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High-throughput single-cell proteome analysis and transcriptome combined analysis method

A single-cell protein and combined analysis technology, which is applied in the field of high-throughput single-cell proteome analysis and its combined analysis with transcriptome, can solve the problems of difficult cell fusion, residual oil phase in the chip channel, and low recovery efficiency. Improve the reaction efficiency, reduce the loss of raw materials, and ensure the effect of matching rate

Pending Publication Date: 2021-11-02
厦门德运芯准科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the natural incompatibility of oil and water, the use of oil phase often leads to unstable formation of oil-water droplets, difficulty in subsequent cell fusion, lysis, residual oil phase in the chip channel, blockage, making it difficult to reuse the chip, oil-water droplets A series of problems such as poor encapsulation and demulsification effect, high cost, and low recovery efficiency make it difficult to realize high-throughput and multi-reaction integration on the chip at the same time

Method used

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  • High-throughput single-cell proteome analysis and transcriptome combined analysis method
  • High-throughput single-cell proteome analysis and transcriptome combined analysis method
  • High-throughput single-cell proteome analysis and transcriptome combined analysis method

Examples

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Embodiment 1

[0092] Based on the method of using the microfluidic chip of the present invention, high-throughput paired capture of single particles is realized. The chip used is such as Figure 1-2 As shown, the chip is processed by standard soft lithography technology, including capture layer, control layer and slide. The capture layer includes two sets of particle capture flow channels arranged in parallel and axisymmetrically. A set of particle capture channels of the capture layer is provided with a cell inlet (1), a buffer inlet (3), a cell gas inlet (4), and a cell outlet (6) for fluid to pass through, and the other set of particle capture channels is provided with Microsphere inlet (2), microsphere gas inlet (5) and microsphere outlet (7). Wherein the cell inlet (1), the buffer inlet (3), and the microsphere inlet (2) are connected to the chip through one side opening of the microparticle capture flow channel, and the cell gas inlet (4), the microsphere gas inlet (5 ), the cell ou...

Embodiment 2

[0100] All reagents used in the following examples are commercially available in the art.

[0101] Exemplary sources of reagents are: anti-EpCAM antibody [VU-1D9] (biotin) purchased from Abcam Company, Cat. No. ab79079; anti-ErbB2 Molecule (biotin) was purchased from Abcam Company, catalog number ab31890; anti-EGFR antibody [EGFR1] (biotin) was purchased from Abcam Company, catalog number ab24293; biotin anti-mouse CD326 (Ep-CAM) antibody was purchased from Biolegend Company, Cat. No. 118203; human IgG control was purchased from Genescript Company, Cat. No. A01006; human colon cancer cell SW480, mouse embryonic stem cell mES, human breast cancer cell MCF7, human breast cancer cell SK-BR-3, human breast cancer cell MDA-MB- 231, all from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences; 0.5% F68 is Pluronic F-68 nonionic surfactant, purchased from Thermo Fisher, Cat. No.: #24040032.

[0102] Schematic diagram of the structure of high-th...

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Abstract

The invention relates to a high-throughput single-cell proteome analysis and transcriptome combined analysis method. The method comprises the following steps: respectively capturing bar code antibody labeled cells and coding microspheres by using a micro-fluidic chip, and introducing gas into the chip, so that the cells and the coding microspheres are respectively wrapped in single liquid drops to respectively form independent reaction units; pairing the cells and the coding microspheres, splitting the cells, and bar code antibodies on the surfaces of the cells and mRNA in the cells being captured by the coding microspheres; sequentially carrying out reverse transcription reaction and excision enzyme treatment reaction on the paired coding microspheres, then introducing gas into the chip, and collecting the coding microspheres; and amplifying the collected microspheres, separating nucleic acid fragments of 100 to 300bp and / or more than 300bp, and sequencing. According to the method, an efficient, rapid, high-throughput and low-cost single-cell proteome and transcriptome combined analysis platform can be realized at the same time.

Description

technical field [0001] The invention relates to the field of microfluidic chips, in particular to a high-throughput single-cell proteome analysis method and its joint analysis method with transcriptome. Background technique [0002] Cells are the basic units that constitute the morphological structure and physiological functions of organisms. Because the process of cell proliferation, differentiation and metabolism is affected by the internal life activities of the cell and the surrounding microenvironment, there is heterogeneity between different cells, even two cells with the same genetic information, the composition and content of the substances in the cell will also vary. There is a large difference between them, resulting in heterogeneity in cell size, chemical composition, biological activity, physiological response objects, and response time. With the great development of amplification technology, sequencing technology and imaging technology, the analysis of single c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/53C12M1/34C12M1/04C12M1/00
CPCG01N33/68G01N33/5308C12Q1/6869C12Q2563/131
Inventor 杨朝勇许醒张明霞邹远张倩倩蔡林峰
Owner 厦门德运芯准科技有限公司
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