An enzymatic amplification Flow Injection Analysis (FIA) system for the sensitive determination of phenol

Abstract

A highly sensitive, enzymatic FIA procedure was developed to determine phenol fluorimetrically. Tyrosinase was immobilized in a packed bed flow reactor. Upon contact with tyrosinase, phenol is oxidized to o-benzoquinone, which oxidizes ascorbic acid to dehydroascorbic acid producing catechol, which is enzymatically reoxidized to o-benzoquinone. Dehydroascorbic acid reacts with o-phenylenediamine forming a highly fluorescent product, which is excited at λ exc=345 nm and detected at λ em=410 nm. Dehydroascorbic acid was detected in the range between 0.5 and 100 μM. The chemoenzymatic substrate recycling enhances the sensitivity of the detection of phenol and catechol with a detection limit of around 0.02 μM in both cases. Amplification factors between 8 and 12 were estimated. Phenol and catechol can be determined in the approximately linear ranges between 0.1 and 2 μM and between 0.02 and 2 μM, respectively. It is possible to perform 20 phenol detections per hour by the automatic FIA procedure with high operational stability.