A recombinant Fab fragment-based electrochemical immunosensor for the determination of testosterone in bovine urine

This work describes the development of an electrochemical, recombinant Fab fragment-based immunosensor for the detection of testosterone in bovine urine. The sensor comprised of a testosterone conjugate on the surface of screen-printed electrodes, and recognition followed by an anti-testosterone Fab fragment. The use of an IgG-horseradish peroxidase conjugate determined the degree of competition. Chronoamperometry at a potential of +100 mV, was chosen to reductively measure the product of the catalysis of 3,3',5,5'-tetramethylbenzidine catalysis. ELISA was primarily used to investigate the assay system, prior to transferring to SPEs. The final Fab-based sensor exhibited the linear range of 300-40,000 pg/ml with limit of detection of 90 +/- 13 pg/ml. Furthermore, the developed Fab sensor allowed for the determination of testosterone in bovine urine directly after dilution, omitting the necessity of extraction and hydrolysis. Comparison of administrated bovine urine samples between the developed Fab sensor and GC-MS data showed quantitative or semi-quantitative results and enabled identification of suspicious samples for further extensive analysis by established analytical techniques. With simple sample preparation, low limit of detection, and good repeatability, the proposed method can offer alternative advantages as a primary screening tool for meat quality control. (c) 2006 Elsevier B.V. All rights reserved.