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

摘要

The streptavidin-biotin interaction was monitored on the surface of a pre-treated carbon paste electrode (CPE). This study has been accomplished for the estimation of the sensitivity of the alkaline phosphatase/3-indoxyl phosphate (AP/3-IP) couple when it was used as a detection scheme in voltammetric affinity devices. The enzymatic turnover of the substrate gives indigo, which shows two reversible electrode processes at potentials around -0.4 and +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 the passive adsorption of streptavidin on the electrode surface and subsequent reaction with AP-labelled biotin (B-AP). The calibration curve obtained for this reaction, and expressed in terms of AP concentration, exhibits a linear dynamic range from 10(-13) to 10(-11) M, with a detection limit of 2x10(-13) M (S/N=3). These results were compared with those calculated when the labelled biotin was adsorbed directly on the electrode surface. The second approach was focused on the development of a carbon paste probe for the detection of biotin hydrazide, based on a sequential competitive assay format. 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. [References: 28]