Renewable fluorometric enzyme immunosensing system using prochlorperazine as the substrate for horseradish peroxidase for the determination of transferrin

Abstract

A renewable fluorometric enzyme immunosensing system using prochlorperazine (PCP) as the substrate for horseradish peroxidase (HRP) has been developed for the determination of transferrin in serum samples. This system is based on paraffin–graphite–transferrin antibody immunocomposite, which permits its surface to be easily regenerated by simply polishing. The renewed surface served as a platform for the competitive immunoreaction of HRP–transferrin and free transferrin (analyte) with the immobilized antibody bound to the surface and for HRP-catalyzed reaction. By using prochlorperazine as the substrate for HRP, the amount of HRP–transferrin bound was quantified fluorimetrically, which was in turn related to free transferrin content in the samples. Prochlorperazine as the substrate, possesses a satisfactory activity towards HRP as compared to p-hydroxyphenylacetic acid. The analytical conditions, such as pH, the amount of labeled transferrin, incubation time, and temperature were studied in detail. Under the optimized experimental conditions, a detection limit of 7.3 ng/ml and the linear detection ranging from 7.3 to 127 ng/ml were obtained. This approach has been applied to clinical samples with satisfactory results.