Detection of NSCLC biomarker miRNAs <i>via</i> localized catalytic hairpin self-assembly and laser-induced fluorescence on a microfluidic platform

This study introduces an innovative microfluidic platform integrating localized catalytic hairpin assembly (LCHA) amplification with laser-induced fluorescence (LIF), for sensitive detection of non-small cell lung cancer (NSCLC) biomarkers miRNA-20a and miRNA-223. The enzyme-free LCHA strategy leverages two hairpin probes (Hp1/Hp2) anchored on a DNA tetrahedron framework, where spatial confinement enhances reaction kinetics by elevating local probe concentrations. Hp2 is dual-labeled with 6-FAM and BHQ-1 for fluorescence quenching control. Target miRNA initiates a cascade reaction through magnetic bead-conjugated capture probes (MBCP): miRNA binding triggers structural unfolding of Hp1 on DTP, which subsequently activates Hp2 to separate 6-FAM from BHQ-1, restoring detectable fluorescence. The self-recycling amplification is achieved <i>via</i> trigger release from MBCP. Magnetic separation replaces capillary electrophoresis, while microfluidic mixing intensifies LCHA efficiency. Demonstrating high specificity, the assay achieves detection limits of 3.96 fmol (miR-20a) and 3.75 fmol (miR-223). The modular design, synergizing spatial confinement and magnetic manipulation, not only offers a robust NSCLC diagnostic tool but also holds adaptability for detecting other disease-related miRNAs through customizable probe engineering.