Electrochemical immunosensors for the detection of 19-nortestosterone and methyltestosterone in bovine urine

Concerns over the use of steroid hormones as growth promoters, have prompted the EU to prohibit their use in food producing animals. Subsequently, rigorous screening procedures have been implemented in all member states to detect the illegal administration of such compounds. In the presented work, disposable immunosensors for the detection of 19-nortestosterone and methyltestosterone in bovine urine have been developed using screen printed electrodes (SPEs). Competitive immunoassays were developed, initially by enzyme linked immunosorbent assay (ELISA), and subsequently transferred to an electrochemical immunosensor format. 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. Assays comprised of an indirect format, with immobilised antigen-protein conjugate. The EC50 values obtained for nortestosterone immunosensor in urine were 936 pg/ml, with an LODs of 10.5 pg/ml. The competitive assay of methyltestosterone in urine yielded an EC50 of 274 pg/ml with an LOD of 14.8 pg/ml. Notable cross-reactivities of anti-nortestosterone were observed with boldenone, testosterone and α-testosterone. Cross-reactivity of anti-methyltestosterone was most significant with methylboldenone. Precision, accuracy and stability studies were satisfactory for both immunosensors.