Development of a Progesterone Immunosensor Based on a Colloidal Gold‐Graphite‐Teflon Composite Electrode

A novel amperometric immunosensor for the determination of progesterone, based on direct attachment of antiprogesterone on a gold nanoparticles‐modified graphite‐Teflon composite electrode is reported. Competitive assay involving progesterone labeled with alkaline phosphatase (AP), and 1‐naphtyl phosphate as the enzyme substrate, was employed. The product of the enzyme reaction, 1‐naphtol was detected amperometrically at +0.3 V vs. Ag/AgCl. Variables involved in the immunosensor preparation such as the colloidal gold loading and the amount of anti‐Prog immobilized, as well as in the immunosensing event (pH, Prog/Prog‐AP ratio and volume) were optimized. A calibration plot was obtained for progesterone with a linear range between 0 and 30 ng mL −1 , and a slope value, −1.2 nA ng −1 mL, which is remarkably higher than that reported for other progesterone biosensors. A good repeatability of the amperometric measurements, RSD =3.5% ( n =10), and an acceptable fabrication reproducicility, RSD =8.2% ( n =3), were obtained. The developed anti‐Prog‐Au coll ‐graphite‐Teflon immunosensor was applied to the progesterone determination in milk. Recovery at the 3.5 ng mL −1 progesterone concentration level was of 101±6% ( n =7).