Micro volume rotating disk electrode (RDE) amperometric detection for a bead-based immunoassay

The application of the rotating disk electrode (RDE) directly on a micro drop of 50 mu l has been developed for electrochemical detection in a bead-based immunoassay for the model analyte mouse IgG. The enzyme label alkaline phosphatase (ALP) was used for enzymatic turnover of the substrate 4-aminophenyl phosphate (PAPP) to the electroactive product 4-aminophenol (PAP), The determination of 4-aminophenol by the RDE has a linear range from 1.9 x 10(-8) to 1.9 x 10(-4) M. Direct analysis in a drop minimizes enzyme product dilution, a key requirement in achieving very low limits of detection. Furthermore, the detection scheme allows enzyme turnover to be monitored continuously, thus reducing errors associated with obtaining data at fixed times as in flow injection analysis. An unoptimized calibration curve for mouse IgG was obtained from 50 to 5000 ng/ml. (C) 1999 Elsevier Science B.V. All rights reserved.