Magnetic separation coupled with gold nanoparticle-assisted hybridization chain reaction for simultaneous detection of exomiR-21 and exomiR-155.

[BACKGROUND] Exosomal miRNA-21 (exomiR-21) and miRNA-155 (exomiR-155) are critical biomarkers for breast cancer diagnosis, reflecting dynamic tumor status. However, simultaneous detection of these low-abundance exosomal miRNAs remains challenging: their trace levels hinder efficient capture, and conventional methods lack adequate signal amplification. Although hybridization chain reaction (HCR) facilitates biomolecule amplification, its standalone use still fails to meet the stringent sensitivity and specificity required for multiplex exosomal miRNA detection. This underscores the urgent demand for innovative biosensing strategies that integrate efficient separation, enhanced amplification, and precise readout to significantly advance breast cancer diagnostics.

[RESULTS] This study presents an innovative dual-miRNA biosensor that integrates magnetic separation with cascade signal amplification for simultaneous detection of miR-21 and miR-155. The system employs a Boolean "AND" logic gate mechanism, where biotinylated cDNA-iDNA-bDNA complexes immobilized on streptavidin-coated magnetic beads ensure highly specific detection requiring concurrent presence of both targets to initiate signal generation. Through a multi-stage amplification strategy involving tetrahedral DNA nanostructure (TDN)-modified electrodes and functionalized gold nanoparticles (AuNPs)/silver nanoparticles (AgNPs), the biosensor achieves exceptional sensitivity with detection limits of 90 aM for miR-21 and 409 aM for miR-155, while maintaining outstanding specificity (<5 % cross-reactivity). The synergistic combination of AuNPs (providing high probe-loading capacity) and AgNPs (enhancing electrochemical signals via redox reactions) enables formation of three-dimensional branched DNA superstructures that amplify signals. Clinical validation demonstrated high accuracy in discriminating breast cancer patients from healthy controls through exosomal miRNA profiling, highlighting the platform's strong potential for point-of-care cancer diagnostics and clinical applications.

[SIGNIFICANCE AND NOVELTY] This work achieves novelty by innovatively integrating magnetic bead separation, logic-gated target recognition (Boolean "AND" operations to distinguish miR-21/miR-155 alone from miR-21/miR-155 co-presence), and multi-stage signal amplification (HCR with AuNPs/AgNPs enhancement) into a unified "separation-amplification-detection" system. Tetrahedral DNA nanostructure (TDN)-modified electrodes form a 3D nano-interface, boosting probe immobilization efficiency and reaction kinetics while lowering background interference. These advances establish a robust platform for multiplexed biomarker detection with high specificity, showing strong potential for clinical precision diagnostics.