A novel method for time-resolved fluorimetric determination and imaging of the activity of peroxidase, and its application to an enzyme-linked immunosorbent assay.

A new type of fluorescence assay for the determination of peroxidase (POx) activity is presented. The assay is based on the indication of the enzymatic consumption of H2O2 (HP), using a fluorescent europium–tetracycline (Eu3TC) complex as indicator. On addition of HP, this complex forms a highly fluorescent adduct (Eu3TC–HP), which is decomposed in the presence of POx to form the weakly fluorescent europium–tetracycline (Eu3TC). Hence, the activity of the enzyme can be directly determined by means of the luminescent Eu3TC complex as indicator. The POx assay demonstrated herein was elaborated starting from a spectral characterization of the complex systems involved. Due to the long lifetime of lanthanide luminescence, both steady-state and time-resolved luminescence assays can easily be performed. The time-resolved assay can quantify POx in the range from 4.0×10−5 to 5.9×10−3 U mL−1, with a limit of detection of 1.0×10−5 U mL−1. The effects of POx inhibitors such as cyanide, hydroxylamine, and azide have also been studied. In addition, a time-resolved fluorescent detection method for a POx-based enzyme-linked immunosorbent assay (ELISA) has been developed, which is demonstrated in a sandwich model assay with bovine IgG serving as analyte. Furthermore, a time-resolved fluorescent imaging method is demonstrated that makes use of a straightforward imaging set-up adjusted to the optical properties of the europium reagent.