Abstract: p-Aminophenyl---galactopyranoside (PAPG) was shown to be a suitable substrate for the amperometric detection of galactosidase activity at neutral pH. The application of this amplification system for immunoassay was demonstrated. The product of the enzyme reaction, p-aminophenol (PAP), was detected at 200 mV, vs. , by flow-injection analysis (FIA), with a 50 nM detection limit. PAPG was hydrolyzed more than 2.5 times faster than p-nitrophenyl---galactopyranoside, by the enzyme. Both PAP and PAPG were stable at pH 7. The galactosidase concentration could be measured down to a concentration of 100 fM, and mouse IgG could be assayed by sandwich immunoassay down to 700 fM. PAPG was found to be a promising reagent for heterogeneous systems, like the one described, and for homogeneous assays of biological fluids.

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 λexc = 345 nm and detected at λem = 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 ~0.02 µM in both cases. Amplification factors 8-12 were estimated Phenol and catechol can be determined in the approximately linear ranges 0.1-2 and 0.02-2 µM, respectively. It is possible to perform 20 phenol detections/h by the automatic FIA procedure with high operational stability.