Dual-signal amplification via high-entropy alloy nanoparticle catalysis and target DNA recycling for ultrasensitive HPV-16/18 detection in human serum

Rapid identification of HPV-16/18 genotypes is critical for cervical cancer prevention, yet remains clinically inaccessible due to inadequate diagnostic sensitivity and specificity. The paper presents an electrochemical DNA biosensor achieving detection of genotypes through an innovative dual-signal amplification cascade. First, an exceptionally active AlCrMnZnAu_0.1 high-entropy alloy was engineered via coordination-driven approach using folic acid and histidine-functionalized boron and nitrogen-doped graphene quantum dot. Synergistic atomic orbital hybridization and tailored support endow unprecedented stability, optimized electron structure and remarkable catalytic activity—outperforming benchmark gold nanoparticle by 6.8-fold. Second, triblock poly-adenine probe (H1) anchored to gold electrode selectively captures HPV-16 or HPV-18 targets. Binding initiates the unfolding of secondary structures: H2 (tagged with AlCrMnZnAu_0.1/thionine) for HPV-16, and H3 (tagged with AlCrMnZnAu_0.1/ferrocene) for HPV-18. Crucially, target DNA is regenerated through toehold-mediated strand displacement, enabling geometric signal amplification. Concurrently, AlCrMnZnAu_0.1 intensifies electrochemical responses by catalytically boosting thionine and ferrocene redox reactions. This orchestrated dual-amplification strategy delivers unmatched specificity and sensitivity for HPV-16/18. The platform exhibits an exceptionally broad linear response (10⁻¹⁸ to 10⁻¹³ M) for both targets and achieves ultralow detection limit of 2.9 × 10⁻¹ ⁹ M, representing a transformative advancement over existing state-of-the-art diagnostic sensors for point-of-care deployment.