Development of an immunosensor for the detection of testosterone in bovine urine

In the presented work, a disposable immunosensor for the detection of testosterone, an endogenous steroid hormone, in bovine urine has been developed using screen-printed electrodes (SPEs). Due to concerns over the use of steroid hormones as growth promoters, the EU prohibits their use in food producing animals. Consequently, rigorous screening procedures have been implemented in all member states to detect the illegal administration of such compounds. Competitive immunoassays were developed, initially by enzyme linked immunosorbent assay (ELISA), and subsequently transferred to an electrochemical immunosensor format using disposable screen-printed carbon electrodes. Horseradish peroxidase (HRP) was the enzyme label of choice and chronoamperometric detection was carried out using a tetramethylbenzidine/hydrogen peroxide (TMB/H2O2) substrate system, at +100 mV. The EC50 values obtained for the assay in buffer and urine gave relatively comparable results, 710 pg mL(-1) and 960 pg mL(-1), respectively. The linear range obtained for the assay in buffer extended from 0.03 ng mL(-1) to 40 ng mL(-1); while that in urine ranged from 0.03 ng mL(-1) to 1.6 ng mL(-1). The corresponding limits of detection (LOD) in buffer and urine were 26 pg mL(-1) and 1.8 pg mL(-1). Cross reactivity profiles of the antibody have been examined, with notable cross reactivities with 19-nortestosterone (11.6%) and boldenone (9.86%). Precision studies for the sensor demonstrated adequate reproducibility (CV<13%, n=3) and repeatability (CV<9%, n=3). Recovery data obtained showed good agreement between spiking studies and known concentrations of analyte. Sensors showed stability for 4 days at +4 degrees C. A sensitive, highly specific, inexpensive, disposable immunosensor, showing excellent overall performance for the detection of testosterone in bovine urine, has been developed.