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Double base mismatches mediated catalytic hairpin assembly for enzyme-free single-base mutation detection: integrating signal recognition and amplification in one

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Abstract

Single nucleotide polymorphism (SNP) biosensors are emerging rapidly for their promising applications in human disease prevention diagnosis, treatment, and prognosis. However, it remains a bottleneck in equipping simple and stable biosensors with the traits of high sensitivity, non-enzyme, and low cost. Double base mismatches mediated chain displacement reactions have attracted fascinating advantages of tailorable thermodynamics stability, non-enzyme, and excellent assembly compliance to involvement in SNP identification. As the base mismatch position and amount in DNA sequence can be artificially adjusted, it provides plenty of selectivity and specificity for exploring perfect biosensors. Herein, a biosensor with double base mismatches mediated catalytic hairpin assembly (CHA) is designed via one base mismatch in the toehold domain and the other base mismatch in the stem sequence of hairpin 1 (H1) by triggering CHA reaction to achieve selective amplification of the mutation target (MT) and fluorescence resonance energy transfer (FRET) effect that is composed of Cy3 and Cy5 terminally attached H1 and hairpin 2 (H2). Depending on the rationally designed base mismatch position and toehold length, the fabricated biosensors show superior SNP detection performance, exhibiting a good linearity with high sensitivity of 6.6 fM detection limit and a broad detection abundance of 1%. The proposed biosensor can be used to detect the KRAS mutation gene in real samples and obtain good recoveries between 106 and 116.99%. Remarkably, these extendible designs of base mismatches can be used for more types of SNP detection, providing flexible adjustment based on base mismatch position and toehold length variations, especially for their thermodynamic model for DNA-strand displacement reactions.

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Funding

This work was supported by the Scientific Research Fund of Hunan Provincial Education Department (22A0348), the Natural Science Foundation of Hunan Province, China (Grant No. 2020JJ5156), and the National College Students’ Innovation and Entrepreneurship Training Program, China (No. S202310534001 and S202310534137).

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Authors and Affiliations

  1. School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan, 411201, Hunan, China

    Lanyue Wang, Sisi Bu, Shijie Xu, Tuo Huang, Fang Yang, Qianglong Tan, Minxin Deng, Wenlin Xie & Jian Chen

  2. Zhejiang Hospital, Hangzhou, 310013, China

    Bobo Cai

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  1. Lanyue Wang
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  2. Sisi Bu
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  3. Shijie Xu
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Correspondence to Wenlin Xie, Bobo Cai or Jian Chen.

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Wang, L., Bu, S., Xu, S. et al. Double base mismatches mediated catalytic hairpin assembly for enzyme-free single-base mutation detection: integrating signal recognition and amplification in one. Microchim Acta 191, 334 (2024). https://doi.org/10.1007/s00604-024-06366-5

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