Triple-signal lateral flow immunoassay for the detection of bovine casein using an M13 phage-displayed nanobody and MnO2 nanozyme conjugate

Cow's milk allergy is a significant global health concern, with bovine casein recognized as its predominant allergen. Rapid, sensitive, and accurate detection of bovine casein in food products is therefore essential. Here, a triple-signal lateral flow immunoassay (LFIA) for casein detection was developed by integrating an M13 phage-displayed nanobody (M13-Nb) with multifunctional MnO 2 nanosheets (MnO 2 NSs) as the signal probe. Anti-casein M13-Nb was selected from a naïve phage-displayed nanobody library through four rounds of biopanning. MnO 2 NSs exhibiting intrinsic colorimetric properties, oxidase-like catalytic activity, and photothermal capability were synthesized and conjugated with M13-Nb, enabling the MnO 2 NSs@M13-Nb-based LFIA to generate colorimetric, amplified colorimetric, and photothermal signals simultaneously. The visual limits of detection (vLOD) for these modes were 125, 15.63, and 31.25 ng/mL, respectively, with corresponding quantitative LODs of 60.97, 6.72, and 23.88 ng/mL. The assay showed high specificity and accuracy, with recovery rates of 87.92%–114.25% in spiked food samples. This triple-signal LFIA provides a rapid and reliable platform for casein detection in foods and highlights the potential of combining M13-Nb and MnO 2 NSs for on-site monitoring of food allergens.