A Two‐Stage Cascading Amplification Strategy Based on Zn ²⁺ ‐Doped WO _X Nanozymes for Ultrasensitive Lateral Flow Immunoassays of Clostridium Difficile Toxin B

Abstract Clostridium difficile is one of the primary causative agents of nosocomial antibiotic‐associated diseases. Early detection and prevention are effective strategies to curb disease transmission, which often requires highly sensitive point‐of‐care testing (POCT) methods. Although lateral flow immunoassay (LFIA) technology—known for its convenience, speed, and cost‐effectiveness—has gained prominence in POCT, its application in early screening remains limited due to relatively low sensitivity. Herein, a two‐stage cascading enhancement strategy based on reductase‐like and peroxidase‐like activities of nanozyme to construct a colorimetrically enhanced LFIA platform is proposed. Specifically, Zn ²⁺ ‐doped WO _X (Zn/WO _X ) loaded with Au nanoparticles (Zn/WO _X @Au) first generates an initial colorimetric signal through immunochromatography. Subsequently, in situ light‐induced deposition of Pt creates Zn/WO _X @Au@Pt, which exhibits enhanced peroxidase‐like activity based on optimal band structure between the components for colorimetric signal amplification. As a result, a 500‐fold enhancement in the visual limit of detection (LOD) is achieved. Moreover, the fitted LOD based on grayscale analysis for Clostridium difficile toxin B (Tcd B) reaches 0.01 ng mL ⁻¹ . This strategy not only maintains the operational simplicity of LFIA but also considerably strengthens the colorimetric signal, offering a novel approach for highly sensitive colorimetric‐enhanced LFIA detection.