DEVELOPMENT OF A SENSITIVE, SELECTIVE ELECTROCHEMICAL IMMUNOASSAY FORPROGESTERONE IN COWS MILK-BASED ON A DISPOSABLE SCREEN-PRINTED AMPEROMETRIC BIOSENSOR

A disposable amperometric biosensor, based on a screen-printed carbon electrode (SPCE) coated with antibodies, has been prepared and evaluat ed for measuring progesterone in cow's milk. This immunosensor was emp loyed in a competitive assay involving progesterone labelled with alka line phosphatase. Initially, phenylphosphate was used as the enzyme su bstrate and the product of the reaction, phenol, was detected by chron oamperometry at +0.70 V vs saturated calomel (SCE). However, this appr oach proved unsatisfactory, since an unidentified component of cow's m ilk adsorbed to the electrode and underwent a variable oxidation at th is potential. This resulted in a calibration curve which did not have the characteristic sigmoidal shape. An alternative enzyme substrate, 4 -aminophenolphosphate, was therefore examined, the product of the reac tion, 4-aminophenol, being detectable at +0.20 V vs SCE; at this lower potential, the interferent in milk did not undergo oxidation. Consist ently low background currents were obtained, enabling construction of a sigmoidal-shaped calibration curve for progesterone concentration in the milk samples investigated. Progesterone levels determined by the proposed electrochemical immunoassay compared favourably with a colori metric immunoassay method. (C) 1998 Elsevier Science Ltd. All rights r eserved.