Abstract
Lipopolysaccharides (LPS) from Gram-negative bacteria represent a significant challenge across various industries due to their prevalence and pathogenicity and the limitations of existing detection methods. Traditional approaches, such as the rabbit pyrogen test (RPT) and the Limulus Amebocyte Lysate (LAL) assay, have served as gold standards for endotoxin detection. However, these methods are constrained by high costs, lengthy processing times, environmental concerns, and the need for significant reagent volumes, which limit their scalability and application in resource-limited settings. In this study, we introduce an innovative microfluidic platform that integrates the LAL assay within microdroplets, addressing the critical limitations of traditional techniques. By leveraging the precise fluid control and reaction isolation offered by microdroplet technology, the system reduces reagent consumption, enhances sensitivity, and enables high-throughput analysis. Calibration tests were performed to validate the platform's ability to detect LPS, using colorimetric measurements. Results demonstrated comparable or improved performance relative to traditional systems, achieving lower detection limits and greater accuracy. This work demonstrates a proof-of-concept miniaturisation of the pharmacopoeial LAL assay. The method yielded low intra-assay variability (σ ≈ 0.002 OD; CV ≈ 0.9% over n = 50 droplets per point) and a LOD estimated from calibration statistics after path-length normalisation. Broader adoption will require additional comparative validation and standardisation. This scalable, cost-effective, and environmentally sustainable approach offers a practical solution for endotoxin detection in clinical diagnostics, biopharmaceutical production, and environmental monitoring. The proposed technology paves the way for advanced LPS detection methods that meet stringent safety standards while improving efficiency, affordability, and adaptability for diverse applications.