Reduction of the Nonspecific Binding of a Target Antibody and of Its Enzyme-Labeled Detection Probe Enabling Electrochemical Immunoassay of an Antibody through the 7 pg/mL-100 ng/mL (40 fM-400 pM) Range

We describe a simple, potentially low-cost, amperometric, enzyme-amplified, sandwich-type immunoassay, monitoring IgG at a concentration as low as ~7 pg/mL with a dynamic range of 104. The assay utilizes a screen-printed carbon electrode on which a redox hydrogel and avidin are coelectrodeposited. To neutralize nonspecifically binding positively charged microdomains of the avidin, two polyanions, poly(acrylic acid-co-maleic acid) and poly(acrylic acid), are applied. These polyanions bind to the film not only electrostatically but also by Michael addition reaction to cysteine, lysine, or arginine functions of the avidin. The electrode is then made specific for the analyte, for which rabbit IgG was chosen, by conjugating the film-bound avidin to biotin-labeled anti-rabbit IgG. After exposure to the tested solution and capture of rabbit IgG, the sandwich is completed by conjugation of horseradish-peroxidase (HRP)-labeled anti-rabbit IgG. Electrical contact between the HRP and the electrode-bound hydrogel results in the formation of an electrocatalyst for the electroreduction of H2O2 to water. The application of the poly(acrylic acidco-maleic acid) and the poly(acrylic acid) reduces the nonspecific adsorption-associated noise, lowers the detection limit from 3 ng/mL (~20 pM analyte antibody concentration) to ~7 pg/mL (~40 fM analyte antibody concentration), and also expands the dynamic range to 104.