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

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.