Single-cell protein quantitative analysis method based on electrophoresis technology

A technology for quantitative analysis and single-cell separation, which is applied in the field of single-cell protein quantitative analysis based on electrophoresis technology. Linear range, wide linear detection range, and the effect of improving efficiency and success rate

Pending Publication Date: 2021-07-30
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The disadvantage of fluorescence color development is mainly that the photochemical stability of fluorescent molecules is relatively poor, and the fluorescent signal is easy to bleach and quench, which makes it difficult to guarantee the accuracy of the detection results; in addition, the sensitivity of fluorescence detection is not very high; on the other hand, due to nitrocellulose The microstructure of the plain film is a disordered porous structure, and the size of the pores is about microns. Therefore, the film has a strong scattering of light, the whole film appears white, the light transmittance is poor, and the scattering is strong. cause a certain impact, and to a certain extent also limit the improvement of sensitivity

Method used

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  • Single-cell protein quantitative analysis method based on electrophoresis technology
  • Single-cell protein quantitative analysis method based on electrophoresis technology
  • Single-cell protein quantitative analysis method based on electrophoresis technology

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Embodiment 1

[0048] An electrophoresis-based single-cell protein quantitative analysis method provided by the present invention is applied to the identification and analysis of circulating tumor cells, including the following steps:

[0049] 1) Through the microwell array chip composed of single cell separation chips, the single cells of circulating tumor cells are physically separated, and the single cells are lysed in situ for electrophoretic separation of proteins. The preparation process of the microwell array chip is as follows:

[0050] ① The photoresist is evenly spin-coated on the silicon wafer, and the photoresist is cured by ultraviolet light;

[0051] ② Form the photoresist microcolumn array by electron beam etching, and use it as the template for the preparation of the single cell separation chip. The cylinder of the microcolumn array has a height of 30 μm and a diameter of 20 μm; the length of the template is 1 mm and the width is 500 μm;

[0052] ③ Cover the template with a ...

Embodiment 2

[0063] A single-cell protein quantitative analysis method based on electrophoresis technology provided by the present invention is also applied to breast cancer cell typing analysis, including the following steps:

[0064] 1) Through the microwell array chip composed of single cell separation chips, the single cells of breast cancer cells are physically separated, and the single cells are lysed in situ for electrophoretic separation of proteins. The preparation of the microwell array chip is as follows:

[0065] ① The photoresist is evenly spin-coated on the copper sheet, and the photoresist is cured by ultraviolet light;

[0066] ② Form the photoresist microcolumn array by ion beam etching, and use it as a template for the preparation of the single-cell separation chip. The height of the cylinder of the microcolumn array is 50 μm, and the diameter is 40 μm; the length of the template is 5 cm, and the width is 4.5 cm;

[0067] ③Cover the template with a quartz glass sheet, an...

Embodiment 3

[0078] A single-cell protein quantitative analysis method based on electrophoresis technology provided by the present invention is applied to oocyte protein analysis, including the following steps:

[0079] 1) Through the microwell array chip composed of single cell separation chips, single cells of circulating tumor cells are physically separated, and the single cells are lysed in situ for electrophoretic separation of proteins. The preparation of the microwell array chip is as follows:

[0080] ① The photoresist is evenly spin-coated on the steel sheet, and the photoresist is cured by ultraviolet light;

[0081] ② Form a photoresist microcolumn array by X-ray etching, and use it as a template for the preparation of a single-cell separation chip. The height of the cylinder of the microcolumn array is 120 μm, and the diameter is 100 μm; the length of the template is 5 cm, and the width is 4.5 cm;

[0082] ③ Cover the template with a polyethylene plastic sheet, and use a pipet...

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Abstract

The invention provides a single-cell protein quantitative analysis method based on an electrophoresis technology, which comprises the following steps: 1) carrying out physical separation through a micropore array chip consisting of single-cell separation chips to obtain single cells, and carrying out in-situ lysis on the single cells to carry out protein electrophoretic separation; 2) transferring a protein strip obtained by electrophoretic separation to an inverse opal structure photonic crystal film; 3) incubating an SERS nano-label developer and the inverse opal structure photonic crystal transfer printing film, and developing protein on a strip through an SERS imaging technology; and 4) performing Raman spectrum analysis, and calculating the concentration of each protein according to the Raman characteristic peak intensity of the nano-label. The method has the beneficial effects that a novel single-cell protein quantitative analysis method is constructed by combining a protein electrophoresis technology, SERS (Surface Enhanced Raman Scattering) and an ordered structure photonic crystal film, and high-sensitivity and wide-linearity quantitative detection requirements can be met at the same time.

Description

technical field [0001] The invention relates to the technical field of single-cell proteomics, in particular to a single-cell protein quantitative analysis method based on electrophoresis technology. Background technique [0002] With the implementation and advancement of the Human Genome Project, life science research has entered the post-genome era. Functional genomics has become the focus of life science research, which mainly includes structural genomics research and proteomics research. Although genomics provides strong and direct evidence of gene activity and disease association, the way genes are expressed is intricate. Under different conditions or at different times, the same gene may express proteins with completely different functions after duplication, transcription and translation. Therefore, in the study of life phenomena, it is far from enough to only understand the structure of the genome, and more in-depth research on proteins, the direct executors of life...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/447G01N21/65B01L3/00
CPCB01L3/5027G01N21/658G01N27/44721
Inventor 刘兵孙斐
Owner NANTONG UNIVERSITY
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