Two kinds of electrochemical immunoassays for the tumor necrosis factor α in human serum using screen-printed graphite electrodes modified with poly(anthranilic acid)

We present two kinds of electrochemical immunoassays for the tumor necrosis factor alpha (TNF-alpha) which is a protein biomarker. The antibody against TNF-alpha was immobilized on a graphite screen-printed electrode modified with poly-anthranilic acid (ASPE). The first is based on impedimetry (and thus label-free) and the target antigen (TNF-alpha) is captured by the surface of the modified electrode via an immunoreaction upon which impedance is changed. This sensing platform has a detection limit of 5.0 pg mL(-1). In the second approach, the monoclonal antibodies on the modified electrode also bind to the target antigen (TNF-alpha), but detection is based on a sandwich immunoreaction. This is performed by first adding secondary anti-TNF-alpha antibodies labeled with horseradish peroxidase, and then detecting the response of the sandwich system by adding hydrogen peroxide and acetaminophen as a probe system for HRP activity. This immunosensor also has a very low detection limit (3.2 pg mL(-1)). The experimental conditions of both assays were studied and optimized via electrochemical impedance spectroscopy and differential pulse voltammetry. The method was then applied to the determination of TNF-alpha in serum samples where it displayed high sensitivity, selectivity and reproducibility.