WO2019218101A1 - 高通量多参数的单细胞源性细胞外囊泡分析芯片及应用 - Google Patents

高通量多参数的单细胞源性细胞外囊泡分析芯片及应用 Download PDF

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WO2019218101A1
WO2019218101A1 PCT/CN2018/000219 CN2018000219W WO2019218101A1 WO 2019218101 A1 WO2019218101 A1 WO 2019218101A1 CN 2018000219 W CN2018000219 W CN 2018000219W WO 2019218101 A1 WO2019218101 A1 WO 2019218101A1
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cell
throughput
array
chip
cells
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French (fr)
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陆瑶
冀雅慧
李林梅
林炳承
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中国科学院大连化学物理研究所
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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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    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/08—Apparatus for tissue disaggregation
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation

Definitions

  • the invention relates to the fields of microfluidic chip technology, single cell analysis technology and immunology, and particularly provides a high-throughput multi-parameter single-cell-derived extracellular vesicle analysis chip and application thereof.
  • Extracellular vesicles are a class of microvesicles of a bilayer lipid membrane structure secreted by cells. In recent years, studies have found that extracellular vesicles play an important role in the communication between cells. Almost all cells can produce extracellular vesicles, which contain a variety of parent cell-derived biologically active components such as lipids, proteins, nucleic acids (DNA, mRNA and microRNA, lncRNA, circRNA, etc.). In the vesicle or carried on the membrane. They participate in inflammatory immune response, intercellular signal communication, cell survival and apoptosis, angiogenesis, thrombosis, autophagy, etc., and play an important role in the maintenance of physiological state and disease.
  • extracellular vesicles On the membrane surface of extracellular vesicles, there is a class of transmembrane proteins such as CD63, CD81, CD9 and the like.
  • the capture of extracellular vesicles can be achieved by the use of a specific immune response in which the capture antibody binds to antigen molecules on the surface of the membrane.
  • the invention relates to a high-throughput multi-parameter single-cell extracellular vesicle analysis chip.
  • the present invention relates to a high throughput, multi-parameter single cell-derived extracellular vesicle (EVs) assay chip.
  • the technical problem solved by the present invention is implemented by the following technical solutions:
  • a high-throughput, multi-parameter single-cell-derived extracellular vesicle assay chip comprising a two-layer structure, one layer being a bioanalytical substrate and the other layer being a chip with a high-throughput array; the bioanalysis The substrate is used for extracellular vesicle capture, detection, and the high throughput array of chips is used for single cell capture.
  • the bioanalytical substrate is a slide coated with protein, antibody or DNA, or a slide coated with a protein array, an antibody array or a DNA array;
  • the slides include, but are not limited to, polylysine slides, epoxy slides.
  • the array is 1-100 strips of specific width and spacing that are not intersecting in parallel, each strip having a width of 5 ⁇ m to 5000 ⁇ m and a pitch of 5 ⁇ m to 5000 ⁇ m.
  • the chip with a high throughput array is a chip with a high throughput microwell array or a chip with a high throughput strip array.
  • the materials of the chip with high-throughput array include, but are not limited to, PDMS or various modified PDMS.
  • the microwell array is a regular polygon, a rectangle or a circular array, and the number of micropores in the array is 1 to 1,000,000/cm 2 .
  • a high-throughput, multi-parameter, single-cell-derived extracellular vesicle assay chip for capturing, detecting, and analyzing different types of extracellular vesicles secreted by single cells of various cells, or cell-cell interactions Different types of extracellular vesicles secreted afterwards,
  • the multi-parametric single cell-derived extracellular vesicle assay includes analysis of 1-100 indicators.
  • the extracellular vesicles include, but are not limited to, microvesicles, exosomes, carcinoma bodies, apoptotic bodies.
  • the various cells include, but are not limited to, mammalian cells, animal cells, tumor cells of tumor patients.
  • the analysis includes qualitative, quantitative detection, correlation analysis, versatility analysis and immunophenotyping analysis of different kinds of extracellular vesicles, and clearly shows the heterogeneity of extracellular vesicles secreted between cells and cells.
  • the invention relates to a high-throughput multi-parameter single-cell-derived extracellular vesicle analysis chip, and the application method for capturing and detecting extracellular vesicles is specifically:
  • the bioassay substrate coated with different kinds of capture antibodies to form an antibody barcode array is taken out; the cells are seeded on the microwell array chip at a certain density; the slides coated with the capture antibody are covered, fixed by a clamp, and placed at 37 ° C, 5 Incubate in a %CO 2 cell incubator for 12-24 hours; take a microscope; remove the clamp, and add extracellular vesicle staining reagent to detect the multiple extracellular vesicles of the same single cell;
  • staining reagents include, but are not limited to, PKH67 or biotinylated antibodies and fluorescently labeled streptavidin.
  • the bioassay substrate is coated with different types of capture antibodies to form an antibody barcode array, and the antibody barcode array can be implemented by, but not limited to, flow fixation, printing, and other methods.
  • the high-throughput microwell array of the chip of the invention can realize simple and efficient high-throughput single cell capture and culture in an in vitro environment
  • the chip can be used to capture different kinds of extracellular vesicles secreted by different kinds of cells
  • the chip can realize the immunophenotyping study of single cell-derived extracellular vesicles
  • the chip can realize multi-parameter simultaneous detection of the research object
  • the chip can be used for the study of multiple secretions of single cells, such as simultaneous detection of a variety of extracellular vesicles and a variety of cytokines;
  • results obtained by the chip can be systematically analyzed by various analytical methods, such as immunophenotyping analysis, which can display the visualization of cell subpopulations;
  • the chip is simple to make, the material price is low, and the amount of antibody is small, which can be generally applied to high-throughput and multi-parameter extracellular vesicle research.
  • FIG. 1 is a schematic structural view of a high-throughput, multi-parameter single-cell-derived extracellular vesicle analysis chip according to Embodiment 1 and Embodiment 2 of the present invention
  • Figure 2 is a magnified view of the bioanalytical substrate of Figure 1;
  • FIG. 3 is a high-throughput microhole array chip of FIG. 1 and a partial enlarged view thereof,
  • FIG. 4 is a schematic view showing the structure of the high-throughput, multi-parameter single-cell-derived extracellular vesicle analysis chip of FIG. 1 applied to single cell extracellular vesicle capture and detection;
  • 1 is a bioanalytical substrate
  • 2 is a chip with a high-throughput array
  • 3 is a fixture
  • 11-19 is a strip beginning segment
  • 21-29 is a strip termination end
  • 20 is a schematic diagram of one of the strips
  • 30 is a schematic diagram of one of the micropores.
  • Figure 5 is a graph showing the detection of antibody uniformity of an antibody barcode array formed on an antibody on a slide.
  • Figure 6 is an atomic force microscope image of extracellular vesicles.
  • Figure 7 is a graph showing the measurement of the size of a single extracellular vesicle in Figure 6.
  • Figure 8 is a graph showing the results of partial scanning of UM-SCC6 single cell extracellular vesicle slides.
  • Figure 9 is a UM-SCC6 single cell heat map.
  • Figure 10 is a scatter plot of UM-SCC6 cells secreting extracellular vesicles and cytokines.
  • Figure 11 is a graph of UM-SCC6 correlation analysis.
  • Figure 12 shows the UM-SCC6 versatility analysis.
  • Figure 13 is a UM-SCC6 single cell immunophenotyping analysis.
  • Figure 14 is a graph showing the results of partial scanning of single-cell extracellular vesicle slides of primary oral squamous cell carcinoma.
  • Figure 15 is a single cell heat map of primary oral squamous cell carcinoma.
  • Figure 16 is a scatter plot of extracellular vesicles and cytokines secreted by primary oral squamous cell carcinoma cells.
  • Figure 17 is a graph showing the correlation analysis of primary oral squamous cell carcinoma.
  • Figure 18 is a versatility analysis of primary oral squamous cell carcinoma.
  • Figure 19 is a graph showing single cell immunophenotyping of primary oral squamous cell carcinoma.
  • Figure 20 is a partial enlarged view of the population cell experimental bioanalytical substrate of Example 3.
  • Example 21 is a fragment of a high-throughput array of a population cell experiment in Example 3 and a partial enlarged view thereof,
  • 1 is a bioanalytical substrate
  • 2 is a chip with a high-throughput array
  • 11-19 is the beginning of the strip
  • 21-29 is the end of the strip
  • 200 is a schematic diagram of one of the strips.
  • 31-39 is the beginning of the channel
  • 41-49 is the end of the channel
  • 300 is a schematic diagram of one of the channels.
  • Figure 22 is a graph showing the results of a partial slide scan of a population cell experiment.
  • Figure 23 is a graph showing the results of a population cell experiment.
  • a high-throughput, multi-parameter single-cell-derived extracellular vesicle assay chip is shown in Figures 1-3.
  • the chip comprises a two-layer structure, one for the bioanalytical substrate 1 and the other for the chip 2 with a high-throughput array, the chip with a high-throughput array being a chip with a high-throughput microwell array.
  • the chip with a high throughput microwell array is a PDMS microwell array chip containing 6343 microwells.
  • the bioassay substrate 1 is a polylysine slide coated with an array of antibody barcodes of different kinds of capture antibodies.
  • the antibody barcode array consists of 9 strips of antibody, each strip having a width of 40 ⁇ m and a strip spacing of 80 ⁇ m.
  • the preparation of the strip requires a corresponding PDMS microchannel chip and a polylysine slide to be hot-bonded, and then Flow Patterning is used to allow the antibody to flow through the channel to be immobilized on the polylysine slide. An antibody barcode array is thus formed.
  • the preparation method of the PDMS microchannel chip is specifically as follows:
  • the PDMS microchannel chip contains 9 channels, each having a width of 40 ⁇ m and a distance between channels of 80 ⁇ m.
  • the wafer is exposed after cooling.
  • the exposure time (light intensity 9.6 mw/mm 2 , exposure time 22 s) was calculated from the ultraviolet light intensity and exposed.
  • the silicon wafer is placed on a hot plate and then baked. The time is about 1 minute. After the pattern appears on the silicon wafer, it stops and is baked.
  • the silicon wafer is developed after cooling.
  • the silicon wafer was immersed in a glassware containing ethyl lactate for 7 minutes, and a portion of the photoresist which should be removed was removed, and then rinsed with isopropyl alcohol.
  • Hard film The wafer is baked on a hot plate. Temperature: 120 ° C, time: 2 h.
  • the structural part of PDMS was cut out, ultrasonically cleaned in a vessel containing absolute ethanol for 5 min, and then dried at 80 °C.
  • the preparation method of the PDMS microwell array chip is as follows:
  • the chip specifications are as follows:
  • the PDMS microwell array contains 6343 micropores 1.44 mm long and 40 ⁇ m wide. Operating procedures:
  • the wafer is exposed after cooling.
  • the exposure time (light intensity 9.6 mw/mm 2 , exposure time 22 s) was calculated from the ultraviolet light intensity and exposed.
  • the silicon wafer is placed on a hot plate and then baked. The time is about 1 minute. After the pattern appears on the silicon wafer, it stops and is baked.
  • the silicon wafer is developed after cooling.
  • the silicon wafer was immersed in a glassware containing ethyl lactate for 7 minutes, and a portion of the photoresist which should be removed was removed, and then rinsed with isopropyl alcohol.
  • Hard film The wafer is baked on a hot plate. Temperature: 120 ° C, time: 2 h.
  • the structural part of PDMS was cut out, ultrasonically cleaned in a vessel containing absolute ethanol for 5 min, and then dried at 80 °C.
  • a high-throughput, multi-parameter single-cell-derived extracellular vesicle assay chip, the PDMS microwell array chip in this example is used to culture head and neck squamous cell carcinoma UM-SCC6, as shown in FIG.
  • Extracellular vesicles were analyzed at the single cell level to demonstrate the heterogeneity of extracellular vesicles secreted between cells and cells. Specifically, the following steps are included:
  • Step 1 Coating the antibody to form a bioanalytical substrate with an array of antibody barcodes
  • Blocking antibody 15 ml of 1% BSA was placed in a Petri dish, and the PDMS microchannel chip was removed from the solution, and the antibody was blocked for 1 h.
  • Step 2 Single cell capture, the cells are seeded on the microwell array chip at a certain density.
  • the polylysine slide is placed over the PDMS microwell array chip, the antibody barcode array is contacted with the cell suspension, and the slide is clamped to the chip with a clamp.
  • Step 3 Single cell large image scan
  • the above chips were incubated in a 37 ° C, 5% CO 2 cell incubator for 1 h, and then scanned with Nikon's fully automated inverted fluorescence microscope under brightfield and fluorescent conditions. Observe the cell distribution and calculate the single cell capture rate. The chip is returned to the incubator for continued incubation.
  • Step 4 Detection of extracellular vesicles and cytokines.
  • Step 5 Data processing, statistical analysis of results.
  • Figures 5-13 The results of this example are shown in Figures 5-13.
  • Figure 5 is a graph showing the uniformity of antibody formation of antibody barcode arrays on slides. FITC-BSA was added to all 9 channels, and the results showed that the strength of the antibody on the slide was highly uniform.
  • Figure 6 is an atomic force microscope image of extracellular vesicles. The extracellular vesicles obtained by the capture and detection methods of the present example were observed under an atomic force microscope.
  • Figure 7 is a graph showing the measurement of the size of a single extracellular vesicle in Figure 6. The horizontal width of a single extracellular vesicle is 82.031 nm, which is consistent with the size range of extracellular vesicles currently reported internationally.
  • Figure 8 is a graph showing the results of partial scanning of UM-SCC6 single cell extracellular vesicle slides. A magnified view of the single cell and its corresponding slide scan results, as well as a superimposed map.
  • Figure 9 is a UM-SCC6 single cell heat map. Each row represents a single cell, and each column represents an extracellular vesicle or a cytokine. The results indicate that there is a significant difference in the secretion of extracellular vesicles and cytokines by single cells.
  • Figure 10 is a scatter plot of UM-SCC6 cells secreting extracellular vesicles and cytokines.
  • FIG 11 is a graph of UM-SCC6 correlation analysis. The results indicate that single cell secretion of CD63+ EVs is associated with CD9+CD63+ EVs.
  • Figure 12 shows the UM-SCC6 versatility analysis. Shows the function of single cells to secrete extracellular vesicles and cytokines.
  • Figure 13 is a UM-SCC6 single cell immunophenotyping analysis. The high-throughput single-cell count was placed in the 2D plane using viSNE software to visualize the subpopulation of cells.
  • a high-throughput, multi-parameter single-cell-derived extracellular vesicle assay chip structure and preparation method are the same as in Example 1.
  • a PDMS microwell array chip was used to culture primary oral squamous carcinoma cells, and the heterogeneity of extracellular vesicles secreted by primary single cells was investigated.
  • the PDMS microchannel chip and the PDMS microwell array chip prepared in the embodiment 1 are used; specifically comprising the following steps:
  • Step 1 Primary cell extraction
  • Rinse the tissue Dip the tissue with a 2% double-antibody DPBS using a pipette, and use ophthalmic surgical scissors to remove fat, connective tissue, blood vessels and other debris.
  • Trypsin digestion add 5-6 times the tissue volume of 0.25% trypsin (or 0.25% trypsin + 0.02% EDTA or collagenase), digest for 20-40min, shake once every 5min, or blow with a pipette to make the cells Separation.
  • Stop trypsin digestion add an equal volume of trypsin to complete medium or trypsin inhibitor, and blow the tissue into a tissue to form a tissue suspension.
  • Type I collagenase digestion The above tissue suspension was centrifuged at a speed of 1000 rpm for 5 minutes. Discard the supernatant after centrifugation. After adding tissue type 5 collagenase 5-10 times, the centrifuge tube was placed on a constant temperature shaking shaker and shaken until the tissue became flocculent.
  • the centrifuge tube containing the flocculent tissue was placed in a 37 ° C, 5% CO 2 incubator for 5 min.
  • the following flocculent precipitate was then uniformly dispersed and spread in a Petri dish coated with type I collagenase.
  • the culture dish was placed in a 37 ° C, 5% CO 2 incubator for 1 h, and the culture solution was added dropwise. Change the fluid regularly and wait until the cells are overgrown with the culture dish for the experiment.
  • Step 2 coating the antibody to form a bioanalytical substrate with an array of antibody barcodes
  • Blocking antibody 15 ml of 1% BSA was placed in a Petri dish, and the PDMS microchannel chip was removed from the solution, and the antibody was blocked for 1 h.
  • Step 3 Single cell capture, the cells are seeded on the microwell array chip at a certain density.
  • the polylysine slide is placed over the PDMS microwell array chip, the antibody barcode array is contacted with the cell suspension, and the slide is clamped to the chip with a clamp.
  • Step 4 Single cell large image scan
  • the above chips were incubated in a 37 ° C, 5% CO 2 cell incubator for 1 h, and then scanned with Nikon's fully automated inverted fluorescence microscope under brightfield and fluorescent conditions. Observe the cell distribution and calculate the single cell capture rate. The chip is returned to the incubator for continued incubation.
  • Step 5 Detection of extracellular vesicles and cytokines.
  • the slide was washed with 2.1% BSA, and 200 ⁇ l of biotinylated antibody was added to the slide for 1 h.
  • the slides were washed with 4.1% BSA, and 200 ⁇ l of 1% BSA (diluted with 1% BSA 1:100) was added dropwise to the glass, and blocked again for 30 min.
  • Step 6 Data processing, statistical analysis of results.
  • Figure 14 is a graph showing the results of partial scanning of single-cell extracellular vesicle slides of primary oral squamous cell carcinoma. A magnified view of the single cell and its corresponding slide scan results, as well as a superimposed map.
  • Figure 15 is a single cell heat map of primary oral squamous cell carcinoma. Each row represents a single cell, and each column represents an extracellular vesicle or a cytokine. The results indicate that there is a significant difference in the secretion of extracellular vesicles and cytokines by single cells.
  • Figure 16 is a scatter plot of extracellular vesicles and cytokines secreted by primary oral squamous cell carcinoma cells.
  • FIG 17 is a graph showing the correlation analysis of primary oral squamous cell carcinoma cells. The results indicate that single cell secretion of CD63+ EVs is associated with CD9+CD63+ EVs.
  • Figure 18 is a versatility analysis of primary oral squamous cell carcinoma cells. Shows the function of single cells to secrete extracellular vesicles and cytokines.
  • Figure 19 is a graph showing single cell immunophenotyping of primary oral squamous cell carcinoma. The high-throughput single-cell count was placed in the 2D plane using viSNE software to visualize the subpopulation of cells.
  • This embodiment is a high-throughput, multi-parameter extracellular vesicle analysis chip, see Figures 20-21.
  • the chip comprises a two-layer structure, one for the bioanalytical substrate 1 and the other for the chip 2 with a high-throughput array, the chip with a high-throughput array being a chip with a high-throughput strip array.
  • the bioassay substrate 1 is a polylysine slide coated with an array of antibody barcodes of different kinds of capture antibodies.
  • the antibody barcode array shown in Fig. 20 was composed of 9 antibody strips with a strip spacing of 1500 ⁇ m.
  • Each strip 200 comprises two branch strips, as shown at 01 and 02 of Figure 20, with a strip width of 100 ⁇ m and a branch strip pitch of 100 ⁇ m.
  • the preparation of the strip requires a corresponding PDMS straight channel chip and a polylysine slide to be thermally bonded, and then an antibody is added to the channel to allow the antibody to flow through the channel to be immobilized on the polylysine slide. An antibody barcode array is thus formed.
  • the chip 2 with the high-throughput strip array is a PDMS straight channel chip with a strip array, as shown in FIG. 21, comprising a total of 9 channels with a channel spacing of 1600 ⁇ m.
  • Each channel 300 includes three branch channels, as shown by 01, 02, and 03 in FIG. 21, the branch channel width is 100 ⁇ m, and the branch channel pitch is 100 ⁇ m.
  • a corresponding PDMS straight channel chip required for the preparation of the bioanalytical substrate 1 and a PDMS straight channel chip having a strip array in the chip 2 having a high-throughput strip array are similar to the embodiment 1.
  • the above-mentioned chip was used to collect head and neck squamous cell carcinoma UM-SCC6 cells for 48 hours containing ultrafiltration serum conditioned medium for population cell experiments, and the results of the above single cell experiments were verified; the following steps were specifically included:
  • Step 1 Collection of conditioned medium
  • the culture medium containing ultrafiltration serum was replaced with the cells, and culture was continued for 48 hours, and the conditioned medium was collected.
  • Conditional culture solution centrifugation 500 ⁇ g 15 min, 2500 ⁇ g 20 min, the supernatant was stored in a -80 ° C refrigerator for use.
  • Step 2 coating the antibody to form a bioanalytical substrate with an array of antibody barcodes
  • PDMS straight channel chip 1 and polylysine slide hotkey and 30 min.
  • the PDMS straight channel chip 1 is as shown by 1 in FIG.
  • Step 3 Incubate the sample
  • the PDMS straight channel chip was treated with a poly-lysine slide after 2 minutes of treatment with an oxygen plasma cleaner. 2. 1% BSA was added to the channel, and the antibody was blocked for 1 h, and 3 ⁇ l was added to each channel. The sample is filled with channels by means of a vacuum pump. Incubate the sample for 18 h.
  • Step 4 Detection of extracellular vesicles and cytokines
  • Step 5 Data processing, statistical analysis of results.
  • Figure 22 shows the results of slide scanning. It can be seen that the population cells secrete extracellular vesicles and cytokine signals.
  • Figure 23 is a statistical analysis of the results of the fluorescence intensity of various signals in Figure 22.

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Abstract

提供一种高通量多参数的单细胞源性细胞外囊泡分析芯片及其应用。该芯片包括两层结构，一层为生物分析基底用于细胞外囊泡捕获、检测，另一层为具有高通量阵列的芯片用于单细胞捕获。将微流控芯片技术、单细胞分析技术应用于细胞外囊泡的研究，利用免疫亲和的方法，可实现高通量、多参数的捕获，检测单个细胞分泌的各种尺寸的细胞外囊泡。进而在单细胞水平对细胞外囊泡的分泌进行了定性、定量检测、相关性分析、多功能性分析、免疫分型分析，清晰展示了细胞与细胞间分泌细胞外囊泡的异质性。

Description

高通量多参数的单细胞源性细胞外囊泡分析芯片及应用 技术领域
本发明涉及微流控芯片技术、单细胞分析技术以及免疫学领域，特别提供了一种高通量多参数的单细胞源性细胞外囊泡分析芯片及应用。
背景技术
细胞外囊泡(EVs)，是一类由细胞分泌的双层脂质膜结构的微小囊泡。近年来研究发现细胞外囊泡在细胞间信息间通信中扮演十分重要的角色。几乎所有的细胞都能产生细胞外囊泡，其中含有脂质、蛋白质、核酸(DNA、mRNA及microRNA、lncRNA、circRNA等noncoding RNA)等多种母细胞来源的生物活性成份，这些信息物质包裹在囊泡中或携带于膜上。他们参与炎症免疫反应、细胞间信号通讯、细胞存活与凋亡、血管新生、血栓形成、自噬等，在生理状态维持及疾病的进程中发挥重要作用。在细胞外囊泡的膜表面，有一类跨膜蛋白，例如CD63、CD81、CD9等。利用特异性免疫反应，捕获抗体与膜表面的抗原分子相结合，可以实现对细胞外囊泡的捕获。
单细胞分析技术目前广泛应用于DNA、RNA、分泌蛋白等研究，但是该技术在细胞外囊泡研究方面尚不成熟。本发明涉及一种高通量多参数的单细胞细胞外囊泡分析芯片。
发明内容
本发明涉及一种高通量、多参数的单细胞源性细胞外囊泡(EVs)分析芯片。构建高通量单细胞捕获及其分泌的细胞外囊泡的捕获、检测平台，以及在单细胞水平分析细胞外囊泡，展示细胞与细胞间分泌细胞外囊泡的异质性。本发明所解决的技术问题采用以下技术方案来实现：
一种高通量、多参数的单细胞源性细胞外囊泡分析芯片，该芯片包括两层结构，一层为生物分析基底，另一层为具有高通量阵列的芯片；所述生物分析 基底用于细胞外囊泡捕获、检测，所述高通量阵列的芯片用于单细胞捕获。
所述生物分析基底为包被了蛋白、抗体或DNA的玻片、或者包被了蛋白阵列、抗体阵列或DNA阵列的玻片；
所述的玻片包括但不限于多聚赖氨酸玻片、环氧树脂玻片。
所述阵列为1-100条平行不交叉的有特定宽度与间距的条带，每条条带宽度为5μm-5000μm，间距为5μm-5000μm。
所述具有高通量阵列的芯片为具有高通量微孔阵列的芯片或具有高通量条带阵列的芯片。
所述具有高通量阵列的芯片的材质包括但不限于PDMS或各种修饰后的PDMS。
所述微孔阵列为正多边形、矩形或圆形阵列，阵列中微孔数目为1～1000000个/cm 2。
一种高通量多参数的单细胞源性细胞外囊泡分析芯片的应用，该芯片用于捕获、检测分析多种细胞的单细胞分泌的不同种细胞外囊泡，或者细胞-细胞相互作用后分泌的不同种细胞外囊泡，
所述的多参数单细胞源性细胞外囊泡分析包括1-100个指标的分析。
所述细胞外囊泡包括但不限于微小囊泡、外泌体、癌小体、凋亡小体。
所述的多种细胞包括但不限于哺乳动物细胞、动物细胞、肿瘤病人病灶细胞。
所述分析内容包括对不同种细胞外囊泡进行定性、定量检测、相关性分析、多功能性分析、免疫分型分析，清晰展示细胞与细胞间分泌细胞外囊泡的异质性。
一种高通量多参数的单细胞源性细胞外囊泡分析芯片的应用，所述的捕获、检测细胞外囊泡的应用方法，具体为：
取出包被了不同种捕获抗体形成抗体条码阵列的生物分析基底；将细胞以一定密度种在微孔阵列芯片上；盖上包被了捕获抗体的玻片，用夹具固定，置 37℃、5％CO 2细胞培养箱中培养12-24小时；显微镜拍照；卸下夹具，滴加细胞外囊泡染色试剂进行同一单细胞多种细胞外囊泡的检测；
所述的染色试剂包括但不限于PKH67或生物素化的抗体和荧光标记的链霉亲合素。
生物分析基底包被不同种捕获抗体形成抗体条码阵列，其抗体条码阵列可采用包括但不限于流动固定、打印及其他方法实现。
本发明的优点在于：
1.本发明芯片的高通量微孔阵列可实现体外环境下简单、高效的高通量单细胞捕获及培养；
2.该芯片可用于捕获不同种细胞分泌的不同种细胞外囊泡；
3.该芯片可实现单细胞源性细胞外囊泡的免疫分型研究；
4.该芯片可实现研究对象的多参数同时检测；
5.该芯片可用于单细胞多种分泌物的研究，如同时检测多种细胞外囊泡和多种细胞因子；
6.该芯片所得结果可用多种分析方法进行系统分析，如免疫分型分析，可展示细胞亚群的形象化等；
7.基于微流控芯片平台的细胞外囊泡研究，芯片制作简便、材料价格低廉，抗体用量少，可以普遍适用于高通量、多参数的细胞外囊泡研究。
附图说明
图1为本发明实施例1和实施例2提出的一种高通量、多参数的单细胞源性细胞外囊泡分析芯片的结构示意图；
图2为图1中生物分析基底及其局部放大图；
图3为图1中高通量微孔阵列芯片及其局部放大图，
图4为图1中高通量、多参数的单细胞源性细胞外囊泡分析芯片应用于单细胞细胞外囊泡捕获、检测时结构示意图；
其中：1为生物分析基底，2为具有高通量阵列的芯片，3为夹具；11-19为条带起始段，21-29为条带终止端，20为其中一条条带的示意图；30为其中一个微孔的示意图。
图5为抗体在玻片上形成抗体条码阵列的抗体均匀性检测图。
图6为细胞外囊泡原子力显微镜图。
图7为图6中单个细胞外囊泡尺寸测量图。
图8为UM-SCC6单细胞细胞外囊泡玻片扫描部分结果图。
图9为UM-SCC6单细胞热图。
图10为UM-SCC6细胞分泌细胞外囊泡及细胞因子散点图。
图11为UM-SCC6相关性分析图。
图12为UM-SCC6多功能性分析。
图13为UM-SCC6单细胞免疫分型分析图。
图14为原代口腔鳞癌单细胞细胞外囊泡玻片扫描部分结果图。
图15为原代口腔鳞癌单细胞热图。
图16为原代口腔鳞癌细胞分泌细胞外囊泡及细胞因子散点图。
图17为原代口腔鳞癌相关性分析图。
图18为原代口腔鳞癌多功能性分析。
图19为原代口腔鳞癌单细胞免疫分型分析图。
图20为实施例3中群体细胞实验生物分析基底及其局部放大图。
图21为实施例3中群体细胞实验高通量阵列的芯片及其局部放大图，
其中：1为生物分析基底，2为具有高通量阵列的芯片，11-19为条带起 始端，21-29为条带终止端，200为其中一条条带的示意图。
31-39为通道起始端，41-49为通道终止端，300为其中一条通道的示意图。
图22为群体细胞实验部分玻片扫描结果图。
图23为群体细胞实验结果分析图。
具体实施方式
为使本发明的目的、技术方法、优点更加清晰明了，下面结合附图对本发明做进一步的详细说明，本发明所使用的实验试剂均为市购，如表1所示。
表1 本实验所用试剂列表。
Figure PCTCN2018000219-appb-000001
Figure PCTCN2018000219-appb-000002
实施例1
本实施例一种高通量、多参数的单细胞源性细胞外囊泡分析芯片，参阅图1-图3。该芯片包括两层结构，一层为生物分析基底1，另一层为具有高通量阵列的芯片2，所述的具有高通量阵列的芯片为具有高通量微孔阵列的芯片。
所述具有高通量微孔阵列的芯片为含有6343个微孔的PDMS微孔阵列芯片。
所述生物分析基底1为包被了不同种捕获抗体形成抗体条码阵列的多聚赖氨酸玻片。所述的抗体条码阵列由9条抗体条带组成，每条条带宽度为40μm，条带间距为80μm，
所述的条带的制备需要相应的PDMS微通道芯片与多聚赖氨酸玻片热键和，之后采用Flow Patterning的方法，使抗体流经通道从而被固定到多聚赖氨酸玻片上，由此形成抗体条码阵列。
所述PDMS微通道芯片的制备方法具体如下：
1.采用光刻操作制备硅片模板。规格如下：PDMS微通道芯片包含9条通道，每条宽度为40μm，通道间距离为80μm。
1)首先将干净的硅片置于热板烘干。温度：105℃，时间：10min。
2)硅片打等离子处理：通氧气plasma cleaner处理2min。
3)调匀胶机转速，根据胶的厚度确定转速和时间。预甩600转，18s；正式甩3000转，30s。
4)将干净的硅片置于真空托盘上，倒入适量的SU-8 3035型光刻胶，抽真空1min后，甩胶。
5)硅片置于热板上前烘。温度95℃，时间：30min。
6)硅片降温后曝光。根据紫外光强度计算曝光时间(光强9.6mw/mm 2，曝光时间为22s)，并曝光。
7)硅片置于热板上后烘。时间约1min。硅片上出现图案后停止后烘。
8)硅片降温后显影。硅片整体浸没于盛有乳酸乙酯的玻璃器皿中清洗7min，除去应去掉的部分光刻胶，再用异丙醇冲洗。
9)坚膜。硅片置于热板上烘烤。温度：120℃，时间：2h。
10)三甲基氯硅烷熏硅片。时间：48h。
2.灌注硅片模板
将RTV615型A胶、B胶10∶1混合并搅拌均匀，用抽真空泵抽净气泡，加入硅片结构区域，80℃热烘1h。
切取结构部分PDMS，置于盛有无水乙醇的器皿中超声清洗5min，之后80℃烘干。
所述PDMS微孔阵列芯片的制备方法如下：
1.采用光刻操作制备硅片模板。芯片规格如下：PDMS微孔阵列包含6343个长1.44mm，宽40μm的微孔。操作流程：
1)首先将干净的硅片置于热板烘干。温度：105℃，时间：10min。
2)硅片打等离子处理：通氧气plasma cleaner处理2min。
3)调匀胶机转速，根据胶的厚度确定转速和时间。预甩600转，18s；正式 甩3000转，30s。
4)将干净的硅片置于真空托盘上，倒入适量的SU-8 3035型光刻胶，抽真空1min后，甩胶。
5)硅片置于热板上前烘。温度95℃，时间：30min。
6)硅片降温后曝光。根据紫外光强度计算曝光时间(光强9.6mw/mm 2，曝光时间为22s)，并曝光。
7)硅片置于热板上后烘。时间约1min。硅片上出现图案后停止后烘。
8)硅片降温后显影。硅片整体浸没于盛有乳酸乙酯的玻璃器皿中清洗7min，除去应去掉的部分光刻胶，再用异丙醇冲洗。
9)坚膜。硅片置于热板上烘烤。温度：120℃，时间：2h。
10)三甲基氯硅烷熏硅片。时间：48h。
2.灌注硅片模板
将RTV615型A胶、B胶10∶1混合并搅拌均匀，用抽真空泵抽净气泡，加入硅片结构区域，80℃热烘1h。
切取结构部分PDMS，置于盛有无水乙醇的器皿中超声清洗5min，之后80℃烘干。
一种高通量、多参数的单细胞源性细胞外囊泡分析芯片的应用，本实施例中的PDMS微孔阵列芯片用于培养头颈部鳞状细胞癌UM-SCC6，如图4所示，在单细胞水平分析细胞外囊泡，展示细胞与细胞间分泌细胞外囊泡的异质性。具体包括以下步骤：
步骤一：包被抗体，形成具有抗体条码阵列的生物分析基底
1.PDMS微通道芯片与多聚赖氨酸玻片热键和2h。
2.Flow Patterning包被抗体。
1)使用移液枪从通道起始端给9条通道各加入3μl抗体。分别为：FITC-BSA、CD63、CD81、CD9、EpCAM、HSP70、IL-6、IL-8、MCP-1。通道起始端位置如图2的11-19所示。
2)从通道起始端给通道通气，使抗体在通道内流动，多余抗体从通道终止端流出后停止通气。通道终止端的位置如图2的21-29所示。
3)封闭抗体：配置15ml 1％BSA置于培养皿中，在溶液中揭下PDMS微通道芯片，封闭抗体1h。
3.清洗、甩干、备用：
首先用移液枪每次吸取1ml DPBS在多聚赖氨酸玻片表面冲洗，然后将玻片依次浸于DPBS、50％DPBS、超纯水中清洗，玻片甩干机甩干。
步骤二：单细胞捕获，将细胞以一定密度种在微孔阵列芯片上
1.PDMS微孔阵列芯片，通氧气plasma cleaner处理2min。
2.将UM-SCC6细胞消化，将其细胞密度稀释为2×10 5cells/ml。细胞经钙黄绿素染色后吸取200μl悬液，散在滴加到PDMS微孔阵列芯片上，静置5min。
3.将多聚赖氨酸玻片盖于PDMS微孔阵列芯片上方，抗体条码阵列与细胞悬液接触，用夹具将玻片与芯片夹紧。
步骤三：单细胞大图扫描
上述芯片在37℃、5％CO 2细胞培养箱中孵育1h后，依次用尼康全自动倒置荧光显微镜分别在明场、荧光两种条件下大图扫描。观察细胞分布情况以及计算单细胞捕获率。芯片放回培养箱中继续孵育。
步骤四：细胞外囊泡、细胞因子检测。
1.上述芯片在37℃、5％CO 2细胞培养箱中孵育12-24h后，拆开夹具，揭下多聚赖氨酸玻片。用1％BSA封闭30min。
2. 1％BSA冲洗玻片，在玻片上滴加生物素化的抗体200μl，反应1h。
3. 1％BSA冲洗玻片，在玻片上滴加荧光标记的链霉亲合素200μl(用1％BSA1∶200稀释)，染色30min。
4. 1％BSA冲洗玻片，在玻上滴加1％BSA 200μl(用1％BSA1∶100稀释)，再次封闭30min。
5.首先用移液枪每次吸取1ml DPBS在玻片表面冲洗，然后将玻片依次浸于DPBS、50％DPBS、超纯水中清洗，玻片甩干机甩干。
6.用四色激光芯片扫描仪(Genepix+4300A)扫描玻片。
步骤五：数据处理，结果统计分析。
本实施例结果参见图5-图13，图5为抗体在玻片上形成抗体条码阵列的抗体均匀性检测图。9条通道都加入FITC-BSA，结果表明抗体在玻片上的强度是高度均匀的。图6为细胞外囊泡原子力显微镜图。将本实施例的捕获、检测方法得到的细胞外囊泡在原子力显微镜下观察。图7为图6中单个细胞外囊泡尺寸测量图。单个细胞外囊泡的水平宽度为82.031nm，符合目前国际报道的细胞外囊泡的尺寸范围。图8为UM-SCC6单细胞细胞外囊泡玻片扫描部分结果图。其放大图展示了单细胞及其相对应玻片扫描结果，以及二者叠加的图。图9为UM-SCC6单细胞热图。每一行代表一个单细胞，每一列代表一种细胞外囊泡或者一种细胞因子，结果表明单细胞分泌细胞外囊泡、细胞因子有显著差异性。图10为UM-SCC6细胞分泌细胞外囊泡及细胞因子散点图。结果表明细胞分泌细胞外囊泡、细胞因子，随着细胞数目增加而增加。尤其是 CD63+EVs、 CD9+CD63+EVs的分泌。图11为UM-SCC6相关性分析图。结果表明单细胞分泌 CD63+EVs与 CD9+CD63+EVs有相关性。图12为UM-SCC6多功能性分析。展示单细胞分泌细胞外囊泡及细胞因子的功能性。图13为UM-SCC6单细胞免疫分型分析图。利用 viSNE软件将高通量的单细胞计数置于2D平面，达到细胞亚群的形象化。
实施例2
本实施例一种高通量、多参数的单细胞源性细胞外囊泡分析芯片结构及制备方法与实施例1相同。
本实施例将PDMS微孔阵列芯片用于培养原代口腔鳞癌细胞，探究原代单细胞分泌细胞外囊泡的异质性。采用实施例1制备的PDMS微通道芯片和PDMS微孔阵列芯片；具体包括以下步骤：
步骤一：原代细胞提取
1.冲洗组织：使用移液枪吸取含2％双抗的DPBS冲洗组织，并使用眼科手术剪剔除脂肪、结缔组织、血管等杂物。
2.剪碎组织：用眼科手术剪将组织剪至约1mm 3小块。将组织由培养皿转移到盛有2％双抗DPBS的离心管，反复用移液枪吹打冲洗组织，更换DPBS，直到DPBS清亮。最后用基础培养基再洗一遍组织。
3.胰蛋白酶消化：加入组织体积5-6倍的0.25％胰蛋白酶(或0.25％胰蛋白酶+0.02％EDTA或collagenase)，消化20-40min，每隔5min振荡一次，或用吸管吹打，使细胞分离。
4.终止胰蛋白酶消化：加入与胰蛋白酶等体积的完全培养基或胰蛋白酶抑制剂，在溶液中吹打组织形成组织悬液。
5.I型胶原酶消化：离心机离心上述组织悬液，转速1000rpm，时间5min。离心后弃掉上清液。加入组织体积5-10倍的I型胶原酶后，将离心管平放于恒温振荡摇床，振荡至组织变为絮状。
6.首先将含有絮状组织的离心管放于37℃，5％CO 2的培养箱中静置5min。然后将下面的絮状沉淀均匀分散的平铺在经I型胶原酶包被过的培养皿中。将培 养皿置于37℃，5％CO 2培养箱中1h，滴加培养液。定期换液，待细胞长满培养皿，用于实验。
步骤二：包被抗体，形成具有抗体条码阵列的生物分析基底
1.PDMS微通道芯片与多聚赖氨酸玻片热键和2h。
2.Flow Patterning包被抗体。
1)使用移液枪从通道起始端给9条通道各加入3μl抗体。分别为：FITC-BSA、CD63、CD81、CD9、EpCAM、HSP70、IL-6、IL-8、MCP-1。通道起始端位置如图2的11-19所示。
2)从通道起始端给通道通气，使抗体在通道内流动，多余抗体从通道终止端流出后停止通气。通道终止端的位置如图2的21-29所示。
3)封闭抗体：配置15ml 1％BSA置于培养皿中，在溶液中揭下PDMS微通道芯片，封闭抗体1h。
3.清洗、甩干、备用：
首先用移液枪每次吸取1ml DPBS在多聚赖氨酸玻片表面冲洗，然后将玻片依次浸于DPBS、50％DPBS、超纯水中清洗，玻片甩干机甩干。
步骤三：单细胞捕获，将细胞以一定密度种在微孔阵列芯片上
1.PDMS微孔阵列芯片，通氧气plasma cleaner处理2min。
2.将原代口腔鳞癌细胞消化，将其细胞密度稀释为2×10 5cells/ml。细胞经钙黄绿素染色后吸取200μl悬液，散在滴加到PDMS微孔阵列芯片上，静置5min。
3.将多聚赖氨酸玻片盖于PDMS微孔阵列芯片上方，抗体条码阵列与细胞悬液接触，用夹具将玻片与芯片夹紧。
步骤四：单细胞大图扫描
上述芯片在37℃、5％CO 2细胞培养箱中孵育1h后，依次用尼康全自动倒 置荧光显微镜分别在明场、荧光两种条件下大图扫描。观察细胞分布情况以及计算单细胞捕获率。芯片放回培养箱中继续孵育。
步骤五：细胞外囊泡、细胞因子检测。
1.上述芯片在37℃、5％CO 2细胞培养箱中孵育12-24h后，拆开夹具，揭下多聚赖氨酸玻片。用1％BSA封闭30min。
2.1％BSA冲洗玻片，在玻片上滴加生物素化的抗体200μl，反应1h。
3.1％BSA冲洗玻片，在玻片上滴加荧光标记的链霉亲合素200μl(用1％BSA1∶200稀释)，染色30min。
4.1％BSA冲洗玻片，在玻上滴加1％BSA 200μl(用1％BSA1∶100稀释)，再次封闭30min。
5.首先用移液枪每次吸取1ml DPBS在玻片表面冲洗，然后将玻片依次浸于DPBS、50％DPBS、超纯水中清洗，玻片甩干机甩干。
6.用四色激光芯片扫描仪(Genepix+4300A)扫描玻片。
步骤六：数据处理，结果统计分析。
本实施例结果参见图14-图19。图14为原代口腔鳞癌单细胞细胞外囊泡玻片扫描部分结果图。其放大图展示了单细胞及其相对应玻片扫描结果，以及二者叠加的图。图15为原代口腔鳞癌单细胞热图。每一行代表一个单细胞，每一列代表一种细胞外囊泡或者一种细胞因子，结果表明单细胞分泌细胞外囊泡、细胞因子有显著差异性。图16为原代口腔鳞癌细胞分泌细胞外囊泡及细胞因子散点图。结果表明细胞分泌细胞外囊泡、细胞因子，随着细胞数目增加而增加。图17为原代口腔鳞癌细胞相关性分析图。结果表明单细胞分泌 CD63+EVs与 CD9+CD63+EVs有相关性。图18为原代口腔鳞癌细胞多功能性分析。展示单细胞分泌细胞外囊泡及细胞因子的功能性。图19为原代口腔鳞癌单细胞免疫分型分 析图。利用viSNE软件将高通量的单细胞计数置于2D平面，达到细胞亚群的形象化。
实施例3
本实施例一种高通量、多参数的细胞外囊泡分析芯片，参阅图20-图21。该芯片包括两层结构，一层为生物分析基底1，另一层为具有高通量阵列的芯片2，所述的具有高通量阵列的芯片为具有高通量条带阵列的芯片。
所述生物分析基底1为包被了不同种捕获抗体形成抗体条码阵列的多聚赖氨酸玻片。所述的抗体条码阵列如图20所示，由9条抗体条带组成，条带间距为1500μm。每条条带200包含两条分支条带，如图20的01和02所示，分支条带宽度为100μm，分支条带间距为100μm。
所述的条带的制备需要相应的PDMS直通道芯片与多聚赖氨酸玻片热键和，之后在通道内加入抗体，使抗体流经通道从而被固定到多聚赖氨酸玻片上，由此形成抗体条码阵列。
所述的具有高通量条带阵列的芯片2为具有条带阵列的PDMS直通道芯片，如图21所示，共包含9条通道，通道间距为1600μm。每条通道300包含三条分支通道，如图21中的01、02、03所示，分支通道宽度为100μm，分支通道间距为100μm。
所述生物分析基底1制备中需要的相应的PDMS直通道芯片，以及具有高通量条带阵列的芯片2中具有条带阵列的PDMS直通道芯片的制备方法与实施例1相似。
采用上述芯片收集头颈部鳞状细胞癌UM-SCC6细胞48h含超滤血清条件培养液用于群体细胞实验，对上述单细胞实验的结果进行验证；具体包括以下步骤：
步骤一：条件培养液的收集
1.用完全培养基培养细胞，待细胞密度长至80％。
2.给细胞更换含超滤血清的培养液，再连续培养48h，收集条件培养液。
3.条件培养液离心：500×g 15min，2500×g 20min，上清液保存到-80℃冰箱备用。
步骤二：包被抗体，形成具有抗体条码阵列的生物分析基底
具体包括以下步骤：
1.PDMS直通道芯片1与多聚赖氨酸玻片热键和30min。PDMS直通道芯片1如图20中1所示。
2.使用移液枪从通道入口给9条通道各加入1.5μl抗体。包括：FITC-BSA、CD63、CD81、CD9、EpCAM、HSP70、IL-6、IL-8、MCP-1。通道入口位置如图20中11-19所示。
3.使用抽真空泵通道出口吸，使抗体充满整个通道。包被抗体4h。通道出口位置如图20中2的31-39所示。
4. 1％BSA冲洗通道5次，加满1％BSA，封闭抗体10min。
5. 1％BSA冲洗通道5次，将溶液吸干，揭下PDMS直通道芯片1。用移液枪每次吸取1ml DPBS在玻片表面冲洗，然后将玻片依次浸于DPBS、50％DPBS、超纯水中清洗，玻片甩干机甩干备用。
步骤三：孵育样本
1.PDMS直通道芯片2通氧气plasma cleaner处理2min后与多聚赖氨酸玻片键合。2. 1％BSA加入通道，封闭抗体1h后加样本，每条通道加3μl。借助抽真空泵使样本充满通道。孵育样本18h。
步骤四：细胞外囊泡、细胞因子检测
1. 1％BSA冲洗通道5次，每条通道加入生物素化的抗体3μl(用1％BSA1∶200稀释)。
2. 1％BSA冲洗通道5次，每条通道加入荧光标记的链霉亲合素3μl(用1％BSA 1∶200稀释)。
3. 1％BSA冲洗通道5次，加满1％BSA再封闭10min。将溶液吸干。
4.将PDMS通道芯片2从玻片上揭下。用移液枪每次吸取1ml DPBS在玻璃片表面冲洗，然后将玻璃片依次浸于DPBS、50％DPBS、超纯水中清洗，玻片甩干机甩干。
5.用四色激光芯片扫描仪(Genepix+4300A)扫描玻片。
步骤五：数据处理，结果统计分析。
本实施例结果参见图22-图23。图22为玻片扫描结果，可见群体细胞分泌细胞外囊泡及细胞因子信号。图23为图22中各种信号荧光强度的结果统计分析。

Claims (12)

  1. 一种高通量多参数的单细胞源性细胞外囊泡分析芯片，其特征在于该芯片包括两层结构，一层为生物分析基底，另一层为具有高通量阵列的芯片；所述生物分析基底用于细胞外囊泡捕获、检测，所述高通量阵列的芯片用于单细胞捕获。
  2. 根据权利要求1所述的一种高通量多参数的单细胞源性细胞外囊泡分析芯片，其特征在于所述生物分析基底为包被了蛋白、抗体或DNA的玻片，或者包被了蛋白阵列、抗体阵列或DNA阵列的玻片；
    所述的玻片包括但不限于多聚赖氨酸玻片、环氧树脂玻片。
  3. 根据权利要求2所述的一种高通量多参数的单细胞源性细胞外囊泡分析芯片，其特征在于所述的阵列为1-100条平行不交叉的有特定宽度与间距的条带，每条条带宽度为5um-5000um，间距为5um-5000um。
  4. 根据权利要求1所述的一种高通量多参数的单细胞源性细胞外囊泡分析芯片，其特征在于所述具有高通量阵列的芯片为具有高通量微孔阵列的芯片或具有高通量条带阵列的芯片；
    所述具有高通量阵列的芯片的材质包括但不限于PDMS或各种修饰后的PDMS。
  5. 根据权利要求4所述的一种高通量多参数的单细胞源性细胞外囊泡分析芯片，其特征在于所述微孔阵列为正多边形、矩形或圆形阵列，阵列中微孔数目为1～1000000个/cm 2。
  6. 根据权利要求1所述的一种高通量多参数的单细胞源性细胞外囊泡分析芯片的应用，其特征在于该芯片用于捕获、检测分析多种细胞的单细胞分泌的不同种细胞外囊泡，或者细胞-细胞相互作用后分泌的不同种细胞外囊泡，
    所述的多参数单细胞源性细胞外囊泡分析包括1-100个指标的分析。
  7. 根据权利要求6所述的一种高通量多参数的单细胞源性细胞外囊泡分析芯片的应用，其特征在于所述细胞外囊泡包括但不限于微小囊泡、外泌体、癌小体或凋亡小体。
  8. 根据权利要求6所述的一种高通量多参数的单细胞源性细胞外囊泡分析 芯片的应用，其特征在于所述的多种细胞包括但不限于哺乳动物细胞、动物细胞或肿瘤病人病灶细胞。
  9. 根据权利要求6所述的一种高通量多参数的单细胞源性细胞外囊泡分析芯片的应用，其特征在于所述分析内容包括对不同种细胞外囊泡进行定性、定量检测、相关性分析、多功能性分析或免疫分型分析，清晰展示细胞与细胞间分泌细胞外囊泡的异质性。
  10. 根据权利要求6所述的高通量多参数的单细胞源性细胞外囊泡分析芯片的应用，其特征在于捕获、检测细胞外囊泡的应用方法具体为：
    取出包被了不同种捕获抗体形成抗体条码阵列的生物分析基底；将细胞以一定密度种在微孔阵列芯片上；盖上包被了捕获抗体的玻片，用夹具固定，置37℃、5％CO 2细胞培养箱中培养12-24小时；显微镜拍照；卸下夹具，滴加细胞外囊泡染色试剂进行同一单细胞多种细胞外囊泡的检测。
  11. 根据权利要求10所述的高通量多参数的单细胞源性细胞外囊泡分析芯片的应用，其特征在于所述的染色试剂包括但不限于PKH67或生物素化的抗体和荧光标记的链霉亲合素。
  12. 根据权利要求10所述高通量多参数的单细胞源性细胞外囊泡分析芯片的应用，其特征在于其特征为生物分析基底包被不同种捕获抗体形成抗体条码阵列，其抗体条码阵列可采用包括但不限于流动固定、打印及其他方法实现。
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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112834754A (zh) * 2019-11-22 2021-05-25 中国科学院大连化学物理研究所 一种便携高通量单细胞外囊泡检测装置及可视化检测方法
WO2022125701A1 (en) * 2020-12-09 2022-06-16 Becton, Dickinson And Company Multiplexed single cell immunoassay
US11492660B2 (en) 2018-12-13 2022-11-08 Becton, Dickinson And Company Selective extension in single cell whole transcriptome analysis
US11525157B2 (en) 2016-05-31 2022-12-13 Becton, Dickinson And Company Error correction in amplification of samples
US11535882B2 (en) 2015-03-30 2022-12-27 Becton, Dickinson And Company Methods and compositions for combinatorial barcoding
US11618929B2 (en) 2013-08-28 2023-04-04 Becton, Dickinson And Company Massively parallel single cell analysis
US11634708B2 (en) 2012-02-27 2023-04-25 Becton, Dickinson And Company Compositions and kits for molecular counting
US11639517B2 (en) 2018-10-01 2023-05-02 Becton, Dickinson And Company Determining 5′ transcript sequences
US11649497B2 (en) 2020-01-13 2023-05-16 Becton, Dickinson And Company Methods and compositions for quantitation of proteins and RNA
US11661625B2 (en) 2020-05-14 2023-05-30 Becton, Dickinson And Company Primers for immune repertoire profiling
US11661631B2 (en) 2019-01-23 2023-05-30 Becton, Dickinson And Company Oligonucleotides associated with antibodies
US11739443B2 (en) 2020-11-20 2023-08-29 Becton, Dickinson And Company Profiling of highly expressed and lowly expressed proteins
US11773441B2 (en) 2018-05-03 2023-10-03 Becton, Dickinson And Company High throughput multiomics sample analysis
US11773436B2 (en) 2019-11-08 2023-10-03 Becton, Dickinson And Company Using random priming to obtain full-length V(D)J information for immune repertoire sequencing
US11782059B2 (en) 2016-09-26 2023-10-10 Becton, Dickinson And Company Measurement of protein expression using reagents with barcoded oligonucleotide sequences
US11845986B2 (en) 2016-05-25 2023-12-19 Becton, Dickinson And Company Normalization of nucleic acid libraries
US11932849B2 (en) 2018-11-08 2024-03-19 Becton, Dickinson And Company Whole transcriptome analysis of single cells using random priming
US11932901B2 (en) 2020-07-13 2024-03-19 Becton, Dickinson And Company Target enrichment using nucleic acid probes for scRNAseq
US11939622B2 (en) 2019-07-22 2024-03-26 Becton, Dickinson And Company Single cell chromatin immunoprecipitation sequencing assay
US11970737B2 (en) 2009-12-15 2024-04-30 Becton, Dickinson And Company Digital counting of individual molecules by stochastic attachment of diverse labels
US12071617B2 (en) 2019-02-14 2024-08-27 Becton, Dickinson And Company Hybrid targeted and whole transcriptome amplification
US12084712B2 (en) 2017-06-05 2024-09-10 Becton, Dickinson And Company Sample indexing for single cells

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110951834A (zh) * 2019-12-10 2020-04-03 中国科学院深圳先进技术研究院 一种基于微流控芯片的细胞荧光原位杂交方法及其应用
CN110954703A (zh) * 2019-12-27 2020-04-03 杭州迪相实业有限公司 一种同时检测外泌体内蛋白和rna、外泌体膜蛋白的方法
CN113030481A (zh) * 2020-06-17 2021-06-25 山东大学 一种单细胞外泌体静态检测试剂盒及检测分析方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101168717A (zh) * 2006-10-25 2008-04-30 中国科学院大连化学物理研究所 一种用于细胞凋亡研究的集成化微流控芯片及其应用
CN101748059A (zh) * 2008-12-08 2010-06-23 中国科学院大连化学物理研究所 一种微流控芯片及研究细胞在三维介质中定向移动的方法
CN104760922A (zh) * 2014-01-03 2015-07-08 中国科学院电子学研究所 一种超微平面电极阵列传感器及其制备方法
CN104884605A (zh) * 2012-08-24 2015-09-02 耶鲁大学 用于高通量多重检测的系统、装置和方法
CN206787889U (zh) * 2017-05-05 2017-12-22 中国科学院半导体研究所 一种分离和富集体液成分的装置

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5804620B2 (ja) * 2010-08-09 2015-11-04 一般財団法人生産技術研究奨励会 電気的機能を持つマイクロチャンバーアレイ装置およびそれを用いた検査対象物解析方法
CN103797131A (zh) * 2011-06-16 2014-05-14 卡里斯生命科学卢森堡控股有限责任公司 生物标志物组合物和方法
EP2900803B1 (en) * 2012-09-26 2019-11-06 Quantumcyte, Inc. Devices and methods for single cell analysis
CN105492621A (zh) * 2013-03-28 2016-04-13 英属哥伦比亚大学 微流控装置及其在多细胞分泌检测中的使用方法
CN103630571B (zh) * 2013-09-12 2015-08-19 中国科学院电子学研究所 一种微纳阵列传感器及其制备方法
EP3227684B1 (en) * 2014-12-03 2019-10-02 Isoplexis Corporation Analysis and screening of cell secretion profiles
US20180264466A1 (en) * 2015-01-08 2018-09-20 The Regents Of The University Of California Position-defined cell culture and characterization platform
CN107667178A (zh) * 2015-03-26 2018-02-06 休斯敦大学系统 细胞的整合功能和分子概况
CN105063168B (zh) * 2015-07-17 2019-01-18 上海交通大学 检测血液中游离上皮细胞器官来源的装置
CN204989194U (zh) * 2015-07-17 2016-01-20 上海交通大学 一种血液中稀有细胞蛋白组的检测装置
EP3147370A1 (en) * 2015-09-28 2017-03-29 Rheinische Friedrich-Wilhelms-Universität Bonn Exosomal microrna in serum as an indicator for the activation of brown and beige fat tissue (bat)
US11702661B2 (en) * 2016-09-21 2023-07-18 The Broad Institute, Inc. Constructs for continuous monitoring of live cells

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101168717A (zh) * 2006-10-25 2008-04-30 中国科学院大连化学物理研究所 一种用于细胞凋亡研究的集成化微流控芯片及其应用
CN101748059A (zh) * 2008-12-08 2010-06-23 中国科学院大连化学物理研究所 一种微流控芯片及研究细胞在三维介质中定向移动的方法
CN104884605A (zh) * 2012-08-24 2015-09-02 耶鲁大学 用于高通量多重检测的系统、装置和方法
CN104760922A (zh) * 2014-01-03 2015-07-08 中国科学院电子学研究所 一种超微平面电极阵列传感器及其制备方法
CN206787889U (zh) * 2017-05-05 2017-12-22 中国科学院半导体研究所 一种分离和富集体液成分的装置

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12060607B2 (en) 2009-12-15 2024-08-13 Becton, Dickinson And Company Digital counting of individual molecules by stochastic attachment of diverse labels
US11993814B2 (en) 2009-12-15 2024-05-28 Becton, Dickinson And Company Digital counting of individual molecules by stochastic attachment of diverse labels
US11970737B2 (en) 2009-12-15 2024-04-30 Becton, Dickinson And Company Digital counting of individual molecules by stochastic attachment of diverse labels
US11634708B2 (en) 2012-02-27 2023-04-25 Becton, Dickinson And Company Compositions and kits for molecular counting
US11702706B2 (en) 2013-08-28 2023-07-18 Becton, Dickinson And Company Massively parallel single cell analysis
US11618929B2 (en) 2013-08-28 2023-04-04 Becton, Dickinson And Company Massively parallel single cell analysis
US11535882B2 (en) 2015-03-30 2022-12-27 Becton, Dickinson And Company Methods and compositions for combinatorial barcoding
US11845986B2 (en) 2016-05-25 2023-12-19 Becton, Dickinson And Company Normalization of nucleic acid libraries
US11525157B2 (en) 2016-05-31 2022-12-13 Becton, Dickinson And Company Error correction in amplification of samples
US11782059B2 (en) 2016-09-26 2023-10-10 Becton, Dickinson And Company Measurement of protein expression using reagents with barcoded oligonucleotide sequences
US12084712B2 (en) 2017-06-05 2024-09-10 Becton, Dickinson And Company Sample indexing for single cells
US11773441B2 (en) 2018-05-03 2023-10-03 Becton, Dickinson And Company High throughput multiomics sample analysis
US11639517B2 (en) 2018-10-01 2023-05-02 Becton, Dickinson And Company Determining 5′ transcript sequences
US11932849B2 (en) 2018-11-08 2024-03-19 Becton, Dickinson And Company Whole transcriptome analysis of single cells using random priming
US11492660B2 (en) 2018-12-13 2022-11-08 Becton, Dickinson And Company Selective extension in single cell whole transcriptome analysis
US11661631B2 (en) 2019-01-23 2023-05-30 Becton, Dickinson And Company Oligonucleotides associated with antibodies
US12071617B2 (en) 2019-02-14 2024-08-27 Becton, Dickinson And Company Hybrid targeted and whole transcriptome amplification
US11939622B2 (en) 2019-07-22 2024-03-26 Becton, Dickinson And Company Single cell chromatin immunoprecipitation sequencing assay
US11773436B2 (en) 2019-11-08 2023-10-03 Becton, Dickinson And Company Using random priming to obtain full-length V(D)J information for immune repertoire sequencing
CN112834754A (zh) * 2019-11-22 2021-05-25 中国科学院大连化学物理研究所 一种便携高通量单细胞外囊泡检测装置及可视化检测方法
CN112834754B (zh) * 2019-11-22 2022-06-03 中国科学院大连化学物理研究所 一种便携高通量单细胞外囊泡检测装置及可视化检测方法
US11649497B2 (en) 2020-01-13 2023-05-16 Becton, Dickinson And Company Methods and compositions for quantitation of proteins and RNA
US11661625B2 (en) 2020-05-14 2023-05-30 Becton, Dickinson And Company Primers for immune repertoire profiling
US11932901B2 (en) 2020-07-13 2024-03-19 Becton, Dickinson And Company Target enrichment using nucleic acid probes for scRNAseq
US11739443B2 (en) 2020-11-20 2023-08-29 Becton, Dickinson And Company Profiling of highly expressed and lowly expressed proteins
WO2022125701A1 (en) * 2020-12-09 2022-06-16 Becton, Dickinson And Company Multiplexed single cell immunoassay

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