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Electrochemical detection of the p53 gene using exponential amplification reaction (EXPAR) and CRISPR/Cas12a reactions

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Abstract

An improved electrochemical sensor has been developed for sensitive detection of the p53 gene based on exponential amplification reaction (EXPAR) and CRISPR/Cas12a. Restriction endonuclease BstNI is introduced to specifically identify and cleave the p53 gene, generating primers to trigger the EXPAR cascade amplification. A large number of amplified products are then obtained to enable the lateral cleavage activity of CRISPR/Cas12a. For electrochemical detection, the amplified product activates Cas12a to digest the designed block probe, which allows the signal probe to be captured by the reduced graphene oxide-modified electrode (GCE/RGO), resulting in an enhanced electrochemical signal. Notably, the signal probe is labeled with large amounts of methylene blue (MB). Compared with traditional endpoint decoration, the special signal probe effectively amplifies the electrochemical signals by a factor of about 15. Experimental results show that the electrochemical sensor exhibits wide ranges from 500 aM to 10 pM and 10 pM to 1 nM, as well as a relatively low limit detection of 0.39 fM, which is about an order of magnitude lower than that of fluorescence detection. Moreover, the proposed sensor shows reliable application capability in real human serum, indicating that this work has great prospects for the construction of a CRISPR-based ultra-sensitive detection platform.

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Funding

This work was supported by the National Natural Science Foundation of China (No. 81772290), Chongqing science and technology commission (cstc2021jcyj-msxmX0608), the Graduate Scientific Research and Innovation Foundation of Chongqing, China (CYB22072), the Fundamental Research Funds for the Central Universities (2021CDJYGRH006), the Sichuan Science and Technology Program (2022YFSY0013), the Chongqing Graduate Tutor Team Construction Project, and the Analytical and Testing Center of Chongqing University (SEM/Raman) and the sharing fund of Chongqing University’s large equipment.

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  1. Shiying Zhou and Liyuan Deng contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory for Biorheological Science and Technology of Ministry of Education, Bioengineering College of Chongqing University, Chongqing, 400044, People’s Republic of China

    Shiying Zhou, Liyuan Deng, Jiangbo Dong, Na Qi, Mei Yang, Danqun Huo & Changjun Hou

  2. Chongqing University, Three Gorges Hospital, Chongqing, 404000, People’s Republic of China

    Peng Lu

  3. Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, SeNA Research Institute and Szostak-CDHT Large Nucleic Acids Institute, Chengdu, 610065, People’s Republic of China

    Zhen Huang

  4. Chongqing Key Laboratory of Bio-Perception & Intelligent Information Processing, School of Microelectronics and Communication Engineering, Chongqing University, Chongqing, 400044, People’s Republic of China

    Changjun Hou

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  1. Shiying Zhou
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Zhou, S., Deng, L., Dong, J. et al. Electrochemical detection of the p53 gene using exponential amplification reaction (EXPAR) and CRISPR/Cas12a reactions. Microchim Acta 190, 113 (2023). https://doi.org/10.1007/s00604-023-05642-0

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