Self-assembled multifunctional hairpin probe for ultrasensitive and mismatch-selective miRNA detection

Baoqiang Chen; Yiping Fan; Qi Wang; Jianguo Xu; Yi Wang; Bingyong Lin; Lee Jia; Longhua Guo

doi:10.1016/j.jpha.2026.101561

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Baoqiang Chen, Yiping Fan, Qi Wang, Jianguo Xu, Yi Wang, Bingyong Lin, Lee Jia, Longhua Guo. Self-assembled multifunctional hairpin probe for ultrasensitive and mismatch-selective miRNA detection[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2026.101561

Citation:

Baoqiang Chen, Yiping Fan, Qi Wang, Jianguo Xu, Yi Wang, Bingyong Lin, Lee Jia, Longhua Guo. Self-assembled multifunctional hairpin probe for ultrasensitive and mismatch-selective miRNA detection[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2026.101561

Citation:

Baoqiang Chen, Yiping Fan, Qi Wang, Jianguo Xu, Yi Wang, Bingyong Lin, Lee Jia, Longhua Guo. Self-assembled multifunctional hairpin probe for ultrasensitive and mismatch-selective miRNA detection[J]. Journal of Pharmaceutical Analysis. doi: 10.1016/j.jpha.2026.101561

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Self-assembled multifunctional hairpin probe for ultrasensitive and mismatch-selective miRNA detection

doi: 10.1016/j.jpha.2026.101561

Baoqiang Chen¹,
Yiping Fan^{2
,
,},
Qi Wang³,
Jianguo Xu^{1
,
,},
Yi Wang⁴,
Bingyong Lin¹,
Lee Jia^{5
,
,},
Longhua Guo¹

1. Provincial Key Laboratory of Multimodal Perceiving and Intelligent Systems, Engineering Research Center of Intelligent Human Health Situation Awareness of Zhejiang Province, Jiaxing Key Laboratory of Molecular Recognition and Sensing, College of Biological and Chemical Engineering, Jiaxing University, Jiaxing, Zhejiang, 314001, China;
2. Key Laboratory of Traditional Chinese Medicine for the Prevention and Treatment of Infectious Diseases in the Elderly in Zhejiang Province (Cultivation), Jiaxing Hospital of Traditional Chinese Medicine, Jiaxing University, Jiaxing, Zhejiang, 314000, China;
3. School of Biological and Food Engineering, Fuyang Normal University, Fuyang, Anhui, 236037, China;
4. Department of Oncology, Hefei First People’s Hospital, Third Affiliated Hospital of Anhui Medical University, Hefei, Anhui, 230032, China;
5. China-Ireland Food & Medicine Innovation Joint Laboratory, The First Affiliated Hospital, Henan University, Kaifeng, Henan, 475004, China

Funds:

This work was financially supported by the Jiaxing Science and Technology Program-Social Development Special Project (Grant No.: 2025BS009), Zhejiang Provincial Natural Science Foundation of China (Grant No.: LZ25C200005), Jiaxing Applied Basic Research Project (Grant No.: 2023AY11033), Zhejiang Provincial Traditional Chinese Medicine Science and Technology Plan (Grant No.: 2023ZL693), National Natural Science Foundation of China (Grant Nos.: 81773063&

81961138017), and Postgraduate Scientific Research and Practice Innovation Project of Jiaxing University (Grant No.: PSRPIP2025001B).

Received Date: Aug. 10, 2025
Accepted Date: Jan. 22, 2026
Rev Recd Date: Jan. 21, 2026
Available Online: Jan. 23, 2026

Abstract

Abstract

Sensitive, specific, and stable detection of microRNAs in complex biological environments remains a formidable challenge in molecular diagnostics. We introduce a novel bidirectional palindromic assembled multifunctional hairpin probe (A-MF-HP)-a molecular tool that integrates target recognition, cascade signal amplification, and fluorescence reporting into a single, compact system. Two palindromic arms drive autonomous, bidirectional self-assembly into a nuclease-resistant architecture, enabling robust operation in serum-rich environments. Upon recognition of miRNA-21, a structural switch in one hairpin triggers polymerase extension, nicking, and strand displacement all without auxiliary probes. This initiates a self-propagating disassembly cascade, unfolding additional hairpins and exponentially amplifying the signal. Under the optimized conditions, the platform demonstrates femtomolar-level sensitivity (∼1 × 10^–15 M) across a six-order dynamic range, with single-nucleotide mismatch discrimination and negligible cross-reactivity to unrelated miRNAs. Notably, A-MF-HP retains full functionality after 12 h in 10% human serum, and clinical application to blood samples from lung cancer patients revealed marked fluorescence elevation compared to healthy controls. By uniting biostability, multifunctionality, and autonomous amplification in a single programmable probe, this novel strategy addresses limitations commonly observed in multi-component isothermal amplification assays, such as poor nuclease tolerance and dependence on multiple separate probes, offering a powerful diagnostic tool for microRNA profiling in biomedical and clinical settings.
- Multifunctional hairpin probe,
- Biostability,
- Palindromic structure,
- DNA assembly,
- miRNA analysis,
- Isothermal amplification

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References(43)

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