Ultrasensitive chromogen system for Horseradish Peroxidase Using 3-Methyl-2-benzothiazolinone Hydrazone andN-Phenyldiethanolamine

An ultrasensitive chromogenic system for horseradish peroxidase (HRP) is described. The chromogenic substrate system consists of 3-methyl-2-benzothiazolinone hydrazone (MBTH) andN-phenyldiethanolamine (PDEA). The oxidative coupling of MBTH and PDEA using H₂O₂ and HRP, respectively, as the oxidant and catalyst, yields a blue chromophore that most likely is an indamine dye. The chromophore has a rather broad absorption band, with an absorption maximum at 602 nm. In 0.1M phosphate, 0.1M citrate buffer, pH 7.5, the optimal concentrations of MBTH, PDEA, and H₂O₂ are, respectively, 0.5, 25, and 8 mM. Using this assay system, HRP can be determined in lower picomolar levels by either a rate or fixed-time method.