Method for detection of telomerase activity based on strand displacement reaction and DNA-modified gold nanoparticles

A technology of gold nanoparticles and chain replacement reaction, which is applied in the field of biosensing, can solve the problems of non-specific amplification of strong background fluorescence signals, and achieve the effect of low cost, simple operation and high sensitivity

Active Publication Date: 2022-05-17
SUN YAT SEN UNIV
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Problems solved by technology

Although somewhat sensitive, nonspecific amplification with strong background fluorescence signals is unavoidable

Method used

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  • Method for detection of telomerase activity based on strand displacement reaction and DNA-modified gold nanoparticles
  • Method for detection of telomerase activity based on strand displacement reaction and DNA-modified gold nanoparticles
  • Method for detection of telomerase activity based on strand displacement reaction and DNA-modified gold nanoparticles

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Embodiment 1

[0052] A method for detecting telomerase activity in urine based on strand displacement reaction (SDA) and DNA-modified gold nanoparticles, specifically comprising the steps of:

[0053] 1) Extraction of telomerase

[0054] Extraction of telomerase from cells:

[0055] MCF-7 cells were cultured in DMEM medium containing 10% fetal calf serum, 100 μg / ml penicillin and 100 μg / ml streptomycin. After extracting cells with trypsin, 7 × 10 6 cells. The cells were dispersed in 1.5 ml EP tubes, centrifuged at 1000 rpm for 5 min, and washed twice with ice-cold PBS. The extract was then suspended in 200 μL of lysis buffer, incubated on ice for 30 minutes, and then centrifuged at 4° C. and 12,000 rpm for 20 minutes. The supernatant was transferred, diluted, and stored at -80°C until use.

[0056] Extraction of telomerase from urine:

[0057] Collect fresh urine samples and centrifuge at 850rpm for 10 minutes at 4°C, wash once with PBS and centrifuge at 2300rpm at 4°C for 5 minutes, ...

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Abstract

The invention discloses a method for detecting telomerase activity based on strand displacement reaction (SDA) and DNA-modified gold nanoparticles. The method is that when the active telomerase exists, the extended product of its substrate will trigger the SDA reaction of the probe Hairpin and Primer to form a long double-stranded DNA product with single strands at both ends. The SDA product can then be hybridized to a DNA-modified gold nanoparticle (DNA‑AuNP) probe, resulting in aggregation of the gold nanoparticles. The change of dynamic light scattering signal (particle size) of gold nanoparticles is measured to realize highly sensitive quantitative analysis of telomerase activity. The method has the advantages of simple operation, low detection cost, less sample required, high sensitivity and good specificity.

Description

technical field [0001] The invention relates to a method for detecting telomerase activity, belonging to the technical field of biosensing, in particular to a method for detecting telomerase activity based on strand replacement reaction and DNA-modified gold nanoparticles. Background technique [0002] Telomeres are nucleic acids of a continuous repeat sequence (TTAGGG) located at the ends of chromosomes. Telomerase is a ribonucleoprotein complex that can maintain telomere length by adding TTAGGG repeats at the end of telomeres through its own inherent RNA template and reverse transcriptase and related proteins. Telomerase activity is inhibited in most normal human tissues, and telomere length decreases with cell cycle division, leading to cell aging and death. However, telomerase activity is activated and expressed in more than 85% of malignant tumor cells, so that cancer cells can continue to proliferate, so telomerase is considered to be a biomarker and therapeutic targe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/48G01N15/02
CPCG01N15/00G01N15/0205C12Q1/48G01N2015/0065
Inventor 凌连生王京李婷婷沈瑞迪
Owner SUN YAT SEN UNIV
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