A kind of electrochemical biosensor and its preparation method based on three-strand linked DNA

A biosensor and electrochemical technology, applied in the direction of material electrochemical variables, scientific instruments, instruments, etc., can solve the problems of high cost, time-consuming, poor repeatability, etc., achieve high versatility, low background signal, and reduce false positives The effect of probability

Inactive Publication Date: 2019-07-09
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although these methods achieve accurate and reliable detection of miRNA, these methods also have certain defects, such as poor reproducibility, time-consuming, high cost and require professional experimental skills

Method used

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  • A kind of electrochemical biosensor and its preparation method based on three-strand linked DNA
  • A kind of electrochemical biosensor and its preparation method based on three-strand linked DNA
  • A kind of electrochemical biosensor and its preparation method based on three-strand linked DNA

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1, an ultrasensitive electrochemical biosensor based on triple-stranded DNA was used to detect different concentrations of target miRNA-21 in solution, and a standard working curve of the sensor's current response was obtained.

[0047] (1) A gold electrode with a diameter of 2mm is covered with Al 2 o 3 Grinding with polishing powder, rinsed with ultrapure water;

[0048] (2) Take 10µL, 1µM capture probe immobilization solution and drop it on the electrode surface, cover it, incubate at room temperature for 180min, wash the electrode surface with buffer solution, N 2 blow dry;

[0049] (3) Take 10µL, 2mM mercaptohexanol solution and drop it on the surface of the electrode obtained in step (2), cover it, incubate at room temperature for 90min, seal the non-specific active sites on the electrode surface, wash the electrode surface with buffer solution, N 2 blow dry;

[0050] (4) Take 10µL of hybridization buffer containing different concentrations of target p...

Embodiment 2

[0069] Example 2, the ultrasensitive electrochemical biosensor based on triple-stranded concatenated DNA is applied to the detection of miRNA-21 content in actual serum samples

[0070] (1) Construct an electrochemical biosensor according to the preparation method of the present invention, and use an electrochemical workstation for testing with a three-electrode system, silver / silver chloride is used as a reference electrode, a platinum wire electrode is used as a counter electrode, and the prepared sensor is used as a working electrode , test in 8ml, 10mM pH 6 electrochemical detection solution (containing serum sample, the volume ratio of serum sample to 10mM electrochemical detection solution is 1:1);

[0071] (2) Use differential pulse voltammetry to detect the above electrochemical detection solution, the potential range is -0.6~0.2, the amplitude is 0.05V, the pulse width is 0.05s, and the pulse period is 0.5s;

[0072] (3) As shown in Figure 4, the expression of miRNA-2...

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Abstract

The invention discloses an electrochemical biosensor based on triplex catenated DNA (deoxyribonucleic acid) and a preparation method. An inner ring probe and an outer ring probe are designed; the twoprobes complement each other to form DNA ringlets; the ringlets are self-assembled through Hoogsteen bonds and Watson-Crick bonds under an appropriate pH (potential of hydrogen) condition to form a catenated nucleic acid chain; a catenated DNA long chain is connected onto a gold electrode with the assistance of an acquisition probe and a connecting probe; hexaammineruthenium is absorbed on a DNA framework by an electrostatic effect; and miRNA (micro ribonucleic acid) is detected by detecting an electrochemical signal of hexaammineruthenium. The biosensor accurately detects target miRNA-21 between 1fM and 10nM, and a limit of detection is 1.63fM. The biosensor can directly detect miRNA in a serum sample without pretreatment, can be used for detecting other biomarkers, and has good application prospects in clinical diagnosis and prognosis.

Description

technical field [0001] The invention belongs to the technical field of electrochemical biosensing and detection, and in particular relates to an electrochemical biosensor based on triple-stranded DNA and a preparation method thereof. Background technique [0002] MicroRNAs (miRNAs) are a class of small endogenous non-coding RNAs, consisting of approximately 22-25 nucleotides, that can negatively regulate gene expression through translational repression, mRNA degradation and protein synthesis repression. Studies have shown that miRNAs play important regulatory roles in a variety of biological processes, such as allergic diseases, hematopoietic differentiation, cardiovascular diseases, and cancer. Furthermore, it has been demonstrated that miRNAs can be released into the circulation in body fluids and can be detected in serum or plasma in a very stable form. miRNA-21 is significantly upregulated in cervical cancer, colon cancer, non-small cell lung cancer, hidradenitis suppur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/327G01N27/26
CPCG01N27/26G01N27/3277
Inventor 陈宪姚乐
Owner FUZHOU UNIV
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