Method for simultaneously detecting protein, RNA and exosome membrane protein in exosome

A technology of exosomes and internal proteins, which is applied in the field of exosome membrane proteins and simultaneously detects exosomes and internal proteins and RNA, can solve the problems that have not been reported, and achieve the effect of improving specificity and sensitivity

Pending Publication Date: 2020-04-03
HANGZHOU DIXIANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] As one of the three major fields of liquid biopsy, exosomes have many potential biological targets, but there are no reports on detection systems that can simultaneously target trace amounts of nucleic acids and proteins, especially in situ detection of proteins contained in exosomes

Method used

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  • Method for simultaneously detecting protein, RNA and exosome membrane protein in exosome
  • Method for simultaneously detecting protein, RNA and exosome membrane protein in exosome
  • Method for simultaneously detecting protein, RNA and exosome membrane protein in exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Chip processing for in situ capture of exosomes

[0035] 1) Use ultra-thin (thickness 0.17-0.19mm), ultra-low fluorescence, ultra-high light transmittance, geometric mean roughness (RMS roughness) <100nm special glass, use high-purity electronic grade alcohol, at 24-200 After repeated cleaning under clean conditions at ℃, blow dry with high-purity nitrogen;

[0036] 2) After repeated washing with piranha solution, keep it clean and dry;

[0037] 3) The glass surface and organosiloxane with functional groups (Organosiloxane with functional groups) are placed in a vacuum container, the vacuum must be <10mmHg, and vapor deposition is carried out at 24-180°C for 6-24 hours;

[0038] 4) Mix the appropriate amount of avidin and biotin at a ratio of 1:7-1:30, mix at a low speed, let it stand for 30-60 minutes, and use PBS, BupH TM Phosphate Buffered Saline and ultrapure water (approximately 18 megohm) to prepare buffer, 0.01-1% Tween 20 repeatedly washed and place...

Embodiment 2

[0041] Example 2: Nanoparticle preparation and antibody coating

[0042] 1) DOTMA (trimethyl-2,3-dioleyloxypropyl ammonium chloride), DSPE-PEG-2000 (1,2-distearoyl-SN-glycerol-3-phosphoethanolamine- Polyethylene glycol 2000) and cholesterol are thoroughly mixed according to 3:1:4, ultrasonicated for 5 minutes, and left at room temperature for 2 hours to obtain stable cationic liposomes;

[0043] 2) Put an appropriate amount of molecular beacons, fluorescent monoclonal antibodies (RNA molecular beacons and antibodies can be mixed and encapsulated with nanoparticles at a molar ratio of 1:2 to 1:5 on the basis of excess) and the above cationic liposomes in PBS After fully blowing and mixing, ultrasonication for 5 minutes, the molecular beacon is that the 5' end stem and loop are completely complementary to the target gene, the 3' end stem is partially complementary to the 5' end stem, and the 5' end and 3' end are respectively separated by fluorescent groups. Group and quenching...

Embodiment 3

[0045] Example 3: A method for simultaneous detection of exosome internal protein and RNA, and exosome membrane protein

[0046] 1) Take the crude extracellular vesicles obtained by the precipitation method, and use the exosome in situ capture chip (prepared by the method in Example 1) under a DC electric field of 15-90V to directly capture the exosomes in the sample;

[0047] 2) Binding the target antibody to the exosome membrane captured in step 1), through the combination of the antigen and the antibody, a labeled type A fluorescent signal is emitted, and the corresponding membrane protein is detected;

[0048] 3) Take the RNA molecular beacon and fluorescent monoclonal antibody nanoparticles (prepared by the method in Example 2) that wrap the target to be detected;

[0049] 4) Fusion the encapsulated RNA molecular beacon and fluorescent monoclonal antibody nanoparticles obtained in step 3) with the exosomes captured in step 1), the RNA molecular beacon and fluorescent mono...

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Abstract

The invention discloses a method for simultaneously detecting protein, RNA and exosome membrane protein in exosome, and belongs to the technical field of exosome detection. The method comprises the following steps: capturing exosome in a sample; detecting the corresponding membrane protein; preparing nano particles; fusing the nanoparticles with the exosome; photographing and imaging by a total internal reflection fluorescence microscope, and analyzing and processing data. Verification is carried out in the aspects of exosome characterization detection, cell supernatant exosome membrane protein and mRNA expression comparison experiments, human plasma exosome specificity detection, human plasma exosome membrane protein comparison experiments and detection of various human plasma exosome intracellular proteins, RNA and membrane proteins. Compared with an existing recognized exosome detection technology, a comparison experiment shows that the same marker detection result of the technologyfor simultaneously detecting the same sample is consistent with that of the recognized technology, and the technology can be used for simultaneously detecting protein, RNA and exosome membrane protein in the exosome and judging the sample difference.

Description

technical field [0001] The invention belongs to the technical field of exosome detection, and in particular relates to a method for simultaneously detecting exosome internal protein, RNA, and exosome membrane protein. Background technique [0002] Exosomes (exosomes) are tiny membrane vesicles that can be secreted by most cells, with a diameter of about 30-150nm. They have a lipid bilayer membrane structure and can well protect their coated substances. The tiny membrane vesicles contain specific proteins, nucleic acids and lipids derived from host cells, which can be transmitted to other cells as signal molecules, and are an important medium for communication between cells, enabling recipient cells to perform various biological functions. Change. All cells can produce exosomes, but the components and contents of exosomes secreted by different cells are different. Specific gene products are selectively loaded into exosomes, and biologically active molecules are transferred b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/569G01N33/53
CPCG01N33/5308G01N33/56966G01N33/577G01N33/68
Inventor 曾恒山
Owner HANGZHOU DIXIANG CO LTD
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