Construction of universal electrochemical biosensor ultra-sensitive detection target based on tetrahedral-tripod-assisted multi-hairpin cascade assembly

A biosensor, tetrahedral technology, applied in the field of biological analysis, can solve problems such as poor stability, cumbersome experimental procedures, and hindering clinical application.

Inactive Publication Date: 2021-04-13
CHONGQING MEDICAL UNIVERSITY
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Problems solved by technology

[0003] Traditional nucleic acid detection methods include PCR, Southern Blot, genotyping, high-resolution melting, etc. Although these methods have high advantages, they are easy to cause false positive signals, and cumbersome experimental procedures and complex reaction systems lead to poor stability, which greatly hinders its wider clinical application

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  • Construction of universal electrochemical biosensor ultra-sensitive detection target based on tetrahedral-tripod-assisted multi-hairpin cascade assembly
  • Construction of universal electrochemical biosensor ultra-sensitive detection target based on tetrahedral-tripod-assisted multi-hairpin cascade assembly
  • Construction of universal electrochemical biosensor ultra-sensitive detection target based on tetrahedral-tripod-assisted multi-hairpin cascade assembly

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Embodiment Construction

[0058] The specific implementation of the present invention will be described in the following example steps, which should be specially explained, all of which fully illustrate the technical invention but are not limited thereto.

[0059] 1. Efficient Construction of Tetrahedral Tripods

[0060] First, the four tetrahedral tripod chain (S1-S4) DNA dry powders were centrifuged at 13000rpm in a centrifuge at 4°C for 10 minutes, and dissolved in TE buffer (10mM Tris HCl, 1mM EDTA, pH=8.0) to A 100 μM stock solution was obtained, dissolved on ice three times for 3 minutes, mixed well, and then stored at -20°C for later use. Add 1 μL of S1-S4 to 86 μL of TM buffer (20 mM Tris, 50 mM MgCl 2 ·6H 2 0, pH=8.0) and 10 μL TCEP (30 mM diluted in ultrapure water) to make the total volume 100 μL, place it at room temperature for 1 hour and fully reduce it in a Bio-Rad T100 thermal cycler ((Bio-Rad ,USA)) reacted at 95°C for 5 minutes and rapidly lowered to 4°C within 30s, took out the pr...

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Abstract

Nucleic acid is an important genetic substance of a human body and plays an important role in storing, copying and transmitting genetic information, methylated DNA is one of main epigenetic modification modes, abnormal DNA sequences, base mutation and epigenetic changes often cause cancers, and in order to realize early target detection, the invention designs a tetrahedron-assisted multi-series hairpin cascade assembly detection target (DNA / RNA / methylated DNA and the like) universal electrochemical sensor, a plurality of hairpin cascade assemblies are incubated to trigger signal amplification reaction, electric signal detection is realized in a RuHex solution, and powerful electrostatic attraction acts on a DNA phosphoric acid skeleton to cause increase of DPV current response. The linear concentration range of target detection is 10 aM to 100 pM, and the lowest detection limit is 1.78 aM. By means of a specific enzyme digestion reaction, methylated DNA is incubated on an electrochemical platform to carry out related experiments, and the superiority of the platform is further proved.

Description

technical field [0001] The invention relates to the technical field of biological analysis, and constructs an electrochemical biosensor in which a plurality of hairpins are gradually serially assembled on a DNA tetrahedron tripod to detect nucleic acid DNA and DNA methylation. Background technique [0002] Nucleic acid, as the main genetic material of the human body, determines the structure of biomolecules, proteins, and cellular components, and plays a vital role in storing, regulating, and transmitting genetic information in the body. Nucleic acid structural abnormalities, gene base mutations and chromosomal abnormalities have greatly increased the incidence of cancer and diseases. DNA methylation is one of the main epigenetic modification methods. The methyl group is covalently bonded to 5-methylcytosine under the catalysis of DNA methyltransferase (DNMT). Occurring at the cytosine-phosphate-guanine site (CpG island), epigenetic changes play a crucial role in the early ...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26G01N27/30G01N27/327C12Q1/6825
CPCC12Q1/6825G01N27/26G01N27/30G01N27/3275C12Q2525/301C12Q2565/607
Inventor 易钢黄玉麒赵书慧张文秀
Owner CHONGQING MEDICAL UNIVERSITY
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