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Paper-based detection method for exosome

A detection method and technology of exosomes, which are used in the determination/inspection of microorganisms, biochemical equipment and methods, etc.

Active Publication Date: 2021-04-06
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with antibodies, nucleic acid aptamers have the advantages of stable chemical properties, easy preparation, easy modification, and easy combination with nucleic acid amplification technology for signal amplification. However, there is no report on the use of nucleic acid LFB for exosome detection.

Method used

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  • Paper-based detection method for exosome
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  • Paper-based detection method for exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Establishment of a paper-based detection method for exosomes

[0064] 1. Experimental materials

[0065] 20×SSC, Tris-HCl, sodium phosphate dodecahydrate, bovine serum albumin (BSA), Tween-20, sucrose and TMB chromogenic solution, etc.

[0066] The sequence design is as follows (SEQ ID NO:1-4):

[0067]

[0068] 2. LFB detection and verification of single-stranded DNA targets

[0069] Dilute the single-stranded DNA target 2-fold concentration gradient, add 50 µL system buffer and 1 µL single-stranded DNA target solution to the sample pool, and drop-coat 1 µL PtAu-NiO on the superposition of the test paper sample pad and the binding pad 2 After the nanozyme signal probe, insert the test paper sample pad into the sample pool, wait for 3 minutes, and observe the result with the naked eye. When both the C line and the T line are black, it is determined to be positive. The color of the T line becomes lighter as the concentration of the ssDNA target decreases,...

Embodiment 2

[0078] Example 2 Practical investigation of detection method

[0079] According to the detection method constructed in Example 1, exosomes extracted from equal volumes of HepG2 and L02 cell supernatants were detected. LFB can effectively distinguish tumor exosomes from non-tumor exosomes ( Image 6 ).

Embodiment 3

[0080] Embodiment 3 Amplification effect verification of detection method

[0081] After conversion and amplification of exosomes, the gray pixel area of ​​each T line in the LFB detection results was substituted into the gray pixel area of ​​the T line obtained by LFB detection after 2-fold gradient dilution of the single-stranded DNA target to perform standard curve fitting. Formula: y =14.829e 0.0005x In , the concentration of the single-stranded DNA target after exosome conversion and amplification was obtained. The exosome concentration was converted to the concentration of the substance and compared with the single-stranded DNA target. The molecular magnification of exosomes by this method is 1000 times (Table 1).

[0082] Table 1 Molecular magnification of exosomes after target conversion and amplification

[0083]

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Abstract

The invention provides a paper-based detection method for exosome. The paper-based detection method employs (1) a recognition and molecular transformation system, (2) an amplification reaction system and (3) an LFB detection and analysis system. According to the invention, the exosome is converted into a single-stranded DNA target; the single-stranded DNA is amplified by a nucleic acid amplification technology; and the amplified single-stranded DNA is further detected by using lateral flow chromatography test paper which uses PtAu-NiO2 nano-enzyme as a signal probe. The nucleic acid lateral flow chromatography test paper is used as a terminal detection platform, so the problem that the exosome cannot be qualitatively and quantitatively detected at the same time is solved, and simple, convenient and low-cost detection of the exosome is realized.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a paper-based detection method for exosomes. Background technique [0002] The nanoscale particle size and ultra-trace concentration of exosomes in body fluids pose great challenges for their identification and analysis. Existing analysis methods not only rely on large and expensive instruments, but also require complex pretreatment processes and professional operators. In addition, traditional exosome detection methods cannot be qualitative and quantitative at the same time. Therefore, the development of fast, simple, low-cost, and highly sensitive exosome detection methods is the key to the rapid development and popularization of clinical disease diagnosis and treatment methods using exosomes as biomarkers. [0003] Lateral Flow Chromatography (LFB) is a sensitive, cheap and simple visual rapid detection method. Early LFBs were based on immune recogni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2525/205C12Q2563/155C12Q2565/625C12Q2565/519C12Q2545/114C12Q2563/137
Inventor 许文涛黄昆仑王馨娴李舒婷程楠朱龙佼杜再慧张博洋
Owner CHINA AGRI UNIV
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