Detection of NSCLC biomarker miRNAs via 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 via 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.