Logic-Gated Fluorescent Biosensor Integrating Aptamer Recognition and Oxidative Cleavage-Responsive DNA Circuit for Myeloperoxidase Detection

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. 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. 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. 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.