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Electrochemical sensor for detecting hepatitis B exosome miRNA as well as preparation and application of electrochemical sensor

An electrochemical and exosome technology, applied in the biological field, can solve the problems of leakage, time-consuming and laborious, low hybridization efficiency, etc.

Active Publication Date: 2021-08-06
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although localized reaction substrates in sensing systems can greatly increase the reaction speed, these strategies suffer from the following drawbacks: i) the construction of localized substrates requires multiple assembly steps, resulting in time-consuming and labor-intensive steps; ii) diffusible reactions The hybridization efficiency of substances with DNA nanowires may be low under physical conditions, directly resulting in incomplete localization structures; iii) the inherent characteristics of CHA and HCR, such as unacceptable cycle leakage and stringent reaction conditions, that is, divalent metal ions ( Such as Mg2+) always adversely affect the analytical performance, limiting their further application in early tumor diagnosis
In addition, the leakage may be worse because the increased number of DNA hairpins in the compact space are more prone to non-specific interactions

Method used

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  • Electrochemical sensor for detecting hepatitis B exosome miRNA as well as preparation and application of electrochemical sensor
  • Electrochemical sensor for detecting hepatitis B exosome miRNA as well as preparation and application of electrochemical sensor
  • Electrochemical sensor for detecting hepatitis B exosome miRNA as well as preparation and application of electrochemical sensor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Preparation of electrochemical sensors and detection of exosomal miRNA

[0074] 1. Materials

[0075] 6-Mercapto-1-hexanol (MCH) was obtained from Sigma-Aldrich (St Louis, MO, USA). HPLC-purified oligonucleotides were synthesized by Shanghai Sangong. Hemin G-four-strand DNAzyme (solution) was purchased from Sangon Bioengineering Co., Ltd. (Shanghai).

[0076] 2. Testing instruments

[0077] Shanghai Chenhua CHI660D electrochemical workstation, the detection system is a three-electrode system, including an Ag / AgCl electrode as a reference electrode, a platinum wire electrode as a counter electrode, and a gold electrode with a diameter of 3mm as a working electrode.

[0078] 3. Detection principle

[0079] Such as figure 1 As shown, the present invention builds an enzyme-free, label-free electrochemical sensor based on the cascade strand displacement reaction (L-TEDCR) and MOF / DNA cascade enzymes for detecting exosomal miRNA. The detection principle is: The chain di...

Embodiment 2

[0110] Validation of the feasibility of detecting exosomal miRNA electrochemical sensors

[0111] Determine whether Exo-miRNA triggers L-TEDCR by PAGE experiment, the results are as follows image 3 shown.

[0112] image 3 The PAGE electrophoresis characterization results of the cascade strand displacement reaction (L-TEDCR) of the present invention are shown. Among them, lane M is the DNA gradient marker, and lanes 1-5 are P strand, R strand, L strand, L strand 0 chain, auxiliary chain Fs, lane 6 is Ts complex, lane 7 is Ts+auxiliary chain Fs, swimming lane 8 is Ts+auxiliary chain Fs, and lane 9 is Ts+auxiliary chain Fs+target substance.

[0113] Depend on image 3 It can be seen that in the absence of Exo-miRNA, almost no P chain was produced (lanes 7, 8), which indicated that the constructed L-TEDCR strategy had negligible background leakage. In the presence of different concentrations of Exo-miRNA, the auxiliary chain Fs chain decreased, and at the same time a new ba...

Embodiment 3

[0115] Take the MOF nanosheets prepared in Example 1, and verify the synthesis of MOF by TEM.

[0116] Figure 4 shows the TEM images of the MOF nanosheets, given by Figure 4 It can be seen that the MOF synthesized in the present invention exhibits an obvious sheet structure, and its lateral size is about 2 μm, indicating that the MOF nanosheet has a large specific surface area, which is conducive to loading more biomolecules. It shows that the MOF we built was successfully synthesized.

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Abstract

The invention discloses an electrochemical sensor for detecting hepatitis B exosome miRNA as well as preparation and application of the electrochemical sensor. The sensor comprises a working electrode, wherein the surface of the working electrode is fixedly provided with a DNA nano bracket marked by capture probe sulfydryl; a target substance T chain induced cascade chain displacement reaction (L-TEDCR) system: a cascade T-type structure compound Ts and a cascade reaction auxiliary chain Fs, the Ts is composed of a DNA single chain P chain, a DNA single chain R chain, a DNA single chain L chain and a DNA single chain L0 chain, and the Fs is composed of a DNA single chain F chain and a DNA single chain L0 chain; and an MOF / DNA cascade enzyme amplification detection system. After the exosome miRNA is added, the L-TEDCR is rapidly triggered, and a large number of DNA nano-scaffolds are generated; the generated DNA nano-scaffold is fixed on an electrode and is combined with MOF / DNA cascade enzyme, so that a remarkably amplified electrochemical signal for detecting exosome miRNA is generated. The sensor has the advantages of high sensitivity, high detection speed, good specificity and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an electrochemical sensor for detecting hepatitis B exosome miRNA and its preparation and application. Background technique [0002] Hepatitis B virus (HBV) infection is a serious threat to human health. There are many markers of HBV infection, including hepatitis B surface antigen (hepatitis B surface antigen, HBsAg) and hepatitis B e antigen ( Hepatitis B e antigen (HBeAg), hepatitis B virus quantification (HBV DNA), and new serological markers in recent years, including hepatitis B core-associated antigen (hepatitis B core associated antigen, HBcrAg), HBV pregenomic RNA (pregenomic RNA, pgRNA) and so on. Although the existing detection methods of virological markers in peripheral blood have their own advantages in diagnosis and curative effect monitoring, they still cannot well replace the detection of covalently closed circular DNA (cccDNA) in liver tissue. Research to explore ...

Claims

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Application Information

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IPC IPC(8): G01N27/327G01N27/48C12Q1/6825
CPCG01N27/3271G01N27/48C12Q1/6825C12Q2525/207C12Q2531/119
Inventor 丁世家刘萍舒晓佳
Owner CHONGQING MEDICAL UNIVERSITY
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