Voltammetric monitoring of the interaction between streptavidin and biotinylated alkaline phosphatase through the enzymatic hydrolysis of 3-indoxyl phosphate

The streptavidin-biotin interaction was monitored on the surface of a pre-t reated carbon paste electrode (CPE). This study has been accomplished for t he estimation of the sensitivity of the alkaline phosphatase/3-indoxyl phos phate (AP/3-IP) couple when it was used as a detection scheme in voltammetr ic affinity devices. The enzymatic turnover of the substrate gives indigo, which shows two reversible electrode processes at potentials around -0.4 an d +0.3 V (versus Ag/AgCl) in a 0.1 M Tris buffer solution pH 7.2. The peak current of the second reduction process constitutes the analytical signal. Two different approaches have been developed. The first one was based on th e passive adsorption of streptavidin on the electrode surface and subsequen t reaction with AP-labelled biotin (B-AP). The calibration curve obtained f or this reaction, and expressed in terms of AP concentration, exhibits a li near dynamic range from 10(-13) to 10(-11) M, with a detection limit of 2x1 0(-13) M (S/N=3). These results were compared with those calculated when th e labelled biotin was adsorbed directly on the electrode surface. The secon d approach was focused on the development of a carbon paste probe for the d etection of biotin hydrazide, based on a sequential competitive assay forma t. A calibration curve in the range of 10(-13)-10(-11) M was obtained with an estimated detection limit of 3 x 10(-13) M. (C) 2000 Elsevier Science B. V. All rights reserved.