Single-Molecule Detection of Lactoferrin and Osteopontin in Infant Formula and Dairy Products Using FraC Nanopores

Abstract Biological nanopore technology enables single-molecule analysis with promising applications in food quality control. Among dairy-derived bioactive components, lactoferrin (LF) and osteopontin (OPN) are two pivotal whey glycoproteins that support infant development and serve as quality indicators for dairy products. However, their folded tertiary structures and heterogeneous charge distributions complicate accurate analysis of these native proteins. Here, we developed a high-resolution nanopore sensing platform utilizing the conical biological nanopore Fragaceatoxin C (FraC), enabling simultaneous qualitative and quantitative analysis of LF and OPN. The platform distinguished and quantified both proteins in mixtures at varying ratios. It also enabled rapid, label-free detection and quantification in commercial dairy products, including infant formula, without chromatographic separation or complicated sample pretreatment. These findings establish a robust theoretical foundation and practical validation for nanopore technology in food quality control, while opening technical avenues for on-site, real-time monitoring of key functional proteins in dairy products, particularly infant formula.