4.6 Article

A label-free and ultrasensitive electrochemical biosensor for oral cancer overexpressed 1 gene via exonuclease III-assisted target recycling and dual enzyme-assisted signal amplification strategies

Journal

ANALYST
Volume 147, Issue 11, Pages 2412-2424

Publisher

ROYAL SOC CHEMISTRY
DOI: 10.1039/d2an00367h

Keywords

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Funding

  1. National Natural Science Foundation of China [22164014, 21864006]
  2. Natural Science Foundation of Guangxi Province [2021GXNSFAA075015]
  3. Thousands of Young Teachers Training Program of Guangxi Province [[2018]18]
  4. BAGUI Scholar Program of Guangxi Province of China

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A label-free and ultrasensitive electrochemical biosensor for oral cancer overexpressed 1 (ORAOV1) gene was constructed using exonuclease III-assisted target recycling and dual enzyme-assisted signal amplification strategies. The biosensor can accurately detect ORAOV1 gene in human saliva samples.
A label-free and ultrasensitive electrochemical biosensor for oral cancer overexpressed 1 (ORAOV1) gene was constructed via exonuclease III-assisted target recycling and dual enzyme-assisted signal amplification strategies. Capture DNA with a sulfhydryl group at its 3 ' terminus was modified onto the surface of a bare gold electrode via an Au-S bond. Assisted DNA hybridized with basal DNA to form hybrid DNA in advance, and ORAOV1 gene hybridized continuously with such a hybrid DNA from the other terminus to construct intact double-stranded DNA. Exonuclease III digested basal DNA in such intact double-stranded DNA specifically, and both ORAOV1 gene and assisted DNA were released into solution. ORAOV1 gene induced another intact double-stranded DNA digestion for target recycling, while assisted DNA hybridized with the capture DNA to form double-stranded DNA on the modified electrode surface. Unhybridized capture DNA on the modified electrode surface was hydrolyzed by RecJf exonuclease to reduce the background electrochemical signal. The 3 ' terminus of double-stranded DNA on the modified electrode surface was prolongated to be guanine-rich oligonucleotides under the catalysis of terminal deoxynucleotidyl transferase. In the presence of K+ ions, hemin adsorbed onto guanine-rich oligonucleotides to construct a G-quadruplex/hemin complex with a large steric hindrance effect to efficiently avoid the charge transfer of the [Fe(CN)(6)](3-/4-) probe toward the electrode surface. The electrochemical impedance value was increased significantly after the addition of ORAOV1 gene via exonuclease III-assisted target recycling and dual enzyme-assisted signal amplification strategies. The electrochemical impedance value was linearly related to the logarithmic concentration of ORAOV1 gene in the range from 0.05 fM to 20 pM, and the detection limit of ORAOV1 gene was low to 0.019 fM. This biosensor was used to detect ORAOV1 gene in complicated human saliva samples with satisfactory results.

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