Method for identifying CD44 and CD24 molecular phenotypes

A CD24, CD44 technology, applied in the direction of analysis materials, measuring devices, instruments, etc., can solve the problems of large amount of data in double-color flow cytometry analysis, increase the workload and difficulty of data analysis, and dope with wrong information, etc., and meet the technical level requirements Low cost, easy operation, and small amount of data analysis

Active Publication Date: 2021-04-30
章毅 +7
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the amount of data generated by two-color flow cytometry analysis is large, and the probability of misinformation is correspondingly increased, which greatly increases the workload and difficulty of data analysis

Method used

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  • Method for identifying CD44 and CD24 molecular phenotypes
  • Method for identifying CD44 and CD24 molecular phenotypes
  • Method for identifying CD44 and CD24 molecular phenotypes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Electrochemical signal labeling of breast cancer stem cells based on CD44 antibody-nucleic acid conjugates

[0058] (a) Take 20 μL of DNA probes A and A at a concentration of 50 μM 1 Put it in a microtube, mix it evenly and put it under the condition of 25 ℃ for 2 hours to get AA 1 Double strand solution.

[0059] (b) Take 10 μL of the AA prepared in step (a) 1 The double-strand solution was placed in a microtube, and then 10 μL of 30 mM ascorbic acid solution, 10 μL of 2 mM copper sulfate solution, and 70 μL of 3-(N-morpholino)propanesulfonic acid (MOPS) buffer ( Containing 20mM MOPS, 300mM NaCl, pH 7.5), reacted at 25°C for 15 minutes, so that A 1 The polycytosine sequence at the 5' end of the chain (T 30 ) as a template to synthesize copper nanoparticles that can be used as electrochemical signal probes.

[0060] (c) Dissolve 5 μL of CD44 antibody with a concentration of 1 μM and 5 μL of Tetrazine-PEG5-NHS with a concentration of 5 μM in 100 μL of phos...

Embodiment 2

[0067] Electrochemical analysis of embodiment 2 breast cancer stem cells

[0068] (a) Take 20 μL of DNA probes A and A at a concentration of 50 μM 1 Put it in a microtube, mix it evenly and put it under the condition of 25 ℃ for 2 hours to get AA 1 Double-stranded solution; take 20 μL of DNA probes B and B at a concentration of 50 μM 1 Put it in a microtube, mix it evenly and put it at 25°C for 2 hours to get BB 1 Double-strand solution: Take 20 μL of DNA probes T and F with a concentration of 100 μM and place them in microtubes respectively to obtain T-strand and F-strand solutions.

[0069] (b) Take 10 μL of the AA prepared in step (a) 1 The double-strand solution was placed in a microtube, and then 10 μL of 30 mM ascorbic acid solution, 10 μL of 2 mM copper sulfate solution, and 70 μL of 3-(N-morpholino)propanesulfonic acid (MOPS) buffer ( Containing 20mM MOPS, 300mM NaCl, pH 7.5), reacted at 25°C for 15 minutes, so that A 1 The polycytosine sequence at the 5' end of t...

Embodiment 3

[0083] Electrochemical analysis of breast cancer stem cells under the coexistence condition of interference cells in embodiment 3

[0084] In order to examine the specificity and anti-interference ability of this technology for the analysis of breast cancer stem cells, we divided 5×10 5 pcs, 5×10 4 and 5×10 3 breast cancer stem cells with 5×10 5 Three different molecular phenotypes (CD44 阳 CD24 阳 , CD44 阴 CD24 阳 , CD44 阴 CD24 阴 ) of interfering cells were mixed, and electrochemical analysis was carried out according to the steps in Example 2. The result is as Figure 5 As shown, the presence of three different molecular phenotypes of interfering cells will not cause changes in the peak current value of the electrochemical response of breast cancer stem cells, indicating that this technology uses antibody-nucleic acid conjugate-based selective electrochemical signal labeling and The background signal erasing strategy based on programmable DNA circuit can effectively e...

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Abstract

A method for identifying CD44 and CD24 molecular phenotypes comprises a first antibody-nucleic acid conjugate specifically binding to CD44 protein on the surface of a cell, a second antibody-nucleic acid conjugate specifically binding to CD24 protein on the surface of the cell, a DNA single-stranded T chain and a DNA single-stranded F chain, wherein the T chain is complementarily bound to the nucleic acid chain of the first antibody-nucleic acid conjugate. The F chain and the nucleic acid chain of the second antibody-nucleic acid conjugate are complementarily combined to form a programmed DNA loop together, the signal marker on the surface of the CD44 positive CD24 positive phenotypic cell is erased, and the signal probe marked to the cell is retained. The method provided by the invention realizes recognition and effective distinguishing of CD44 and CD24 molecular phenotype cells, and has the characteristics of simple operation, accurate result, low technical level requirement, small data analysis amount and the like.

Description

technical field [0001] The invention relates to a method for separating cells according to molecular phenotypes, in particular to a method for identifying CD44 and CD24 molecular phenotypes. Background technique [0002] In 2003, A 1 -Hajj et al first isolated CD44 from primary and metastatic breast cancer tissue according to the expression level of cell surface proteins 阳 CD24 阴 cell subgroups. This subpopulation differentiates slower than other breast cancer cells, but has stronger tumorigenic ability, and has similar self-renewal, proliferation and differentiation abilities as normal stem cells, so it is called breast cancer stem cells. Studies in recent years have shown that breast cancer stem cells can maintain the vitality of breast cancer cell groups through self-renewal and unlimited proliferation, and their migration and differentiation capabilities make the recurrence and metastasis of breast cancer possible, which is the key to the occurrence, development and m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/569
CPCG01N33/57415G01N33/56966
Inventor 章毅伍婷赵婧陈亮曹亚
Owner 章毅
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