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Method for high-throughput analysis of single cell inclusions by using paired micro-fluidic chip

A technology of microfluidic chips and inclusions, applied in the field of molecular and cell biology, can solve the problems that high-throughput analysis is not involved, single-cell inclusions cannot be analyzed, effective analysis targets are reduced, and the occupancy rate is achieved. High, high pairing efficiency, high-throughput effect

Active Publication Date: 2017-08-04
HANGZHOU WEIZHU BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The capture of cells and microspheres in this method is based on the principle of Poisson distribution. Most of the droplets have no cells, and only ~1% of the droplets contain single cells. Combined with the Poisson distribution of microspheres, the effective analysis target is further reduced. Only a small number of cells in a large number of actual samples can be analyzed, which may ignore some important individual cells in the sample
In addition, this strategy is only suitable for samples with a large number of analysis objects. For some rare cells (such as circulating tumor cells), the number of cells in the sample is too small (10-100 / mL blood), and this method cannot be used to achieve single-cell analysis.
These techniques are limited to the analysis of single-cell mRNA, and other single-cell contents cannot be analyzed, such as genome, miRNA, proteome, methylated DNA, metabolites, liposomes, phospholipids, etc.
None of the currently disclosed technologies involve high-throughput analysis

Method used

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  • Method for high-throughput analysis of single cell inclusions by using paired micro-fluidic chip
  • Method for high-throughput analysis of single cell inclusions by using paired micro-fluidic chip
  • Method for high-throughput analysis of single cell inclusions by using paired micro-fluidic chip

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Such as Figure 1 to Figure 3 As shown, the present invention is used for high-throughput paired capture of single microspheres and single cells. The chip is processed by standard soft lithography technology and includes three parts: a capture layer, a control layer and a slide. The capture layer is composed of three parts: cell flow channel (10), microsphere flow channel (11) and connection channel (13), and the control layer is composed of isolation channel (12). Wherein the cell flow path contains a plurality of cell capture units (8) connected in series, and each unit consists of three parts: a flow path (10), a liquid storage chamber (14), and a capture channel (16); see image 3 , the flow path (10) comprises a U-shaped tube, the left arm end of the U-shaped tube of the previous unit is connected to the right arm end of the U-shaped tube of the next unit, the connecting part between the U-shaped tubes is a straight tube, and the liquid storage chamber The chamber...

Embodiment 2

[0062] High-throughput single-cell transcriptome analysis

[0063]a. Single cell capture: Fill the isolation pump inlet (3) with solution, increase the pressure of the syringe pump, the isolation pump (12) deforms, and the diaphragm (18) squeezes the uppermost layer of the connecting channel until the connecting channel (13) is completely blocked Open, pass through the cell A549 suspension from the channel inlet (1) with a syringe pump, when the single cell enters the capture unit (8), because the path that the fluid passes through from the flow channel (10) is greater than from the liquid storage chamber (14), The cells will enter the liquid storage chamber (14) first, because the capture channel (16) is smaller than the cells, the cells are stuck in front of the capture channel (16), and at the same time block the capture channel (16), and flow through the liquid storage chamber (14) The fluid flow rate approaches zero, and at this time, subsequent cells cannot enter the liq...

Embodiment 3

[0087] Example 3: Single-cell genome analysis

[0088] High-throughput single-cell genome analysis

[0089] a. Single cell capture: Fill the isolation pump inlet (3) with solution, increase the pressure of the syringe pump, the isolation pump (12) deforms, and the diaphragm (18) squeezes the uppermost layer of the connecting channel until the connecting channel (13) is completely blocked Open, pass through the cell A549 suspension from the channel inlet (1) with a syringe pump, when the single cell enters the capture unit (8), because the path that the fluid passes through from the flow channel (10) is greater than from the liquid storage chamber (14), The cells will enter the liquid storage chamber (14) first, because the capture channel (16) is smaller than the cells, the cells are stuck in front of the capture channel (16), and at the same time block the capture channel (16), and flow through the liquid storage chamber (14) The fluid flow rate approaches zero, and at this ...

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Abstract

The invention relates to a method for high-throughput analysis of single cell inclusions by using a paired micro-fluidic chip. The paired micro-fluidic chip is the micro-fluidic chip for high-throughput capturing single microspheres / single cells. The method comprises the steps of (1) carrying out high-throughput pairing capturing on single microspheres / single cells; (b) carrying out parallel control on a pairing unit, for example, isolation, cultivation and pyrolysis of a lot of single cells, and capturing of various inclusions in the single cells; and (c) converting information of the single cell inclusions into DNA sequence information through encoding the microspheres, analyzing a lot of single cell inclusions, such as a transcriptome, a genome, an miRNA, a proteome, a methylated DNA, a metabolite, a lipidosome and phospholipid by adopting a high-throughput sequencing technology and a bioinformatics method. According to the method, the single cell inclusion information can be comprehensively and completely analyzed, and the method has the advantages of high throughput, high accuracy, low cost and wide analyzable target.

Description

technical field [0001] The invention relates to molecular cell biology, in particular to a technology for high-throughput analysis of single-cell content by using paired microfluidic chips. Background technique [0002] Analysis of cell contents is the basis of molecular biology research and the analysis target of modern medical diagnosis. The traditional method of analyzing cell content analyzes and processes the average signal of a large number of cells, which makes the average signal blur people's understanding of the heterogeneity between single cells. Current cutting-edge biological research and medical research have shown that heterogeneity exists between different tissues of the same individual, between different parts of the same tissue, and between different cells in the same part. There may also be great heterogeneity between individuals, and these cells with the greatest differences can often reveal important biological phenomena or suggest major diseases such as...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/02
CPCC12Q1/6834C12Q1/6869G01N33/5005C12Q2563/149C12Q2535/122
Inventor 杨朝勇邹远张明霞朱志
Owner HANGZHOU WEIZHU BIOLOGICAL TECH CO LTD
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