Development of a Rapid Spore-Based Paper-Strip Assay for Detection of Antibiotic Residues in Milk

In this study, a spore-based paper-strip assay was developed for rapid, on-site screening of antibiotics in milk. An α-d-glucosidase was identified as the optimal marker enzyme, producing a visible color signal within 30 min. Standardized production of Geobacillus stearothermophilus 331 spores served as a stable biological sensing element. Optimization of germinants, nutrient supplements, sporulation conditions, and strip-loading parameters enabled efficient spore germination and enzyme release, reducing assay time to 60-90 min. Thermal activation of spores at 100°C for 20 min improved sensitivity while maintaining signal clarity, and the final device was assembled on Whatman Grade 3 filter paper with immobilized chromogenic substrate and activated spores. The assay detected 42 antibiotics across 14 classes, including β-lactams, aminoglycosides, tetracyclines, macrolides, sulfonamides, fluoroquinolones, polymyxins, nitrofurans, glycopeptides, ionophores, rifamycins, lincosamides, trimethoprims, and others, with limits of detection at or below regulatory maximum residue limits (MRLs). Field validation on 529 raw milk samples showed 15.63% positivity rate with 100% agreement with the commercial spore-based kit and CHARM II and ROSA assays. The assay functions as a qualitative to semi-quantitative screening tool based on visual color inhibition (where complete or partial lack of color development indicates residue levels at or near regulatory MRLs) and is not intended for exact quantification or identification of individual antibiotic concentrations, which requires instrument-based confirmatory analytical methods. The assay also performed reliably on pasteurized and dried milk products. The developed strip assay offers a rapid, cost-effective, user-friendly, field-deployable tool for routine screening of antibiotic residues in milk. PRACTICAL APPLICATIONS: The developed spore-based paper-strip assay provides a rapid, cost-effective, and portable tool for on-site screening of antibiotic residues in milk without requiring sophisticated laboratory equipment. Its ability to detect 42 antibiotics from 14 classes within 60-90 min makes it suitable for routine monitoring at dairy farms, milk collection centers, and processing plants. The assay can facilitate an early identification of contaminated milk, thereby enhancing food safety, regulatory compliance, and antimicrobial stewardship across the dairy value chain.