Enzyme-Linked Immunoassay of α-1-Fetoprotein in Serum by Differential Pulse Voltammetry

A method for the determination of α-1-fetoprotein (AFP) in human serum by using a horseradish peroxidase (HRP) label in an enzyme-linked immunosorbent assay (ELISA) is proposed. The method is based on the electrochemical determination of enzymatic reaction product with differential pulse voltammetry at a gold disk electrode. The assay consists of two successive steps. The first step is a conventional HRP-mediated ELISA for the formation of electroactive 2,2′-diaminoazobenzene by means of the o-phenylenediamine–H2O2–HRP system. At the second step, 2,2′-diaminoazobenzene exhibits a sensitive voltammetric response at −0.19 V in pH 2.0 PBS. The peak current is proportional to the concentration of AFP in the range of 0.5–400 ng/mL (R =0.9993) under optimum conditions. The sensitivity of this method is higher than that of the traditional spectrophotometric ELISA procedure. The proposed methodhas been applied to the clinical determination of AFP in human serum with satisfactory precision and accuracy.