Fiber optic monooxygenase biosensor for toluene concentration measurement in aqueous samples

Measurements of pollutants such as toluene are critical for the characterization of contaminated sites and for the monitoring of remediation processes and wastewater treatment effluents. Fiber optic enzymatic biosensors have the potential to provide cost-effective, real time, continuous, in situ measurements. In this study, a fiber optic enzymatic biosensor was constructed and characterized for the measurement of toluene concentrations in aqueous solutions. The biological recognition element was toluene ortho-monooxygenase (TOM), expressed by Escherichia coli TG1 carrying pBS(Kan)TOM, while an optical fiber coated with an oxygen-sensitive ruthenium-based phosphorescent dye served as the transducer. Toluene was detected based on the enzymatic reaction catalyzed by TOM, which resulted in the consumption of oxygen and changes in the phosphorescence intensity. The biosensor was found to have a limit of detection of 3 μM, a linear signal range up to 100 μM, and a response time of 1 h. The performance was reproducible with different biosensors (RSD=7.4%, n=8). The biosensor activity declined with each measurement and with storage time, particularly at elevated temperatures. This activity loss could be partially reversed by exposure to formate, suggesting that NADH consumption was the primary factor limiting lifetime. This is the first report of an enzymatic toluene sensor and of an oxygenase-based biosensor. Since many oxygenases have been reported, the design concept of this oxygenase-based biosensor has the potential to broaden biosensor applications in environmental monitoring.