Femtomolar miRNA-21 detection in serum via a Y-shaped DNA assembly amplified catalytic hairpin assembly and Bi@PtAg nanocatalyst-driven electrochemical biosensor

MicroRNA-21 (miRNA-21) is a key oncogenic biomarker for early cancer diagnosis, yet its ultrasensitive detection in serum remains challenging due to limitations of conventional methods such as low sensitivity, high background, and poor biocompatibility. This study develops an electrochemical biosensor for femtomolar miRNA-21 detection by integrating Y-shaped DNA scaffold-enhanced catalytic hairpin assembly (Y-CHA) and Bi@PtAg nanoclusters (NCs). This design achieved an ultra-low detection limit, approximately 2.8-fold lower than that of the conventional sensor. The Y-CHA strategy spatially confines hairpin probes (H1 and H2) within a Y-DNA scaffold, significantly enhancing signal amplification and hybridization efficiency. Meanwhile, the core-shell structured Bi@PtAg NCs exhibit 2.1-fold higher H₂O₂ reduction catalytic activity than monometallic nanoparticles and demonstrate low cytotoxicity (approximately 80% cell viability at 200 μg·mL⁻¹). The biosensor achieves an ultra-low detection limit of 0.05 fM with a wide linear range from 1 fM to 100 pM (R² = 0.9982). It also displays excellent specificity in distinguishing miRNA-21 from mismatched and non-complementary RNAs, good stability (87.1% signal retention after 8 days), and reliable performance in serum samples (93.0–99.7% recovery, RSD < 5%). This study develops a versatile electrochemical biosensing platform for miRNA analysis and disease biomarker detection.