Click chemistry-empowered multi-channel biosensing for highly-efficient detection of antibiotic resistance genes in aquatic environments

Antibacterial resistance in microbial infection poses a serious global threat to both human health and environmental safety. Accurate, sensitive and rapid detection of antibiotic resistant genes (ARGs) is essential for comprehensive evaluation of aquatic environmental risks. However, conventional ARG detection methods strongly rely on nucleic acid amplification based on complete DNA template, failing to capture highly degraded ARG fragments, particularly free ARGs in complex environmental matrices. In this study, we present a novel biolayer interferometry-based multi-channel biosensing platform for ARGs detection that breaks through the framework of nucleic acid amplification. By leveraging a click chemistry-mediated ligation chain reaction and enzyme labeling signal amplification strategy, our platform achieves highly-efficient detection of tetracycline- and sulfonamide-related ARGs (tetA, tetC, tetG, tetM and sul1) at a detection limit of 602 copies/µL, with a response time of less than 30 min. This platform demonstrates excellent accuracy, sensitivity, selectivity, and repeatability using renewable biosensors, enabling direct detection of ARG levels in practical wastewater from farms, hospitals and wastewater treatment plants. The simple “dip-and-read” operation allows for real-time monitoring of dynamic changes in ARG levels in aquatic environments. This BLI platform provides a powerful tool for early warning, risk assessment, and improved management of ARGs, offering substantial potential for safeguarding public and ecological health.