A Spore-Based Biosensor-on-Pillar Platform for Detecting ß-Lactam Antibiotics in Milk

Antimicrobial resistance (AMR) is increasingly becoming a major global public health concern, as antibiotics are losing their effectiveness at an alarming rate due to drug resistance. The ß-lactam group of antibiotics are widely used in dairy farms to treat animal infections, and their presence in the food chain is a significant concern. Addressing this issue requires the development of effective analytical tools for the rapid detection of antibiotics. In this work, a miniaturized Biosensor-on-Pillar platform was developed for detecting ß-lactam antibiotics in milk, which operates in a rapid, cost-effective, and user-friendly format, making it particularly suitable for resource-limited settings. The platform employs an enzyme induction-based approach, wherein Bacillus cereus spores germinate in the presence of β-lactam antibiotics, leading to the production of β-lactamase enzyme, which is then recognized using a chromogenic substrate functionalized on paper associated with the pillar platform. The developed biosensor can detect 12 β-lactam antibiotics with limits of detection (LODs) ranging from 1 to 1000 ppb, achieving sensitivity at or below the maximum residue limits (MRLs) set by regulatory bodies (FSSAI/CODEX) for the majority of the tested antibiotics. The performance of the platform, including the design, fabrication, and working principle, was further evaluated by analyzing six blind milk samples, yielding significant results compared to the commercially available AOAC-approved gold-standard method. Hence, the developed biosensor demonstrates promising potential for the rapid, cost-effective and high-throughput screening of milk samples for β-lactam antibiotics, benefiting the dairy industry and ensuring food safety.