Protein-free colorimetric biosensor for sensitive detection of HPV-18 mRNA via MNAzyme-driven catalytic hairpin assembly

Cervical cancer is significantly associated with persistent infection of human papillomavirus (HPV) type 18, making accurate detection of low-abundance HPV-18 mRNA crucial for diagnosis and treatment. Current nucleic acid detection methods are often limited by complex instrumentation, time-consuming procedures, or fluorophore modifications. To address this, we developed an enzyme-free colorimetric biosensor by integrating a multicomponent nucleic acid enzyme (MNAzyme) with catalytic hairpin assembly (CHA) for signal amplification. In this strategy, target recognition triggers the formation of an active MNAzyme that cleaves a substrate hairpin, initiating a CHA cascade which releases G-rich sequences. These sequences form G-quadruplexes with peroxidase-like activity, catalyzing the oxidation of colorless TMB into a soluble blue product, enabling both visual detection and spectrophotometric quantification at 650 nm. Under optimized conditions, the biosensor achieved a detection limit of 6.92 pM for HPV-18 mRNA, allowing naked-eye identification of target levels as low as 10 pM. The platform exhibited high specificity and could be adapted to detect other HPV mRNA subtypes by simply modifying the recognition sequence. This work presents a universal, simplified, and sensitive colorimetric strategy with great promise for future development of point-of-care testing and viral analysis platforms.