Fluorescence‐Enhanced Catalytic Hairpin Assembly‐Driven Nanobiosensor for Ultrasensitive Detection of HPV16 E7 mRNA

ABSTRACT Early detection of human papillomavirus type 16 (HPV16), the primary etiological agent of cervical cancer, is essential for effective clinical management. Specifically, detecting HPV16 E7 mRNA expression provides superior prognostic value, identifying transcriptionally active infections most likely to progress to precancerous lesions. In this study, we developed a fluorescence‐based nanobiosensor that integrates catalytic hairpin assembly (CHA) with Fe 3 O 4 @Au core–shell nanoparticles (Fe 3 O 4 @Au NPs). This novel design combines magnetic enrichment and enzyme‐free signal amplification, distinguishing it from prior HPV biosensors by enabling ultrasensitive detection in complex matrices like first‐void urine (FVU). The biosensor showed linear fluorescence responses from 0.002 to 1 pM in PBS and 0.1–1 pM in FVU, with excellent specificity for single‐base mismatch discrimination. It remained stable for 45 days. Validation using RNA from HPV16 plasmid‐transformed E. coli and CaSki cells confirmed robust performance, while clinical swab specimens matched commercial assays completely. This biosensor offers a promising tool for early HPV16 detection in screening programs.