Triple-step amplified and quadruple-mode readout lateral flow immunoassay based on multifunctional Fe3O4@Ru@Pt nanozyme for enhanced sensitivity and reliability

A multifunctional core-shell Fe 3 O 4 @Ru@Pt nanozyme was synthesized. • A cascade multimodal LFIA for sensitive and reliable detection of β-CG was developed. • A 151-fold sensitivity improvement was achieved through cascade amplification. • Four detection modes provided flexible readout and cross-validation. Traditional lateral flow immunoassays (LFIAs) suffer from limited sensitivity and insufficient reliability, which restrict their practical application. To address these challenges, we synthesized a multifunctional core-shell Fe 3 O 4 @Ru@Pt nanozyme with integrated magnetic, optical, catalytic, and photothermal properties. This nanozyme enables a three-step cascade that enhances sensitivity through sequential magnetic enrichment, catalytic amplification, and photothermal amplification. In addition, four detection modes (direct colorimetric, magnetic-enriched colorimetric, catalytic colorimetric, and photothermal) provide flexible readout and cross-validation to improve reliability. Using a specific phage-displayed nanobody as the recognition element, we developed a Fe 3 O 4 @Ru@Pt-based cascade multimodal LFIA for β-conglycinin detection. The photothermal mode achieved a limit of detection as low as 2.02 pg mL −1 , representing a 151-fold improvement over the direct colorimetric mode. The assay also exhibited excellent specificity and satisfactory accuracy in spiked real samples, with recoveries of 83.21%–114.72%. This multifunctional nanozyme-mediated cascade multimodal platform substantially improves both sensitivity and reliability, offering a promising and practical tool for point-of-care testing applications.