Novel electrochemical assay for H2O2 determination in aqueous solutions: A non time-critical method for H2O2 trace level detection

A two-step method for H2O2 trace level determination has been developed, utilizing, for the first time, the fluorogenic peroxidase substrate, Amplex Red (N-acetyl-3,7-dihydroxyphenoxazine), in conjunction with an electroanalytical detection scheme: Upon peroxidase catalyzed turnover of H2O2 with Amplex Red, the fluorescent and electroactive product resorufin (7-hydroxy-3H-phenoxazin-3-one) was detected amperometrically in a thin-layer flow cell with high selectivity. A linear detection range from 100 nmol L-1 through 1000 nmol L-1 was obtained. The performance was validated using exhaled breath condensates (EBC) with and without H2O2 added, as real samples. Due to the improved chemical stability of resorufin as compared to H2O2, processing larger numbers of EBC samples is greatly facilitated with the new assay, since the enzymatic reaction can be performed up to 18 hours before the actual measurement.