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Hypersensitive microRNA electrochemical detection method of incision enzyme driven multi-legged DNA molecular machine

A DNA molecule and molecular machine technology, applied in the field of ultrasensitive microRNA electrochemical detection, to achieve the effect of improving the detection and analysis performance

Active Publication Date: 2021-02-19
GUIZHOU PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the deficiencies in the prior art, the present invention provides an ultrasensitive microRNA electrochemical detection method of endonuclease-driven multi-legged DNA molecular machines, which has the advantages of no need for precise control The thermal PCR instrument can achieve amplification under isothermal conditions, which has the advantage of simplicity. The DNA molecular machine is highly efficient and has single-molecule controllability, which improves the performance of detection and analysis. The electrochemical transmission of the gene polypod molecular machine Sensitive technology provides a promising simple and sensitive candidate technology for the detection of circulating microRNA. Problems with constraints such as primer design

Method used

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  • Hypersensitive microRNA electrochemical detection method of incision enzyme driven multi-legged DNA molecular machine
  • Hypersensitive microRNA electrochemical detection method of incision enzyme driven multi-legged DNA molecular machine
  • Hypersensitive microRNA electrochemical detection method of incision enzyme driven multi-legged DNA molecular machine

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Embodiment 1

[0044] Such as figure 1 It is a design diagram of the electrochemical sensing detection principle based on the three-fork walking DNA molecular machine in Example 1 of the present invention.

[0045] (1) The biological probe sequences used in the electrochemical detection process of microRNA in this embodiment are shown in Table 1:

[0046] Table 1: Biological probe sequences used in the electrochemical detection of microRNA

[0047]

[0048] (2) Biological probe preparation Gold electrode interface preparation:

[0049] The gold electrode was first scanned in 0.5M dilute sulfuric acid for 12 weeks, and then polished on the buckskin with 0.3 μM and 0.05 μM aluminum powder for 10 min, the surface of the gold electrode was washed with water, and then the surface of the gold electrode was washed with piranha solution (H 2 SO 4 :H 2 o 2 =3:1) for 3 times, 5 min each time, then, wash the surface of the gold electrode with water, and dry the surface of the gold electrode wit...

Embodiment 2

[0055] (1) Feasibility verification results of nucleic acid testing:

[0056] Agarose gel (3%) electrophoresis verification polypod DNA nanostructure construction, the results are as follows figure 2 Shown: Lane 1 is a 500bp DNA marker; Lanes 2, 3, 4, and 5 are target microRNA and detection probes (probe1, 2, 3); Lane 6 is the assembled chain microRNA+probe1; Lane 7 is the assembled chain microRNA+probe1 +probe2; lane 8 is the assembled chain microRNA+probe1+probe2+probe3; lane 9 is probe1+probe2+probe, the maker lane is 500bp DNA ladder (Takara), lanes 1 to 4 are assembled chains, as an electrophoresis control, lanes 5 to 7 is the DNA assembly process. Compared with the control band, the band is gradually reared. This is because the assembled chain is gradually assembled into a large-molecular-weight DNA hybrid, which leads to a slowdown in the electrophoretic movement rate. The result suggests that the polypod DNA nanostructure The construction is feasible, and the respons...

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Abstract

The invention relates to the technical field of molecular diagnosis, and discloses a hypersensitive microRNA electrochemical detection method of an incision enzyme driven multi-legged DNA molecular machine. The method comprises the following steps: aiming at target microRNA, designing and synthesizing detection probes 1, 2, 3 (probe 1, 2, 3) as recognition elements, designing and synthesizing a sulfhydrylation modified probe 4 (probe 4) as a report element, adding the sulfhydrylated probe 4 on the surface of a golden electrode and subjected to incubating overnight in a refrigerator at 4 DEG Cso that a report probe is anchored on the surface of the gold electrode and further a molecular orbit is prepared, identifying through a target sequence and a detection probe, and carrying out cascadeprogrammed reaction to form a trident DNA nano structure, designing a short nucleic acid restriction endonuclease site at each end of the structure as a leg of a molecular machine, and further forming the endonuclease driven multi-legged molecular machine. According to the present invention, the accurate temperature control PCR instrument is not required, the amplification can be achieved under the isothermal condition, and the advantages of simpleness, convenience and hypersensitivity are provided.

Description

technical field [0001] The invention relates to the technical field of molecular diagnosis, in particular to a supersensitive microRNA electrochemical detection method for an endonuclease-driven multi-legged DNA molecular machine. Background technique [0002] Tumor cell subclonal populations evolve in response to various selective pressures that promote their proliferation and dissemination and reduce their susceptibility to therapy. To achieve accurate diagnosis and monitoring and avoid ineffective treatment, molecular detection technologies that continuously explore the dynamic molecular evolution information of tumor cells are urgently needed. Circulating microRNAs (18-24 nucleotides) play key roles in tumor proliferation, differentiation, and apoptosis and serve as potential biomarkers for therapy monitoring, drug resistance assessment, and quantification of minimal residual disease. At the same time, the detection of circulating microRNAs in blood has the advantages o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26C12Q1/6825C12Q1/682
CPCG01N27/26C12Q1/6825C12Q1/682
Inventor 许永杰罗洁达静静郑翔李学英张志敏闫旭东
Owner GUIZHOU PROVINCIAL PEOPLES HOSPITAL
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