Electrochemical sensor construction method based on DNA (deoxyribonucleic acid) dual characters, and telomerase activity detection application thereof

A construction method and activity detection technology, applied in the field of electrochemical biosensing, to achieve the effect of sensitivity and selectivity

Active Publication Date: 2018-12-11
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, polyadenine DNA has not been reported for the detection of telomerase activity

Method used

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  • Electrochemical sensor construction method based on DNA (deoxyribonucleic acid) dual characters, and telomerase activity detection application thereof
  • Electrochemical sensor construction method based on DNA (deoxyribonucleic acid) dual characters, and telomerase activity detection application thereof
  • Electrochemical sensor construction method based on DNA (deoxyribonucleic acid) dual characters, and telomerase activity detection application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] CeO 2 -TiO 2 Preparation and Characterization of Composite Nanorods

[0031] Preparation of CeO 2 -TiO 2 Composite nanorods: Dissolve 1.736g of cerous nitrate hexahydrate, 0.2400g of titanium sulfate and 19.20g of sodium hydroxide in 10mL, 5mL and 65mL of ultrapure water respectively, mix the above solutions and stir for 1h, then reflux in a water bath at 90°C 24h, naturally cooled to room temperature, the product was centrifuged at 8000rpm and washed three times with ultrapure water and absolute ethanol successively, and the collected precipitate was vacuum-dried at 60°C to make a precursor. Mix with water and place in a high-pressure reactor for hydrothermal reaction at 160°C for 12 hours, cool naturally to room temperature, centrifuge the product at 8000rpm and wash it with ultrapure water and absolute ethanol for 3 times, and store the collected precipitate at 60°C Vacuum dried to make CeO 2 -TiO 2 composite nanorods.

[0032] CeO was studied by transmission ...

Embodiment 2

[0035] Construction and Characterization of Electrochemical Sensors

[0036] The gold electrode was polished successively with 1.0, 0.3 and 0.05 μm aluminum oxide paste, and then washed with 0.1M nitric acid, absolute ethanol and ultrapure water, and the surface of the electrode was blown dry with nitrogen; 5 μL of telomerase extract, 2.5 μL 10 mM telomerase primer DNA (nucleotide sequence is 5'-AAT CCG TCG AGC AGA GTT-3'), 5 μL 10 mM deoxyribonucleoside triphosphates (dNTPs) and 37.5 μL 20 mM Tris-HCl reaction buffer solution ( pH 8.3 with 1.5mM MgCl 2 , 63mM KCl, 0.005% Tween 20, 1mM EGTA, 0.1mg / mL BSA) mixed, incubated at 37°C for 1.5h, then added 4μM hairpin DNA (the nucleotide sequence is 5'-AGG GAA AAA AAC CCT AACT-3 ') and 100U exonuclease III (Exo III), incubate the reaction solution at 37°C for 2.5h, soak the cleaned gold electrode in the reaction solution overnight, add 4μL 10mg / mL CeO 2 -TiO 2 The composite nanorod solution was drop-coated on the surface of the g...

Embodiment 3

[0041] Detection of Telomerase Activity by Electrochemical Sensor

[0042] 5 μL of telomerase extracts extracted from different numbers of cells, 2.5 μL of 10 mM telomerase primer DNA, 5 μL of 10 mM dNTPs, and 37.5 μL of 20 mM Tris-HC1 reaction buffer solution (pH 8.3, containing 1.5 mM MgCl 2 , 63mM KCl, 0.005% Tween 20, 1mM EGTA, 0.1mg / mL BSA) were mixed, incubated at 37°C for 1.5h, after that, 4μM hairpin DNA and 100U of Exo III were added, and the reaction solution was incubated at 37°C for 2.5h, Soak the gold electrode in the reaction solution overnight, add 4 μL of 10 mg / mL CeO 2 -TiO 2 The composite nanorod solution was drop-coated on the surface of the gold electrode and dried at room temperature to form CeO 2 -TiO 2 / DNA-modified gold electrode; will use CeO 2 -TiO 2 A three-electrode system consisting of a DNA-modified gold electrode as the working electrode, a platinum wire as the counter electrode, and a saturated calomel electrode as the reference electrode w...

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Abstract

The invention discloses an electrochemical sensor construction method based on DNA (deoxyribonucleic acid) dual characters, and telomerase activity detection application thereof. When telomerase is inthe presence, the product, which extends, of a telomerase primer and hairpin DNA are hybridized to form dual chains, exonuclease III gradually shears the mononucleotide of the hybridization part of the hairpin DNA in the dual chains and a telomerase extension product, meanwhile, a telomerase product extension product and a short DNA single chain rich in an A basic group are released, the releasedtelomerase extension product can be hybridized with the hairpin DNA again and is sheared by the exonuclease III so as to circularly release more short DNA single chains rich in the A basic group, andthe short DNA single chain rich in the A basic group can be modified on a gold electrode through the mutual function of the A basic group and gold. The phosphate group of the short DNA single chain can adsorb CeO2-TiO2 compound nanorods so as to increase the steric hindrance of an electrode surface and electrostatic repulsion for [Fe(CN)6]<3-/4-> is increased, the electrochemistry signal of [Fe(CN)6]<3-/4-> is weakened, and the sensitive detection of telomerase activity in cells can be realized according to the electrochemistry signal.

Description

technical field [0001] The invention relates to a method for constructing an electrochemical sensor based on DNA double-sidedness and its application for detecting telomerase activity, belonging to the technical field of electrochemical biosensing. Background technique [0002] As a ribonucleoprotein reverse transcriptase, telomerase can bind to the ends of human chromosomes and add a hexameric telomeric sequence (TTAGGG) based on its endogenous RNA template n , to protect the inherent genetic material from unwanted degradation, recombination, and end-to-end fusion. Studies have shown that more than 85% of human tumor cells overexpress telomerase, while no overexpression of telomerase is found in adjacent normal human cells. Therefore, telomerase is considered to be an important biomarker for early cancer diagnosis and treatment. At present, many methods for quantitative detection of telomerase activity have been developed, for example, polymerase chain reaction-based telo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327
CPCG01N27/3278
Inventor 邱建丁王艺梁汝萍
Owner NANCHANG UNIV
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