Fondaparinux-Enabled Competitive Lateral Flow Assay for Monitoring Lactoferrin Bioactivity during Dairy Processing

The assessment of lactoferrin (LF) bioactivity is pivotal for determining the nutritional quality and processing history of dairy products; however, current detection methods cannot effectively distinguish between active and thermally denatured forms. Herein, a competitive lateral flow assay (LFA) based on specific electrostatic binding was developed using fondaparinux, a synthetic heparin-like pentasaccharide, as a nonantibody recognition element. To address immobilization challenges, a “BSA-bridging” strategy was employed to construct stable AuNPs-BSA-fondaparinux nanoprobes. Under optimized conditions, the LFA achieved quantitative LF detection within 15 min (LOD 0.94 μg/mL; linear range: 1–200 μg/mL; R 2 = 0.973) with high specificity. Comparisons with high-performance liquid chromatography confirmed the good accuracy of the proposed method. Furthermore, the assay selectively detected the loss of bioactive LF due to thermal denaturation in pasteurized milk. Overall, this work establishes a rapid platform utilizing a synthetic receptor for monitoring lactoferrin bioactivity changes during thermal processing in the dairy industry.