Capillary electrophoretic enzyme immunoassay with electrochemical detection for thyroxine

Abstract

A capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED) has been developed. In this method, antigen (Ag) competes with horseradish peroxidase (HRP)-labeled antigen (HRP–Ag) for a limited number of antibody (Ab) binding sites. The free HRP–Ag and the bound HRP–Ag–Ab complex are separated by capillary electrophoresis in a separation capillary. Then they catalyze the oxidation of their enzyme substrate 3,3 ′,5,5 ′-tetramethylbenzide (TMB (reduced form)) with H 2O 2 in a reaction capillary, which follows the separation capillary. The reaction product (TMB (oxidized form)) is amperometrically determined using a carbon fiber microdisk bundle electrode at the outlet of the reaction capillary. Due to the amplification of the enzyme, the concentration of TMB(Ox) is much higher than those of free HRP–Ag and the bound HRP–Ag–Ab complex. Therefore, the limit of detection (LOD) of CE-EIA-ED is very low. The method has been used to determine thyroxine in human serum. A concentration of LOD of 3.8×10 −9 mol/L, which corresponds to a mass LOD of 23.2 amol, was achieved.