Chemiluminescent measurement of peroxidase activity and its application using a lucigenin CT-complex

Luminol (3-aminophthaloylhydrazine) is well known as a chemiluminescent reagent that can be used to measure peroxidase (POD) activity. We have developed a more sensitive assay for POD using a new chemiluminescent reagent based on 10,10'-dimethyl-9, 9'-biacridinium nitrate (lucigenin). This new reagent was obtained from lucigenin by light irradiation in an organic polar solvent such as N,N-dimethylacetamide (DMAc). The detection limit for POD activity using this reagent is 10(-19) mol/assay. In an application for enzyme immunoassay based on a POD label, we were able to detect human alpha-fetoprotein (AFP) at 1pg/mL. The characterization of this chemiluminescent reagent was obtained by optical measurements, and the formation of lucigenin charge transfer complex (CT-complex) was confirmed. We also investigated the stability of lucigenin CT-complex as a chemiluminescent reagent for the measurement of POD activity, and confirmed that the reagent was stable for more than a year.