Development and validation of a novel fluorometric sensor for hydrogen peroxide monitoring in exhaled breath condensate

Abstract

A novel fluorometric sensor based on catalyzed 5-aminosalicylic acid (5-ASA)/H₂O₂ systems was developed to detect trace levels of H₂O₂ in exhaled breath condensate (EBC). It relies on the oxidative conversion of the fluorophore (5-ASA) into its non-fluorescent product (5-ASA_ox). The fluorescence intensity of 5-ASA was lost by the reaction with H₂O₂ in the presence of horseradish peroxidase (HRP) or CuO nano-particles (NPs). As the decrease in the fluorescence intensity is proportional to H₂O₂ concentration, new methods were developed for the monitoring of H₂O₂ in EBC samples using the HRP- and CuO NP-catalyzed 5-ASA/H₂O₂ assays. The methods were optimized and validated according to the standard validation techniques in EBC samples. Under the optimized experimental conditions, linear calibration graphs were obtained in the range of 200–8000 nmol L⁻¹ and 50–500 nmol L⁻¹ for the HRP-catalyzed and CuO NP-catalyzed 5-ASA/H₂O₂ assays, respectively. The limits of detection, evaluated as 3S_b/m (where S_b and m are the standard deviation of the blank and slope of the calibration curves, respectively), were found to be 137.0 nmol L⁻¹ and 33.6 nmol L⁻¹ for HRP-catalyzed and CuO NP-catalyzed 5-ASA/H₂O₂ systems, respectively. These developed systems are promising for application in the detection of trace amounts of H₂O₂ in oxidative stress studies.