biostudies-literatureUnknown15(9)Maksin IVLateral 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 β-lactams, 1-10 ng/mL for tetracyclines, 50 ng/mL for streptomycin, and 0.3 ng/mL for chloramphenicol.Biosensors592https://www.ebi.ac.uk/biostudies/studies/S-EPMC12467014biostudies-literatureDevelopment of a Multiplex Lateral Flow Immunoassay for the Detection of Antibiotics in Milk Utilizing Lyophilized Gold Nanoparticle Conjugates.PMC12467014Kesareva VAKuandykova APolyakova DIKhunteev GAMaksin IVKirillova YGSimonova EIIvanov VSLuzyanin TAfalseDevelopment 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 β-lactams, 1-10 ng/mL for tetracyclines, 50 ng/mL for streptomycin, and 0.3 ng/mL for chloramphenicol.2025-01-01T00:00:00Z2025 Sep2026-05-01T03:19:23.499Z2026-05-01T03:10:46.373ZS-EPMC124670144100233210.3390/bios15090592