A Dual-Mode Fluorescence-Surface-Enhanced Raman Scattering Probe Integrated with Magnetic Separation and DSN Amplification for miR-10b Detection in Cancer Serum

Abstract Developing highly sensitive and reliable methods for microRNA detection is crucial for early cancer diagnosis. Herein, a dual-mode fluorescence (FL) and surface-enhanced Raman scattering (SERS) probe was constructed for the ultrasensitive detection of miR-10b, integrating magnetic separation and duplex-specific nuclease (DSN)-assisted target recycling amplification. The probe consists of Fe3O4@AuNPs modified with Cy5-labeled hairpin DNA (H1). Without miR-10b, Cy5 is fluorescence resonance energy transfer (FRET)-quenched by AuNPs (low FL) but SERS-enhanced by localized surface plasmon resonance (LSPR) (strong SERS). Upon target recognition, H1 opens and forms a DNA-RNA duplex, which DSN cleaves, releasing Cy5 fragments and intact miR-10b for recycling. This disrupts FRET and LSPR, causing FL recovery and SERS decrease. Under optimized conditions, the probe exhibited excellent linear responses over a concentration range of 101 to 105 pM in both modes, with detection limits as low as 18.20 fM (FL) and 20.33 fM (SERS). The probe demonstrated outstanding selectivity, excellent reproducibility (batch-to-batch RSD < 2.26%), and good stability over 28 days of storage. Furthermore, the practical applicability was validated by analyzing miR-10b in spiked human serum (recoveries: 99.50%–104.80% for FL; 101.30%–104.09% for SERS) and clinical serum samples from lung, breast, and thyroid cancer patients, where the probe successfully distinguished cancer patients from healthy controls. This dual-mode strategy provides a sensitive and reliable platform for early cancer diagnosis via mutual signal cross-validation and effective suppression of false positives.