Four-in-one multifunctional CoCu-NC@AuPt nanozyme integrated M13 phage-displayed nanobody based multimodal lateral flow immunoassay for bovine lactoferrin detection

Bovine lactoferrin (LF) is widely used as a nutritional additive in infant formula and functional foods, but is also a milk allergen. Therefore, rapid and sensitive detection of LF is crucial for ensuring food quality and protecting public health. Here, we report a multimodal lateral flow immunoassay (LFIA) for LF detection that integrates an M13 phage-displayed nanobody (M13-Nb) with a “four-in-one” multifunctional nanozyme, namely an AuPt nanoparticle-loaded CoCu-embedded nitrogen-doped carbon nanocomposite (CoCu-NC@AuPt). The specific M13-Nb against LF was isolated from a naïve nanobody library. The synthesized CoCu-NC@AuPt exhibits magnetic, colorimetric, peroxidase-mimetic, and photothermal properties, enabling magnetic enrichment and triple-mode sensing (colorimetric, catalytic colorimetric, and photothermal modes). The proposed assay achieved detection limits of 98 pg/mL, 8.49 pg/mL, and 37 pg/mL for the three detection modes, respectively. Practical applicability was verified in pasteurized milk, UHT milk, and yogurt samples, with recoveries of 86.2%–112.5% and coefficients of variation below 15%. This work provides a reliable and sensitive platform for on-site LF detection and demonstrates the potential of combining CoCu-NC@AuPt nanozyme with phage-displayed nanobodies to improve point-of-care testing performance.