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Electrochemiluminescence sensor for detecting hepatitis B virus DNA and preparation and application thereof

A hepatitis B virus, electrochemical technology, applied in the biological field, can solve the problems of limited sensitivity, limited application, unfavorable early diagnosis and treatment of HBV, etc., and achieve the effect of improving sensitivity, good reproducibility, and excellent ECL performance

Active Publication Date: 2021-08-10
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the real-time fluorescence quantitative PCR method has been widely used in the clinical detection of HBV, the demand for expensive equipment and well-trained technicians limits its application in primary hospitals and areas with limited resources, and the entire detection process takes about 2 hours and The sensitivity is limited, which is not conducive to the early diagnosis and treatment of HBV

Method used

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  • Electrochemiluminescence sensor for detecting hepatitis B virus DNA and preparation and application thereof
  • Electrochemiluminescence sensor for detecting hepatitis B virus DNA and preparation and application thereof
  • Electrochemiluminescence sensor for detecting hepatitis B virus DNA and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Preparation of Electrochemiluminescence Sensor and Detection of HBV DNA

[0069] 1. Materials

[0070] Tetrakis[4-(4'-carboxyphenyl)phenyl]ethylene (H 4 ETTC) was purchased from Beijing Huaruike Chemical Co., Ltd.; zirconium chloride (ZrCl 4 ), N, N dimethylformamide (DMF), formic acid (HCOOH) and triethylamine (TEA) were provided by Aladdin Biochemical Technology Co., Ltd.; HPLC purified oligonucleotides were synthesized by Shanghai Sangong; EnGen Lba Cas12a and NEBuffer 3.0 solution were purchased from New England Biolabs; DNA Marker, enzyme-free water and RNase inhibitor were purchased from Dalian Takara Company.

[0071] 2. Testing instruments

[0072] Xi'an Remax MPI-E electrochemical luminescence workstation, the detection system is a three-electrode system, including the reference electrode is Ag / AgCl electrode, the counter electrode is platinum wire electrode, and the working electrode is a glassy carbon electrode with a diameter of 4mm.

[0073] 3. Detectio...

Embodiment 2

[0098] Characterization of Zr-ETTC

[0099] 1. SEM and TEM characterization

[0100] Such as figure 2 As shown, Zr-ETTC presents a wrinkled-like ultrathin nanosheet morphology with a large specific surface area, which facilitates the loading of biomolecules.

[0101] 2. ECL signal characterization

[0102] Such as image 3 As shown, Zr-ETTC has an excellent ECL signal in the presence of co-reactant triethylamine (TEA). This is attributed to the following reasons: On the one hand, the rigid MOF framework can confine the H 4 The free intramolecular motion of ETTC and reduced nonradiative relaxation enable strong ECL emission. At the same time, due to the use of H 4 ETTC acts as a bridging ligand, making the emitter H 4 ETTC can be massively immobilized in the MOF framework, further enhancing the ECL emission. On the other hand, due to the ultrathin porous structure of Zr-ETTC, co-reactants can be enriched in the micropores, thus shortening the co-reactant TEA and H 4 T...

Embodiment 3

[0104] Crosslinking Characterization of Zr-ETTC and Fc-SP

[0105] 1. Zeta potential characterization

[0106] In order to demonstrate the successful combination of Zr-ETTC and Fc-SP, the Zeta potential measurement was carried out on the nanomaterials before and after crosslinking in this example. Such as Figure 4 As shown in A, after binding to Fc-SP, because Fc-SP is negatively charged, the Zeta potential shifts from +3.05mV to -11.01mV, which shows that Zr-ETTC@Fc-SP was successfully prepared.

[0107] 2. ECL signal characterization

[0108] Such as Figure 4 As shown in B, under the same experimental conditions, Zr-ETTC first exhibited a very strong ECL signal, but when Fc was added, the ECL signal decreased significantly, indicating that Fc could indeed quench the ECL signal of Zr-ETTC.

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Abstract

The invention discloses an electrochemiluminescence sensor for detecting hepatitis B virus DNA and preparation and application thereof. The electrochemiluminescence sensor is constructed based on an aggregation-induced electrochemiluminescence body, a toehold-mediated strand displacement reaction (TSDR) and a CRISPR / Cas12a system. A Zr-ETTC@ Fc-SP compound is prepared and fixed to the surface of an electrode, and an ECL signal of Zr-ETTC can be quenched by Fc; a TSDR system is constructed by using a target substance T chain, an A-B compound and a single chain C, and a displacement reaction is carried out to obtain a large amount of Cas12a activator; and the Cas12a-crRNA compound can recognize the Cas12a activator, activate the cleavage activity of the Cas12a activator and perform trans-cleavage on SP, so that Fc is separated from the surface of the electrode, the ECL signal is recovered, and HBV DNA detection is realized. The HBV DNA detection method disclosed by the invention has the advantages of high sensitivity, strong stability, good reproducibility, high reaction speed and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an electrochemiluminescent sensor for detecting hepatitis B virus DNA and its preparation and application. Background technique [0002] Hepatitis B Virus (HBV) is a double-stranded DNA virus belonging to the family Hepadnaviridae (hepadnaviruses). my country's HBV infection rate is high, with more than 700 million people infected, much higher than other infectious diseases such as tuberculosis and HIV. Vaccines and antiviral drugs are currently the mainstays of prevention and treatment, but high mutation rates and drug resistance pose major problems. HBV infection not only seriously affects the health of patients, but also imposes a great burden on the medical system. Therefore, early detection and diagnosis of HBV is crucial. HBV DNA can exist in the serum of patients before the appearance of HBV surface antigen, and a high viral load indicates a strong infectious infection. In...

Claims

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Application Information

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IPC IPC(8): G01N27/26G01N21/76C12Q1/682C12Q1/70
CPCG01N27/26G01N21/76C12Q1/682C12Q1/701
Inventor 丁世家甘秀锋蔡晓颖
Owner CHONGQING MEDICAL UNIVERSITY
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