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A Bifunctional-Blocker-Aided Hybridization Chain Reaction Lighting-Up Self-calibrating Nanocluster Fluorescence for Reliable Nucleic Acid Detection

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Abstract

In this work, we proposed a ratiometric silver nanoclusters (AgNCs) fluorescent assay by designing a bifunctional-blocker-aided hybridization chain reaction (HCR). Hairpin probe 1 (HP1) containing two special DNA fragments (5′-CACCGCT-3′ and 5′-ATTTGCCTTTTGGGGACGGATA-3′) at two terminals creates a red-emitting AgNC nucleation sequence (rNS, 5′-CACCGCTATTTGCCTTTTGGGGACGGATA-3′). We found that the presence of a toehold fragment (5′-TGCCC-3′) in HP1 could silence the rNS. Upon the addition of a target nucleic acid, HCR of HP1 and hairpin probe 2 (HP2) could be initiated, resulting in the formation of long chain of DNA duplexes with multibranched rNS. As the toehold fragment in HP1 participated in generating duplexes, a strong emission of rNS-templated AgNCs was observed at 670 nm. More significantly, a bifunctional blocker was introduced not only to reduce the background red-emitting fluorescence but also to play as an internal green-emitting AgNCs nucleation sequence. On the one hand, the blocker could increase the signal-to-noise-ratio of the constructed biosensor, and on the other hand, the blocker also helped to prepare ratiometric HCR-AgNCs assay with self-calibrating ability to strengthen its reproducibility. Compared with the traditional HCR-AgNCs sensors, the developed ratiometric assay based on the bifunctional-blocker-aided HCR has higher reliability, which is important for the fabrication of biosensors in various fields for practical biosensing applications.

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Data Availability

All the relevant data are available from the authors upon reasonable request.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (22304062), the Zhejiang Provincial Natural Science Foundation of China (LTGY24B050002), the Program for Science and Technology of Jiaxing (2023AY40028) and the Baiqing Foundation of Jiaxing University (CD70621010).

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Authors and Affiliations

  1. College of Chemistry and Materials Science, Zhejiang Normal University, Jinhua, 321004, China

    Dan Zhang, Guobao Zhou, Hongyan Yang, Lei Li & Longhua Guo

  2. Jiaxing Key Laboratory of Molecular Recognition and Sensing, College of Biological, Chemical Sciences and Engineering, Jiaxing University, Jiaxing, 314001, China

    Dan Zhang, Guobao Zhou, Hongyan Yang, Yi Wang, Lijun Shen, Yuxuan Qiu, Lei Li & Longhua Guo

  3. College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, 314000, China

    Yi Wang

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  1. Dan Zhang
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Correspondence to Guobao Zhou, Lei Li or Longhua Guo.

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Zhang, D., Zhou, G., Yang, H. et al. A Bifunctional-Blocker-Aided Hybridization Chain Reaction Lighting-Up Self-calibrating Nanocluster Fluorescence for Reliable Nucleic Acid Detection. J. Anal. Test. (2024). https://doi.org/10.1007/s41664-024-00297-z

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