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Electrochemical sensor for detecting microRNA-21, preparation method and application thereof

An electrochemical and sensor technology, applied in the fields of life and health and medical analysis, can solve the problems of low expression, poor specificity, time-consuming, etc., and achieve the effect of good detection sensitivity and excellent specificity

Active Publication Date: 2019-03-01
安徽深蓝医疗科技股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these detection strategies still have some unavoidable disadvantages
For example, qRT-PCR technology is time-consuming and expensive; Northern blot technology has a high risk of cross-contamination and operational complexity; microarray methods have problems such as low sensitivity and poor specificity
In addition, due to the inherent characteristics of microRNA such as short sequence, low expression level, high sequence homology of family members, and susceptibility to degradation, the direct analysis and detection of microRNA is greatly limited.
Therefore, the quantitative and qualitative detection of microRNA is still very challenging.

Method used

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  • Electrochemical sensor for detecting microRNA-21, preparation method and application thereof
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  • Electrochemical sensor for detecting microRNA-21, preparation method and application thereof

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preparation example Construction

[0025] One aspect of the embodiments of the present invention provides a method for preparing an electrochemical sensor for detecting microRNA-21, the electrochemical sensor includes a working electrode, a reference electrode and a counter electrode, and the preparation method includes:

[0026] contacting the activated capture probe with a gold electrode as a working electrode and incubating, and then applying mercaptohexanol to the gold electrode and incubating to block the blank site;

[0027] Constructing DNA concatemers by hybridization chain reaction, and preparing G-quadruplex-rich lines by extending DNA with terminal deoxyribonucleotidyl transferase;

[0028] applying the DNA concatemer to the surface of the gold electrode for incubation, and interacting with the streptavidin solution;

[0029] A biotin-labeled G-quadruplex-rich line is applied on the surface of the gold electrode, the biotin can specifically recognize the streptavidin, and the G-quadruplex-rich line i...

Embodiment 1

[0056] (1) Preliminary preparation of the gold electrode: ① The gold electrode was polished and polished with 0.05 μm alumina slurry for 5 minutes, and then rinsed thoroughly with ultrapure water to remove the alumina powder non-specifically adsorbed on the surface of the gold electrode; The electrodes were soaked in the prepared piranha solution (H 2 SO 4 / H 2 o 2 , volume ratio 3:1) for 30 min, then thoroughly rinsed with ultrapure water and sonicated with ethanol and ultrapure water for 5 min to remove any remaining impurities; ③ in 0.5M H 2 Set the scanning potential to -0.2 and 1.5V in SO4 solution, and perform electrochemical scanning cleaning on the electrode at a scanning rate of 100mV / s until a stable voltammetric peak is obtained; Blow dry; ⑤ Add the pre-activated probe (at room temperature, incubate 2.5 μL 1 mMTCEP with 5 μL 1 μM capture probe CP for 30 min) dropwise on the treated Au working electrode, and incubate at 4 °C for 12 h; ⑥ Put 5μL of 1.0mM mercaptoh...

Embodiment 2

[0064] (1) Preliminary preparation of the gold electrode: ① The gold electrode was polished and polished with 0.05 μm alumina slurry for 5 minutes, and then rinsed thoroughly with ultrapure water to remove the alumina powder non-specifically adsorbed on the surface of the gold electrode; The electrodes were soaked in the prepared piranha solution (H 2 SO 4 / H 2 o 2 , volume ratio 3:1) for 30min, then thoroughly rinsed with ultrapure water and sonicated with ethanol and ultrapure water for 5min to remove any remaining impurities; ③ at 0.5MH 2 Set the scanning potential to -0.2 and 1.5V in SO4 solution, and perform electrochemical scanning cleaning on the electrode at a scanning rate of 100mV / s until a stable voltammetric peak is obtained; Blow dry; ⑤ Add the pre-activated probe (at room temperature, incubate 2.5 μL 1 mMTCEP with 5 μL 1 μM capture probe CP for 30 min) onto the treated Au working electrode, and incubate at 6 °C for 16 h; ⑥ Put 5μL of 1.0mM mercaptohexanol (MC...

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Abstract

The invention discloses an electrochemical sensor for detecting microRNA-21, a preparation method and application thereof. The method firstly anchors a DNA tandem with a biotin at the 5' end to the surface of an electrode, the DNA tandem is used as a main structure, and a G-rich quadruplex line is assembled by specific recognition of streptavidin-biotin as a branching structure; G tetra-chain monomer and G-quad-chain line pi-pi are stacked, self-assembly is conducted to form multi-branched DNA nanostructure, and the obtained DNA nano-assembly can be tightly combined with hemin to generate an oxidation reduction signal for electrochemical detection of miRNA-21. The method has good miRNA-21 detection sensitivity, has excellent specificity, can distinguish the single nucleotide mutation of the target nucleic acid analyte from the completely matched target RNA, analyzes an actual biological sample, and is a universal DNA nanometer biosensor platform.

Description

technical field [0001] The invention belongs to the field of life health and medical analysis, in particular to a method for detecting microRNA-21 in cancer cells, in particular to a multi-branched DNA nano assembly based on DNA concatemers and G-rich quadruplex lines for ultrasensitive detection of microRNA-21 21 electrochemical sensors and detection methods. Background technique [0002] microRNAs are a class of small endogenous non-coding RNAs that are key players in various biological processes such as gene expression, cell cycle and biological development. In the past two decades, research on microRNA in the field of biology has been extensively carried out. Basic research shows that the change of microRNA may lead to the occurrence of many diseases, especially for cancer, microRNA as a tumor suppressor or oncogene can become a new type of biomarker, which has very important research value in the early diagnosis and clinical application of tumors. Therefore, reliable ...

Claims

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Application Information

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IPC IPC(8): G01N27/30G01N27/327
CPCG01N27/30G01N27/3278
Inventor 徐建国陈伟闫超秦盼柱姚丽
Owner 安徽深蓝医疗科技股份有限公司
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