Development of electrochemical immunosensor for progesterone analysis in milk

A disposable electrochemical biosensor, based on a screen-printed carbon electrode (SPE) coated with a progesterone (prog)-BSA conjugate, has been prepared and evaluated for measuring progesterone in cows' milk. The immunosensor was employed in an indirect competitive assay format involving anti-progesterone monoclonal antibody and anti-species antibody labeled with the enzyme alkaline phosphatase (AP). Differential pulse voltammetry (DPV) and amperometry were used as electrochemical means to detect the product of the enzymatic reaction [p-aminophenol(p-AP)]. Amperometric detection was carried out at +350mV vs. Ag/ AgCl reference electrode. The DPV detection was in the potential range of + 100 to + 500 mV vs. Ag/AgCl reference electrode. Progesterone was detectable in milk matrix by sigmoidal curve (variable slope) method from 13 to 189ng/mL and from a linear calibration between 16 and 256 ng/mL with an associated limit of detection (LOD) of 3 +/- 2 ng/mL progesterone. The use of DPV improved the accuracy of our measurements over conventional amperometric detection by electrode background correction. Errors were significantly lower by this method and conditions. Assays can be performed directly in full fat milk, with a C.V. of 4% and total analysis time of less than 30 minutes.