Whole-Cell Fiber-Optical Biosensor for Detecting P. aeruginosa Via Secreted Quorum-Sensing Molecule Using Bioluminescent Bioreporters

The opportunistic Gram-negative bacterium Pseudomonas aeruginosa poses significant challenges in both clinical and environmental settings due to its pathogenicity and resilience. Quorum-sensing (QS) molecules have emerged as promising biomarkers for the detection of P. aeruginosa. This study focused on the development of an alginate-based bioreporter encapsulation system designed for the identification of QS molecules, utilizing a custom-engineered coating formed through the assembly of poly-lysine (PLL). This PLL coating facilitates the passive diffusion of external QS molecules, resulting in a measurable, dose-dependent bioluminescent response. The microbeads reinforced with PLL exhibited substantial stability in the presence of cation scavengers, enabling prolonged shelf life and functionality. The encapsulated bacterial system demonstrated consistent, dose-dependent detection of QS molecules in media containing synthetic autoinducers and in cell-free supernatants derived from wild-type P. aeruginosa (PAO1) cultures. These bioreporter beads maintained their stability during extended storage at both 4 °C and -80 °C, allowing for immediate, on-site sensing without the need for recovery processes. The RhlR-based bioreporter displayed a dynamic detection range of 10 μM to 0.1 nM, with a sensitivity threshold of 50 pM for the designated QS molecules. The LasR-based bioreporter demonstrated a broader range of 5 μM to 0.2 nM and a lower detection threshold of 0.1 nM for 3-oxo-C12-HSL. Furthermore, the bioreporter beads effectively detected the presence of the synthetic QS inhibitor furanone C-30 in a dose-dependent manner. This proof-of-concept optical-fiber-based whole-cell biosensor illustrates the feasibility of employing an encapsulated bioreporter system for the detection of bacteria through specific QS molecules. The results of this study hold promise for potential applications in diagnostics, environmental monitoring, and screening for quorum-sensing inhibitors.