Magnetic microbead-based enzyme immunoassay for ovalbumin using hydrodynamic voltammetry and fluorometric detection

A paramagnetic microbead-based enzyme immunoassay was demonstrated for detecting ovalbumin (OVA). The immunoassay sandwich was made by attaching a biotinylated antibody to the streptavidin coated beads as a mobile solid phase, capturing antigen, and then exposing the antigen to an antibody conjugated with β-galactosidase. β-Galactosidase converts p-aminophenyl galactopyranoside (PAPG) and fluorescein di-β-d-galactopyranoside (FDG) to p-aminophenol (PAP) and fluorescein, respectively. The current response of PAP generated by the enzymatic reaction of β-galactosidase was detected with hydrodynamic voltammetry in a droplet using a rotating disk electrode (RDE) system. The performance of this electrochemical assay was compared with fluorometric detection of fluorescein produced by the same assay system. The limits of detection for OVA determined by hydrodynamic amperometry and fluorometry were 2.1 nM (43 fmol) and 2.9 nM (58 fmol), respectively. Furthermore, the effects of the conditions commonly found in drinking water supply systems on the OVA assay were also evaluated.