APPLICATION OF SCREEN-PRINTED ELECTRODES AS TRANSDUCERS IN AFFINITY FLOW-THROUGH SENSOR SYSTEMS

An affinity flow-through sensor system based on a heterogeneous compet itive affinity assay for the determination of low molecular weight com pounds is described using the examples of biotin and atrazine determin ation. The binding proteins, either streptavidin or a biotinylated mon oclonal antibody, were immobilized on a biotinylated screen-printed el ectrode, where the competition between the analyte and an analyte-enzy me-conjugate took: place. Determination of the bound enzyme was done t hrough the supply of suitable enzyme substrates and electrochemical de termination of an enzyme reaction product. In the assays described her e, peroxidase was used as enzyme label. As hydrogen peroxide and hydro quinone were used as enzyme substrates, the amount of enzyme retained at the screen-printed graphite electrode was determined amperometrical ly at a reducing potential of - 600 mV vs a screen-printed platinum el ectrode. The activation of the electrode by biotinylation was done in a batch procedure outside the system, before the electrode was inserte d. All following steps of the assay were performed automatically in an unsegmented flow-through system through an appropriate delivery of re quired reagents. The system was optimized mainly through the determina tion of biotin. This assay was based on the competition between biotin and biotinylated peroxidase for the binding sites of streptavidin. Th e method showed a linear range from 0.045 to 2 mu g/l (r(2) = 0.9997, n = 7) with RSD lower than 3.8%. The system was modified further by us ing a biotinylated monoclonal antibody against atrazine for analyte re cognition and performing a competitive assay between atrazine and a tr iazine-peroxidase-conjugate. The linear range was from 0.01 to 10 mu g /l, with IC50 = 0.4 mu g/l and RSD lower than 4.6%. The method was als o applied to atrazine spiked water samples. Regeneration of the sensor surface was based on removal of streptavidin in both assays. (C) 1998 Elsevier Science S.A. All rights reserved.