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Electrochemical biosensor strategy combining DNA entropy-driven technology to activate CRISPR-Cas13a activity and triple-stranded nucleic acids to detect SARS-CoV-2 RdRp gene

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Abstract

By merging DNA entropy-driven technology with triple-stranded nucleic acids in an electrochemical biosensor to detect the SARS-CoV-2 RdRp gene, we tackled the challenges of false negatives and the high cost of SARS-CoV-2 detection. The approach generates a CRISPR-Cas 13a-activated RNA activator, which then stimulates CRISPR-Cas 13a activity using an entropy-driven mechanism. The activated CRISPR-Cas 13a can cleave Hoogsteen DNA due to the insertion of two uracil (-U-U-) in Hoogsteen DNA. The DNA tetrahedra changed on the electrode surface and can therefore not construct a three-stranded structure after cleaving Hoogsteen DNA. Significantly, this DNA tetrahedron/Hoogsteen DNA-based biosensor can regenerate at pH = 10.0, which keeps Hoogsteen DNA away from the electrode surface, allowing the biosensor to function at pH = 7.0. We could use this technique to detect the SARS-CoV-2 RdRp gene with a detection limit of 89.86 aM. Furthermore, the detection method is very stable and repeatable. This technique offers the prospect of detecting SARS-CoV-2 at a reasonable cost. This work has potential applications in the dynamic assessment of the diagnostic and therapeutic efficacy of SARS-CoV-2 infection and in the screening of environmental samples.

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Funding

We gratefully acknowledge the financial support of the National Natural Science Foundation of China (21964018, 81860851), Guangxi Medical High-level Leading Talents Training 139 Project (GWKJ2018-22), Special Funding for Guangxi Special Experts (GRCT2019-13), the Natural Science Foundation of Guangxi Province (2018GXNSFDA281017), the Jiangsu Provincial Health Care Commission Scientific Research Project (M2021035), and the Wuxi “Taihu Light” science and technology (medical and health technology) research (Y20212049).

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Authors and Affiliations

  1. The Affiliated Hospital of Youjiang Medical University for Nationalities, Guangxi, Baise, 533000, China

    Chenyi Zhuo, Yuanxun Gong & Qianli Tang

  2. West Guangxi Key Laboratory for Prevention and Treatment of High-Incidence Diseases, Youjiang Medical University for Nationalities, Guangxi, Baise, 533000, China

    Zichun Song, Jiuying Cui & Xianjiu Liao

  3. School of Chemistry and Materials Science, Nanjing University of Information Science and Technology, 210044, Nanjing, People’s Republic of China

    Kai Zhang

  4. NHC Key Laboratory of Nuclear Medicine, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu, 214063, China

    Kai Zhang

  5. Maternity & Child Care Center of Dezhou, Dezhou, 25300, China

    Xinlei Song

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  1. Chenyi Zhuo
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Zhuo, C., Song, Z., Cui, J. et al. Electrochemical biosensor strategy combining DNA entropy-driven technology to activate CRISPR-Cas13a activity and triple-stranded nucleic acids to detect SARS-CoV-2 RdRp gene. Microchim Acta 190, 272 (2023). https://doi.org/10.1007/s00604-023-05848-2

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