Immunosensing with amperometric detection, using galactosidase as label and P-aminophenyl-β-D-galactopyranoside as substrate

p-Aminophenyl-β-D-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. Ag/AgCl, by flow-injection analysis (FIA), with a 50 nM detection limit. PAPG was hydrolyzed more than 2.5 times faster than p-nitrophenyl-β-D-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 homogenous assays of biological fluids.