Development of a Multiplex Lateral Flow Immunoassay for the Detection of Antibiotics in Milk Utilizing Lyophilized Gold Nanoparticle Conjugates

Lateral flow immunoassays (LFAs) are widely recognized as a powerful and versatile analytical platform. Nevertheless, the development of multiplex formats remains a distinct challenge. The aim of this study was to develop a multiplex LFA using gold nanoparticles (GNPs) as a label, selected for their ease of synthesis and functionalization with biomolecules. We provide practical recommendations regarding protein–hapten synthesis, membrane selection, application buffer composition, and methods to improve the long-term stability of the freeze-dried gold conjugate. The developed assay shows good tolerance to high-fat milk, stability at elevated temperatures, and promising sensitivity, with visual detection limits of 4–100 ng/mL for (Formula presented.) -lactams, 1–10 ng/mL for tetracyclines, 50 ng/mL for streptomycin, and 0.3 ng/mL for chloramphenicol. © 2025 by the authors.