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Exosome detecting method based on adapter and rolling ring amplification

A technology of rolling circle amplification and detection method, which is applied in measuring devices, material analysis through electromagnetic means, instruments, etc., can solve the problems of low sensitivity, high background signal, detection interference, etc., and achieve the effect of improving detection sensitivity

Inactive Publication Date: 2019-04-19
NANJING DRUM TOWER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention solves the problem of low sensitivity in the method of using exosome-specific antibodies or aptamers as recognition probes due to the interference of detection by exosomes produced by normal cells and high background signal

Method used

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  • Exosome detecting method based on adapter and rolling ring amplification
  • Exosome detecting method based on adapter and rolling ring amplification
  • Exosome detecting method based on adapter and rolling ring amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Preparation of circular templates and rolling circle amplification products.

[0032] 1. Hybridize the phosphorylated padlock probe (100 μM) and 1 μL ligation probe (100 μM) in T4 ligase buffer; add 60 U of T4 DNA ligase, and incubate the mixture overnight at 16°C; keep T4 ligase at 65°C Inactivate for 15 minutes, add exonuclease I 20U, exonuclease III 50U to digest the remaining single-stranded DNA and double-stranded DNA; the enzyme is denatured by heating at 85°C for 10 minutes.

[0033] The sequence of the padlock probe and connection probe is as follows:

[0034] Padlock Probe:

[0035] 5’-TAGCACGGACATTTTCCCAACCCGCCCTACCCCTTTTTTTTTTCCCAACCCGCCCTACCCTTTTGTAACTGTTTCCTTC-3’

[0036] Ligation probe: 5'-TGTCCGTGCTAGAAGGAAACAGTTAC-3'

[0037] 2. Add 1μL phi29DNA polymerase buffer, 0.5μL 10mM dNTPs, 0.2μL 0.1mg / mL BSA, 10U phi29DNA polymerase, 10μL 100nM aptamer-primer probe and 10μL 120nM circular template, incubate the electrode at 37℃ 2 hours.

[0038] ...

Embodiment 2

[0041] Example 2: Modifying antibodies on the surface of gold electrodes and using them to capture gastric cancer exosomes and start rolling circle amplification.

[0042] 1. The gold electrode was polished with 0.05mm alumina slurry, rinsed with ultrapure water, and dried at room temperature; the pretreated gold electrode was soaked in PBS solution containing 15mM cysteamine overnight to generate the self-activation of cysteamine. Assemble the monolayer; soak the gold electrode in 5% glutaraldehyde aqueous solution and react at 37°C for 6 hours; incubate the gold electrode with 10 μL 20 μg / mL anti-CD63 antibody at 37°C for 1 hour; Incubate in BSA solution for 1 hour at 37°C, rinse with ultrapure water.

[0043] 2. Incubate the antibody-modified electrode with the gastric cancer exosome solution resuspended in PBS for 1 hour at 37°C, and wash the electrode with PBS solution; modify 10 μL of 100 nM aptamer-primer probe to the surface of the electrode, and incubate at 37°C 1 ho...

Embodiment 3

[0045] Embodiment 3: Electrode working process

[0046] 1. Prepare the gold electrode of the modified antibody: (1) the gold electrode is polished with 0.05mm alumina slurry, the electrode is rinsed with ultrapure water, and dried at room temperature; Soak in PBS solution overnight to produce a self-assembled monolayer of cysteamine; (3) soak the gold electrode in 5% glutaraldehyde aqueous solution and react at 37°C for 6 hours; (4) mix the gold electrode with 10 μL 20 μg / mL The anti-CD63 antibody was incubated at 37°C for 1 hour; (5) The electrode was soaked in 5 mg / mL BSA solution, incubated at 37°C for 1 hour, and rinsed with ultrapure water.

[0047] 2. Circular template preparation: (1) Phosphorylated padlock probe (100 μM) and 1 μL ligation probe (100 μM) were hybridized in T4 ligase buffer; (2) 60 U T4 DNA ligase was added, and the mixture was incubated at 16 Incubate overnight at ℃; (3) Keep T4 ligase at 65℃ for 15 minutes to inactivate, add exonuclease I 20U, exonucl...

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Abstract

The invention relates to an exosome detecting method based on adapter and rolling ring amplification. An electrochemical sensor method is applied for constructing an adapter probe based on exosome singularity. A signal is generated by means of H2O2 reaction catalysis by G-tetrad-Hemin simulated peroxidase. A large number of G-tetrads are synthesized through rolling ring amplification for performing signal amplification. The method realizes quantitative detection for the exosomes and is used for detecting an actual serum sample. According to the method of the invention, the adapter probe basedon the exosome singularity is constructed through applying an electrochemical sensor method; the signal is generated through H2O2 catalysis by the G-tetrad-Hemin simulated peroxidase; and furthermorea large number of G-tetrads are synthesized through rolling ring amplification for realizing signal amplification, thereby greatly improving detecting sensitivity of the exosome.

Description

technical field [0001] The invention relates to the fields of biochemical detection and molecular diagnosis, in particular to an exosome detection method based on aptamers and rolling circle amplification. Background technique [0002] Exosomes are bilayer membranous vesicles secreted by various types of cells, with a diameter of 30-100 nm, which contain cell-specific proteins, lipids, nucleic acids and other components. Exosomes can be effectively detected in almost all types of human body fluids, including peripheral blood, saliva, urine, etc. The important signaling molecules carried and transmitted by exosomes form a new intercellular information transmission system, which affects the physiological state of cells and is closely related to the occurrence and process of various diseases. For example, exosomes secreted by tumor cells can enter Capillaries in the lymphatic system and tumor tissue play a role in the regulation of malignant tumors. In addition, the character...

Claims

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Application Information

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IPC IPC(8): G01N27/327
CPCG01N27/3275
Inventor 李智洋何农跃黄蓉蓉
Owner NANJING DRUM TOWER HOSPITAL
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