Capillary electrophoretic enzyme immunoassay with electrochemical detection for cortisol

A novel capillary electrophoretic enzyme immunoassay with electrochemical detection has been developed and used for the determination of cortisol. In this method, after the competitive enzyme immunoreaction, the free enzyme (horseradish peroxidase, HRP)-labeled cortisol (HRP-cortisol) and the bound enzyme-labeled cortisol (HPR-cortisol–anti-cortisol) were separated in the separation capillary and then catalyzed the enzyme substrate [3,3,5,5-tetramethyl-benzidine dihydrochloride, TMB(Red)] in the reaction capillary. The product of the enzymatic catalysis reaction [TMB(Ox)] was amperometrically detected on a carbon fiber microdisk bundle electrode. A concentration limit of detection (LOD) of 1.7×10−9 mol/l, which corresponds to a mass LOD of 7.8 amol, was achieved with the relative standard deviation of 3.3%. The method has been verified using the cortisol controls.