Logic-gated fluorescent biosensor integrating aptamer recognition and oxidative cleavage-responsive DNA circuit for myeloperoxidase detection.

BACKGROUND: Precise discrimination between protein abundance and catalytic activity of proteases remains a critical yet challenging objective in biomedical diagnostics due to overlapping biological functions, intricate regulatory mechanisms, and extensive interference from endogenous biomolecules. METHODS: Herein, we report a novel dual-lock DNA biosensing platform, exemplified through myeloperoxidase (MPO), which concurrently integrates aptamer-mediated molecular recognition and hypochlorous acid (HOCl)-triggered oxidative cleavage to rigorously assess both MPO protein expression and enzymatic functionality. Specifically, MPO interaction with a conformationally structured DNA aptamer facilitates selective release of a trigger strand, while HOCl, produced enzymatically by active MPO, cleaves a strategically phosphorothioate-modified hairpin structure. Only upon simultaneous fulfillment of these two molecular conditions does the sensing mechanism activate a downstream catalytic hairpin assembly (CHA), achieving significant signal amplification. RESULTS: This stringent AND logic gate configuration markedly suppresses false positives and nonspecific background signals, demonstrating exceptional reliability across diverse and complex biological samples including serum, saliva, and cellular lysates. CONCLUSIONS: The proposed biosensing strategy thus provides a versatile, accurate, and broadly applicable analytical tool for simultaneous quantification of protease content and functional activity, holding considerable promise for advancing clinical diagnostics and pathological investigations.