, detection, and another layer is that the chip with high throughput array is used for unicellular capture.The present invention by microfluidic chip technology, single cell analysis technology be applied to extracellular vesica research, using affine in immunity method, it can be achieved that high throughput, multi-parameter capture, detection individual cells secrete various sizes extracellular vesica.And then qualitative and quantitative analysis, correlation analysis, multi-functional analysis, immunophenotyping analysis have been carried out to the secretion of extracellular vesica in individual cell level, clearly illustrate the heterogeneity of cell Yu the extracellular vesica of intercellular secretory.
Description
Technical field
The present invention relates to microfluidic chip technology, single cell analysis technology and field of immunology, specifically provide one kind
The extracellular vesica analysis chip of the unicellular source property of high flux multiparameter and application.
Background technique
Extracellular vesica (EVs) is the small vesica of a kind of lipid bilayer membrane structure secreted by cell.Recent study
It was found that extracellular vesica communicated between cell-tocell in play the part of highly important role.Almost all of cell can generate carefully
Extracellular vesica, wherein containing lipid, protein, nucleic acid (DNA, mRNA and microRNA, lncRNA, circRNA etc.
Noncoding RNA) etc. a variety of mother cell sources biological activity, these information substances are wrapped in vesica or are carried on
On film.They participate in inflammatory and immune response, intercellular signal communication, cell survival and apoptosis, angiogenesis, thrombosis, from
It bites, plays a significant role in the process of physiological status maintenance and disease.In the film surface of extracellular vesica, there is a kind of cross-film
Albumen, such as CD63, CD81, CD9 etc..Using specific immune response, captures antibody and is combined with the antigen molecule of film surface,
The capture to extracellular vesica may be implemented.
Single cell analysis technology is now widely used for the research such as DNA, RNA, secretory protein, but the technology is extracellular
Vesica research aspect is still immature.The present invention relates to vesica analysis chips outside a kind of unicellular cells of high flux multiparameter.
Summary of the invention
The present invention relates to a kind of high-throughput, multi-parameter the extracellular vesica of unicellular source property (EVs) analysis chips.Building is high
The capture of the extracellular vesica of the unicellular capture of flux and its secretion, detection platform, and it is extracellular in individual cell level analysis
Vesica shows the heterogeneity of cell and the extracellular vesica of intercellular secretory.Technical problem solved by the invention uses following skill
Art scheme is realized:
A kind of high-throughput, multi-parameter extracellular vesica analysis chip of unicellular source property, the chip include double-layer structure, and one
Layer is bioanalysis substrate, and another layer is the chip with high throughput array；The bioanalysis substrate is used for extracellular vesica
The chip of capture, detection, the high throughput array is used for unicellular capture.
The bioanalysis substrate is to be coated with the slide of albumen, antibody or DNA or be coated with protein arrays, antibody
The slide of array or DNA array；
The slide includes but is not limited to poly-D-lysine slide, epoxy resin slide.
The array is the parallel Uncrossed band for having specific width and spacing of 1-100 item, and every rule bandwidth is
5um-5000um, spacing 5um-5000um.
The chip with high throughput array is the chip with high-throughput microwell array or has high-throughput band battle array
The chip of column.
The material of the chip with high throughput array includes but is not limited to the PDMS after PDMS or various modifications.
The microwell array is regular polygon, rectangle or circular array, in array micropore number be 1~1000000/
cm2。
A kind of application of the extracellular vesica analysis chip of the unicellular source property of high flux multiparameter, the chip for capture,
It is secreted after the extracellular vesica or cell not of the same race of the slender intracrine of detection and analysis various kinds of cell-cell interaction
Extracellular vesica not of the same race,
The extracellular vesica analysis of the multi-parameter single cell source property includes the analysis of 1-100 index.
The extracellular vesica includes but is not limited to small vesica, excretion body, cancer-bodies, apoptotic body.
The various kinds of cell includes but is not limited to mammalian cell, zooblast, tumour patient lesion cell.
The analysis content includes that qualitative and quantitative analysis, correlation analysis, multi-functional is carried out to extracellular vesica not of the same race
Property analysis, immunophenotyping analysis, the clear heterogeneity for showing cell and the extracellular vesica of intercellular secretory.
A kind of application of the extracellular vesica analysis chip of the unicellular source property of high flux multiparameter, the capture, detection
The application method of extracellular vesica, specifically:
It takes out and has been coated with the bioanalysis substrate that capture antibody not of the same race forms antibody bar code array；By cell with certain close
Degree kind is on microwell array chip；The slide for being coated with capture antibody is covered, is fixed with fixture, sets 37 DEG C, 5%CO2Cell training
It supports and is cultivated 12-24 hours in case；Microscope is taken pictures；Fixture is unloaded, it is same unicellular that extracellular vesica staining reagent progress is added dropwise
The detection of the outer vesica of various kinds of cell；
The staining reagent includes but is not limited to the strepto- affinity of PKH67 or biotinylated antibody and fluorescent marker
Element.
Bioanalysis substrate coating capture antibody not of the same race forms antibody bar code array, and packet can be used in antibody bar code array
It includes but is not limited to fixed flowing, printing and other methods and realize.
The present invention has the advantages that
1. the high-throughput microwell array of chip of the present invention can realize under vitro simple, efficient high-throughput unicellular catch
It obtains and cultivates；
2. the extracellular vesica not of the same race that the chip can be used for capturing not allogenic cell secretion；
3. the immunophenotyping research that the chip can realize the extracellular vesica of unicellular source property；
4. the chip can realize the multi-parameter of research object while detect；
5. the chip can be used for the research of unicellular a variety of secretion, the outer vesica of various kinds of cell and a variety of thin is such as detected simultaneously
Intracellular cytokine；
6. the chip acquired results can carry out network analysis with various analysis, if immunophenotyping is analyzed, can show thin
The visualization etc. of born of the same parents' subgroup；
7. the extracellular vesica based on micro-fluidic chip platform is studied, chip manufacturing is easy, material price is cheap, and antibody is used
Amount is few, can be generally applicable to high-throughput, multi-parameter extracellular vesica research.
Detailed description of the invention
Fig. 1 is that a kind of high throughput, the unicellular source property of multi-parameter that Example 1 and Example 2 of the present invention proposes are extracellular
The structural schematic diagram of vesica analysis chip；
Fig. 2 is bioanalysis substrate and its partial enlarged view in Fig. 1；
Fig. 3 is high-throughput microwell array chip and its partial enlarged view in Fig. 1,
Fig. 4 is that high-throughput, multi-parameter the extracellular vesica analysis chip of unicellular source property is applied to unicellular cells in Fig. 1
Structural schematic diagram when outer vesica capture, detection；
Wherein: 1 is bioanalysis substrate, and 2 be the chip with high throughput array, and 3 be fixture；11-19 is band starting
Section, 21-29 are band clearing end, and 20 be the schematic diagram of a wherein band；30 be the schematic diagram of one of micropore.
Fig. 5 is the antibody uniformity detection figure that antibody forms antibody bar code array on slide.
Fig. 6 is extracellular vesica atomic force microscopy diagram.
Fig. 7 is the outer vesica dimensional measurement figure of individual cells in Fig. 6.
Fig. 8 is the outer vesica slide sweep test result figure of UM-SCC6 unicellular cells.
Fig. 9 is the unicellular thermal map of UM-SCC6.
Figure 10 is the outer vesica of UM-SCC6 cell secretory cell and cell factor scatter plot.
Figure 11 is UM-SCC6 correlation analysis figure.
Figure 12 is the analysis of UM-SCC6 multifunctionality.
Figure 13 is the unicellular immunophenotyping analysis chart of UM-SCC6.
Figure 14 is the outer vesica slide sweep test result figure of primary oral squamous cell carcinomas unicellular cells.
Figure 15 is the unicellular thermal map of primary oral squamous cell carcinomas.
Figure 16 is the outer vesica of primary Dental clinic secretory cell and cell factor scatter plot.
Figure 17 is primary oral squamous cell carcinomas correlation analysis figure.
Figure 18 is the analysis of primary oral squamous cell carcinomas multifunctionality.
Figure 19 is the unicellular immunophenotyping analysis chart of primary oral squamous cell carcinomas.
Figure 20 is group's cell experiment bioanalysis substrate and its partial enlarged view in embodiment 3.
Figure 21 is the chip and its partial enlarged view of group's cell experiment high throughput array in embodiment 3,
Wherein: 1 is bioanalysis substrate, and 2 be the chip with high throughput array, and 11-19 is a tape beginning end, 21-29
It is the schematic diagram of a wherein band for band clearing end, 200.
31-39 is channel starting point, and 41-49 is channel clearing end, and 300 be the schematic diagram in a wherein channel.
Figure 22 is group's cell experiment part slide scanning result figure.
Figure 23 is group's cell experiment result analysis chart.
Specific embodiment
It is clear to be more clear the purpose of the present invention, technical method, advantage, the present invention is done into one with reference to the accompanying drawing
The detailed description of step, experiment reagent used in the present invention be it is commercially available, as shown in table 1.
1 experiment agents useful for same list of table.
Embodiment 1
A kind of high-throughput, multi-parameter extracellular vesica analysis chip of unicellular source property of the present embodiment, refering to fig. 1-Fig. 3.
The chip includes double-layer structure, and one layer is bioanalysis substrate 1, and another layer is the chip 2 with high throughput array, the tool
The chip for having high throughput array is the chip with high-throughput microwell array.
The chip for having high-throughput microwell array is the PDMS microwell array chip containing 6343 micropores.
The bioanalysis substrate 1 is the poly-D-lysine glass for being coated with capture antibody not of the same race and having formed antibody bar code array
Piece.The antibody bar code array is made of 9 antibody bands, and every rule bandwidth is 40um, and band spacing is 80um,
The preparation of the band need corresponding PDMS micro-channel chip and poly-D-lysine slide hot key and, adopt later
With the method for Flow Patterning, so that antibody is flowed through channel to be fixed on poly-D-lysine slide, be consequently formed anti-
Body bar code array.
The preparation method of the PDMS micro-channel chip is specific as follows:
1. preparing silicon wafer template using lithography operations.Specification is as follows: PDMS micro-channel chip includes 9 channels, every wide
Degree is 40um, and interchannel distance is 80um.
1) clean silicon wafer is placed in hot plate drying first.Temperature: 105 DEG C, the time: 10min.
2) silicon wafer beats plasma treatment: logical oxygen plasma cleaner handles 2min.
3) spin coating machine speed is adjusted, revolving speed and time are determined according to the thickness of glue.600 turns are got rid of in advance, 18s；3000 turns formally are got rid of,
30s。
4) clean silicon wafer is placed on vacuum tray, pours into suitable 3035 type photoresist of SU-8, vacuumizes 1min
Afterwards, whirl coating.
5) silicon wafer is placed in front baking on hot plate.95 DEG C of temperature, the time: 30min.
6) silicon wafer cooling post-exposure.Time for exposure (light intensity 9.6mw/mm is calculated according to ultraviolet ray intensity2, the time for exposure is
22s), it and exposes.
7) baking after silicon wafer is placed on hot plate.Time about 1min.Occur drying after stopping after pattern on silicon wafer.
8) develop after silicon wafer cooling.Silicon wafer, which is integrally immersed in the glassware for fill ethyl lactate, cleans 7min, removes
The part photoresist that should remove, then rinsed with isopropanol.
9) post bake.Silicon wafer is placed on hot plate and toasts.Temperature: 120 DEG C, the time: 2h.
10) trim,ethylchlorosilane smokes silicon wafer.Time: 48h.
2. silicon wafer template is perfused
RTV615 type A glue, B glue 10:1 are mixed and stirred for uniformly, bubble is pumped with vacuum pumping pump, silicon wafer structural area is added
Domain, 80 DEG C of heat dry 1h.
Structure division PDMS is cut, is placed in the vessel for fill dehydrated alcohol and is cleaned by ultrasonic 5min, later 80 DEG C of drying.
The PDMS microwell array chip the preparation method is as follows:
1. preparing silicon wafer template using lithography operations.Chip scale is as follows: PDMS microwell array includes 6343 long
1.44mm, the micropore of wide 40um.Operating process:
1) clean silicon wafer is placed in hot plate drying first.Temperature: 105 DEG C, the time: 10min.
2) silicon wafer beats plasma treatment: logical oxygen plasma cleaner handles 2min.
3) spin coating machine speed is adjusted, revolving speed and time are determined according to the thickness of glue.600 turns are got rid of in advance, 18s；3000 turns formally are got rid of,
30s。
4) clean silicon wafer is placed on vacuum tray, pours into suitable 3035 type photoresist of SU-8, vacuumizes 1min
Afterwards, whirl coating.
5) silicon wafer is placed in front baking on hot plate.95 DEG C of temperature, the time: 30min.
6) silicon wafer cooling post-exposure.Time for exposure (light intensity 9.6mw/mm is calculated according to ultraviolet ray intensity2, the time for exposure is
22s), it and exposes.
7) baking after silicon wafer is placed on hot plate.Time about 1min.Occur drying after stopping after pattern on silicon wafer.
8) develop after silicon wafer cooling.Silicon wafer, which is integrally immersed in the glassware for fill ethyl lactate, cleans 7min, removes
The part photoresist that should remove, then rinsed with isopropanol.
9) post bake.Silicon wafer is placed on hot plate and toasts.Temperature: 120 DEG C, the time: 2h.
10) trim,ethylchlorosilane smokes silicon wafer.Time: 48h.
2. silicon wafer template is perfused
RTV615 type A glue, B glue 10:1 are mixed and stirred for uniformly, bubble is pumped with vacuum pumping pump, silicon wafer structural area is added
Domain, 80 DEG C of heat dry 1h.
Structure division PDMS is cut, is placed in the vessel for fill dehydrated alcohol and is cleaned by ultrasonic 5min, later 80 DEG C of drying.
A kind of application of high-throughput, multi-parameter the extracellular vesica analysis chip of unicellular source property, in the present embodiment
PDMS microwell array chip is for cultivating head and neck squamous cell carcinoma UM-SCC6, as shown in figure 4, thin in individual cell level analysis
Extracellular vesica shows the heterogeneity of cell and the extracellular vesica of intercellular secretory.Specifically includes the following steps:
Step 1: coated antibody forms the bioanalysis substrate with antibody bar code array
1.PDMS micro-channel chip and poly-D-lysine slide hot key and 2h.
2.Flow Patterning coated antibody.
1) 3ul antibody is respectively added to 9 channels from channel starting point using liquid-transfering gun.Be respectively as follows: FITC-BSA, CD63,
CD81,CD9,EpCAM,Hsp-70,IL-6,IL-8,MCP-1.Channel originates end position as shown in the 11-19 of Fig. 2.
2) flow antibody in channel to way vent from channel starting point, Excess antibody is flowed out from channel clearing end
Stop ventilation afterwards.The position of channel clearing end is as shown in the 21-29 of Fig. 2.
3) blocking antibody: configuration 15ml 1%BSA is placed in culture dish, takes PDMS micro-channel chip off in the solution, is sealed
Close antibody 1h.
3. cleaning dries, is spare:
It draws 1ml DPBS every time with liquid-transfering gun first to rinse in poly-D-lysine surface of glass slide, then successively soaks slide
It is cleaned in DPBS, 50%DPBS, ultrapure water, the drying of slide dryer.
Step 2: unicellular capture, by cell with certain density kind on microwell array chip
1.PDMS microwell array chip leads to oxygen plasma cleaner and handles 2min.
2. its cell density is diluted to 2 × 10 by UM-SCC6 cell dissociation5cells/ml.Cell is contaminated through calcein
200ul suspension is drawn after color, is dispersed in and is added drop-wise on PDMS microwell array chip, stands 5min.
3. poly-D-lysine slide is placed on above PDMS microwell array chip, antibody bar code array connects with cell suspension
Touching is clamped slide and chip with fixture.
Step 3: unicellular big figure scanning
Said chip is in 37 DEG C, 5%CO2After being incubated for 1h in cell incubator, it is aobvious that fluorescence is successively automatically inverted with Nikon
The micro mirror figure scanning greatly under the conditions of light field, two kinds of fluorescence respectively.It observes cell distribution situation and calculates unicellular capture rate.Core
Piece, which is put back to, to be continued to be incubated in incubator.
Step 4: extracellular vesica, cytokines measurement.
1. said chip is in 37 DEG C, 5%CO2After being incubated for 12-24h in cell incubator, fixture is dismantled, poly is taken off and relies ammonia
Sour slide.30min is closed with 1%BSA.
2. 1%BSA rinses slide, biotinylated antibody 200ul is added dropwise on slide, reacts 1h.
3. 1%BSA rinses slide, the streptavidin 200ul that fluorescent marker is added dropwise on slide (uses 1%BSA1:200
Dilution), dye 30min.
4. 1%BSA rinses slide, 1%BSA200ul (being diluted with 1%BSA1:100) is added dropwise on glass, closes again
30min。
5. drawn every time with liquid-transfering gun first 1ml DPBS surface of glass slide rinse, then slide is successively dipped in DPBS,
It is cleaned in 50%DPBS, ultrapure water, the drying of slide dryer.
6. scanning slide with four color laser chip scanners (Genepix+4300A).
Step 5: as a result data processing statisticallys analyze.
For the present embodiment result referring to Fig. 5-Figure 13, Fig. 5 is uniform for the antibody that antibody forms antibody bar code array on slide
Property detection figure.FITC-BSA is all added in 9 channels, the results showed that intensity of the antibody on slide is high uniformity.Fig. 6 is thin
Extracellular vesica atomic force microscopy diagram.The extracellular vesica that the capture of the present embodiment, detection method are obtained is in atomic force microscopy
Under the microscope.Fig. 7 is the outer vesica dimensional measurement figure of individual cells in Fig. 6.The horizontal width of vesica is outside individual cells
82.031nm meets the size range of the extracellular vesica of current International reporting.Fig. 8 is the outer vesica of UM-SCC6 unicellular cells
Slide sweep test result figure.Its enlarged drawing illustrate unicellular and its corresponding slide scanning result and the two superposition
Figure.Fig. 9 is the unicellular thermal map of UM-SCC6.Every a line represent one it is unicellular, each column represent a kind of extracellular vesica or one
Kind of cell factor, the results showed that the outer vesica of unicellular secretory cell, cell factor there were significant differences property.Figure 10 is that UM-SCC6 is thin
The extracellular vesica of intracrine and cell factor scatter plot.The result shows that cell secretory cell outer vesica, cell factor, with cell
Number increases and increases.Especially CD63+、CD9+The secretion of extracellular vesica.Figure 11 is UM-SCC6 correlation analysis figure.As a result
Show slender intracrine CD63+With CD9+Extracellular vesica has correlation.Figure 12 is the analysis of UM-SCC6 multifunctionality.It shows slender
The functionality of the extracellular vesica of intracrine and cell factor.Figure 13 is the unicellular immunophenotyping analysis chart of UM-SCC6.It utilizes
High-throughput unicellular counting is placed in 2D plane by viSNE software, reaches the visualization of cell subsets.
Embodiment 2
The present embodiment a kind of high-throughput, multi-parameter the extracellular vesica analysis chip structure of unicellular source property and preparation method
It is same as Example 1.
PDMS microwell array chip is used to cultivate primary Dental clinic by the present embodiment, probes into primary slender intracrine
The heterogeneity of extracellular vesica.The PDMS micro-channel chip and PDMS microwell array chip prepared using embodiment 1；It specifically includes
Following steps:
Step 1: primary cell extracts
1. rinsing tissue: being drawn using liquid-transfering gun and rinse tissue containing 2% dual anti-DPBS, and cut rejecting using ophthalmologic operation
The sundries such as fat, connective tissue, blood vessel.
2. shredding tissue: being cut with ophthalmologic operation by tissue shear to about 1mm3Fritter.Tissue is transferred to by culture dish and is filled
The centrifuge tube of 2% dual anti-DPBS is blown and beaten with liquid-transfering gun repeatedly and rinses tissue, DPBS is replaced, until DPBS is limpid.Finally with basis
Culture medium washes a tissue again.
3. trypsin digestion: be added 5-6 times of tissue volume 0.25% trypsase (or 0.25% trypsase+
0.02%EDTA or collagenase), 20-40min is digested, it is primary every 5min oscillation, or blown and beaten with suction pipe, make cell point
From.
4. terminating trypsin digestion: addition and trypsase isometric complete medium or trypsin inhibitor,
Piping and druming organizes the formation of tissue suspension in the solution.
The digestion of 5.I Collagenase Type: centrifuge is centrifuged above-mentioned tissue suspension, revolving speed 1000rpm, time 5min.It is abandoned after centrifugation
Fall supernatant.After 5-10 times of tissue volume of collagenase type I is added, centrifuge tube is lain against into constant temperature oscillation shaking table, oscillation to group
Knitting becomes cotton-shaped.
6. the centrifuge tube containing cotton-shaped tissue is put in 37 DEG C first, 5%CO2Incubator in stand 5min.Then will
Following flocculent deposit is evenly dispersed to be laid in the culture dish being coated with through collagenase type I.Culture dish is placed in 37 DEG C,
5%CO2Culture solution is added dropwise in 1h in incubator.Liquid periodically is changed, culture dish is covered with to cell, for testing.
Step 2: coated antibody forms the bioanalysis substrate with antibody bar code array
1.PDMS micro-channel chip and poly-D-lysine slide hot key and 2h.
2.Flow Patterning coated antibody.
1) 3ul antibody is respectively added to 9 channels from channel starting point using liquid-transfering gun.Be respectively as follows: FITC-BSA, CD63,
CD81,CD9,EpCAM,Hsp-70,IL-6,IL-8,MCP-1.Channel originates end position as shown in the 11-19 of Fig. 2.
2) flow antibody in channel to way vent from channel starting point, Excess antibody is flowed out from channel clearing end
Stop ventilation afterwards.The position of channel clearing end is as shown in the 21-29 of Fig. 2.
3) blocking antibody: configuration 15ml 1%BSA is placed in culture dish, takes PDMS micro-channel chip off in the solution, is sealed
Close antibody 1h.
3. cleaning dries, is spare:
It draws 1ml DPBS every time with liquid-transfering gun first to rinse in poly-D-lysine surface of glass slide, then successively soaks slide
It is cleaned in DPBS, 50%DPBS, ultrapure water, the drying of slide dryer.
Step 3: unicellular capture, by cell with certain density kind on microwell array chip
1.PDMS microwell array chip leads to oxygen plasma cleaner and handles 2min.
2. primary Dental clinic is digested, its cell density is diluted to 2 × 105cells/ml.Cell is through calcium Huang
200ul suspension is drawn after green uniformly dyeing color, is dispersed in and is added drop-wise on PDMS microwell array chip, stands 5min.
3. poly-D-lysine slide is placed on above PDMS microwell array chip, antibody bar code array connects with cell suspension
Touching is clamped slide and chip with fixture.
Step 4: unicellular big figure scanning
Said chip is in 37 DEG C, 5%CO2After being incubated for 1h in cell incubator, it is aobvious that fluorescence is successively automatically inverted with Nikon
The micro mirror figure scanning greatly under the conditions of light field, two kinds of fluorescence respectively.It observes cell distribution situation and calculates unicellular capture rate.Core
Piece, which is put back to, to be continued to be incubated in incubator.
Step 5: extracellular vesica, cytokines measurement.
1. said chip is in 37 DEG C, 5%CO2After being incubated for 12-24h in cell incubator, fixture is dismantled, poly is taken off and relies ammonia
Sour slide.30min is closed with 1%BSA.
2. 1%BSA rinses slide, biotinylated antibody 200ul is added dropwise on slide, reacts 1h.
3. 1%BSA rinses slide, the streptavidin 200ul that fluorescent marker is added dropwise on slide (uses 1%BSA1:200
Dilution), dye 30min.
4. 1%BSA rinses slide, 1%BSA 200ul (being diluted with 1%BSA1:100) is added dropwise on glass, closes again
30min。
5. drawn every time with liquid-transfering gun first 1ml DPBS surface of glass slide rinse, then slide is successively dipped in DPBS,
It is cleaned in 50%DPBS, ultrapure water, the drying of slide dryer.
6. scanning slide with four color laser chip scanners (Genepix+4300A).
Step 6: as a result data processing statisticallys analyze.
The present embodiment result is referring to Figure 14-Figure 19.Figure 14 is the outer vesica slide scanning of primary oral squamous cell carcinomas unicellular cells
Partial results figure.Its enlarged drawing illustrates the figure of unicellular and its corresponding slide scanning result and the two superposition.Figure 15 is
The primary unicellular thermal map of oral squamous cell carcinomas.Every a line represent one it is unicellular, each column represent a kind of extracellular vesica or one kind
Cell factor, the results showed that the outer vesica of unicellular secretory cell, cell factor there were significant differences property.Figure 16 is primary oral squamous cell carcinomas
The outer vesica of cell secretory cell and cell factor scatter plot.The result shows that cell secretory cell outer vesica, cell factor, with thin
Born of the same parents' number increases and increases.Figure 17 is primary Dental clinic correlation analysis figure.The result shows that slender intracrine CD63+With
CD9+Extracellular vesica has correlation.Figure 18 is the analysis of primary Dental clinic multifunctionality.Show unicellular secretory cell
The functionality of outer vesica and cell factor.Figure 19 is the unicellular immunophenotyping analysis chart of primary oral squamous cell carcinomas.Utilize viSNE software
High-throughput unicellular counting is placed in 2D plane, reaches the visualization of cell subsets.
Embodiment 3
A kind of high-throughput, multi-parameter extracellular vesica analysis chip of unicellular source property of the present embodiment, refering to Figure 20-figure
21.The chip includes double-layer structure, and one layer is bioanalysis substrate 1, and another layer is the chip 2 with high throughput array, described
The chip with high throughput array be the chip with high-throughput band array.
The bioanalysis substrate 1 is the poly-D-lysine glass for being coated with capture antibody not of the same race and having formed antibody bar code array
Piece.The antibody bar code array is as shown in figure 20, is made of 9 antibody bands, and band spacing is 1500um.Every band
200 include Liang Tiao branch band, and as shown in the 01 of Figure 20 and 02, branch's strip width is 100um, and branch's band spacing is
100um。
The preparation of the band need corresponding PDMS straight channel chip and poly-D-lysine slide hot key and, Zhi Hou
Antibody is added in channel, so that antibody is flowed through channel to be fixed on poly-D-lysine slide, antibody bar code battle array is consequently formed
Column.
The chip 2 with high-throughput band array is the PDMS straight channel chip with band array, such as Figure 21
It is shown, it altogether include 9 channels, interchannel is away from for 1600um.Every channel 300 includes three branched bottoms, as in Figure 21 01,
02, shown in 03, branched bottom width is 100um, and branched bottom spacing is 100 microns.
The corresponding PDMS straight channel chip needed in the preparation of bioanalysis substrate 1, and there is high-throughput band
The preparation method of PDMS straight channel chip in the chip 2 of array with band array is similar to Example 1.
The head and neck squamous cell carcinoma UM-SCC6 cell 48h culture solution of serum condition containing ultrafiltration is collected using said chip to use
In group's cell experiment, the result of above-mentioned unicellular experiment is verified；Specifically includes the following steps:
Step 1: the collection of conditioned medium
1. long to 80% to cell density with complete medium culture cell.
2. to the culture solution of cell replacement serum containing ultrafiltration, then continuous culture 48h, collection condition culture solution.
3. conditioned medium is centrifuged: 500 × g 15min, 2500 × g 20min, it is standby that supernatant is saved in -80 DEG C of refrigerators
With.
Step 2: coated antibody forms the bioanalysis substrate with antibody bar code array
Specifically includes the following steps:
1.PDMS straight channel chip 1 and poly-D-lysine slide hot key and 30min.1 in such as Figure 20 of PDMS straight channel chip 1
It is shown.
2. 1.5ul antibody is respectively added to 9 channels from feeder connection using liquid-transfering gun.Include: FITC-BSA, CD63,
CD81,CD9,EpCAM,Hsp-70,IL-6,IL-8,MCP-1.Feeder connection position is as shown in 11-19 in Figure 20.
3. inhaling using vacuum pumping pump channel outlet, make antibody full of entire channel.Coated antibody 4h.Channel outlet position is such as
In Figure 20 shown in 2 31-39.
4. 1%BSA irrigation channel 5 times, filling it up with 1%BSA, blocking antibody 10min.
5. 1%BSA irrigation channel 5 times, solution is blotted, PDMS straight channel chip 1 is taken off.It is drawn every time with liquid-transfering gun
1ml DPBS is rinsed in surface of glass slide, and then slide is successively dipped in DPBS, 50%DPBS, cleaned in ultrapure water, slide drying
Machine drying is spare.
Step 3: it is incubated for sample
1.PDMS straight channel chip 2 is bonded after leading to oxygen plasma cleaner processing 2min with poly-D-lysine slide.
2. channel is added in 1%BSA, it is loaded this after blocking antibody 1h, every channel adds 3ul.Make sample full of logical by vacuum pumping pump
Road.It is incubated for sample 18h.
Step 4: extracellular vesica, cytokines measurement
1. 1%BSA irrigation channel 5 times, it is (dilute with 1%BSA1:200 that biotinylated antibody 3ul is added in every channel
It releases).
2. 1%BSA irrigation channel 5 times, every channel be added fluorescent marker streptavidin 3ul (use 1%BSA 1:
200 dilutions).
3. 1%BSA irrigation channel 5 times, filling it up with the re-closed 10min of 1%BSA.Solution is blotted.
4. PDMS channel chip 2 is taken off from slide.1ml DPBS is drawn every time with liquid-transfering gun to rush in glass sheet surface
It washes, then sheet glass is successively dipped in DPBS, 50%DPBS, cleaned in ultrapure water, the drying of slide dryer.
5. scanning slide with four color laser chip scanners (Genepix+4300A).
Step 5: as a result data processing statisticallys analyze.
The present embodiment result 2- Figure 23 referring to fig. 2.Figure 22 is slide scanning result, it is seen that group's cell secretory cell external capsule
Bubble and cytokine signaling.Figure 23 is the result statistical analysis of various signal fluorescence intensities in Figure 22.
Claims (12)
1. a kind of extracellular vesica analysis chip of the unicellular source property of high flux multiparameter, it is characterised in that the chip includes two layers
Structure, one layer is bioanalysis substrate, and another layer is the chip with high throughput array；The bioanalysis substrate is used for cell
Outer vesica capture, detection, the chip of the high throughput array are used for unicellular capture.
2. a kind of extracellular vesica analysis chip of the unicellular source property of high flux multiparameter according to claim 1, special
Sign is that the bioanalysis substrate is the slide for being coated with albumen, antibody or DNA, or has been coated with protein arrays, antibody battle array
The slide of column or DNA array；
The slide includes but is not limited to poly-D-lysine slide, epoxy resin slide.
3. a kind of extracellular vesica analysis chip of the unicellular source property of high flux multiparameter according to claim 2, special
Sign is that the array is the parallel Uncrossed band for having specific width and spacing of 1-100 item, and every rule bandwidth is
5um-5000um, spacing 5um-5000um.
4. a kind of extracellular vesica analysis chip of the unicellular source property of high flux multiparameter according to claim 1, special
Sign is the chip with high throughput array for the chip with high-throughput microwell array or has high-throughput band array
Chip；
The material of the chip with high throughput array includes but is not limited to the PDMS after PDMS or various modifications.
5. a kind of extracellular vesica analysis chip of the unicellular source property of high flux multiparameter according to claim 4, special
Sign is that the microwell array is regular polygon, rectangle or circular array, and micropore number is 1~1000000/cm in array2。
6. a kind of extracellular vesica analysis chip of unicellular source property of high flux multiparameter according to claim 1 is answered
With, it is characterised in that the chip is used to capture, test and analyze the extracellular vesica not of the same race of the slender intracrine of various kinds of cell, or
The extracellular vesica not of the same race secreted after person's cell-cell interaction,
The extracellular vesica analysis of the multi-parameter single cell source property includes the analysis of 1-100 index.
7. a kind of extracellular vesica analysis chip of unicellular source property of high flux multiparameter according to claim 6 is answered
With, it is characterised in that the extracellular vesica includes but is not limited to small vesica, excretion body, cancer-bodies or apoptotic body.
8. a kind of extracellular vesica analysis chip of unicellular source property of high flux multiparameter according to claim 6 is answered
With, it is characterised in that the various kinds of cell includes but is not limited to that mammalian cell, zooblast or tumour patient lesion are thin
Born of the same parents.
9. a kind of extracellular vesica analysis chip of unicellular source property of high flux multiparameter according to claim 6 is answered
With, it is characterised in that the analysis content include extracellular vesica not of the same race is carried out qualitative and quantitative analysis, correlation analysis,
Multifunctionality analysis or immunophenotyping analysis, the clear heterogeneity for showing cell and the extracellular vesica of intercellular secretory.
10. the application of the extracellular vesica analysis chip of the unicellular source property of high flux multiparameter according to claim 6,
It is characterized in that capturing, detecting the application method of extracellular vesica specifically:
It takes out and has been coated with the bioanalysis substrate that capture antibody not of the same race forms antibody bar code array；By cell with certain density kind
On microwell array chip；The slide for being coated with capture antibody is covered, is fixed with fixture, sets 37 DEG C, 5%CO2Cell incubator
Middle culture 12-24 hours；Microscope is taken pictures；Fixture is unloaded, it is same unicellular a variety of that extracellular vesica staining reagent progress is added dropwise
The detection of extracellular vesica.
11. the application of the extracellular vesica analysis chip of the unicellular source property of high flux multiparameter according to claim 10,
It is characterized in that the staining reagent includes but is not limited to the strepto- affinity of PKH67 or biotinylated antibody and fluorescent marker
Element.
12. the application of the extracellular vesica analysis chip of the unicellular source property of high flux multiparameter according to claim 10,
It is characterized in that it is characterized in that bioanalysis substrate coating capture antibody not of the same race forms antibody bar code array, antibody bar code array
Fixed including but not limited to flowing, printing and other methods can be used to realize.
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| PCT/CN2018/000219 WO2019218101A1 (en) | 2018-05-17 | 2018-06-11 | High-throughput multiparametric single cell-derived extracellular vesicle analysis chip and use thereof |
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