Electrochemical immunosensor using p-aminophenol redox cycling by hydrazine combined with a low background current

Signal amplification and noise reduction are crucial for obtaining low detection limits in biosensors. Here, we present an electrochemical immunosensor in which the signal amplification is achieved using p-aminophenol (AP) redox cycling by hydrazine, and the noise level is reduced by implementing a low background current. The redox cycling is obtained in a simple one-electrode, one-enzyme format. In a sandwich-type heterogeneous immunosensor for mouse IgG, an alkaline phosphatase label converts p-aminophenyl phosphate into AP for 10 min. This generated AP is electrooxidized at an indium tin oxide (ITO) electrode modified with a partially ferrocenyl-tethered dendrimer (Fc-D). The oxidized product, p-quinone imine (QI), is reduced back to AP by hydrazine, and then AP is electrooxidized again to QI, resulting in redox cycling. Moreover, hydrazine protects AP from oxidation by air, enabling long incubation times. The small amount of ferrocene in a 0.5% Fc-D-modified ITO electrode, where 0.5% represents the ratio of ferrocene groups to dendrimer amines, results in a low background current, and this electrode exhibits high electron-mediating activity for AP oxidation. Moreover, there is insignificant hydrazine electrooxidation on this electrode, which also results in a low background current. The detection limit of the immunosensor using a 0.5% Fc-D-modified electrode is 2 orders of magnitude lower than that of a 20% Fc-D-modified electrode (10 pg/mL vs 1 ng/mL). Furthermore, the presence of hydrazine reduces the detection limit by an additional 2 orders of magnitude (100 fg/mL vs 10 pg/mL). These results indicate that the occurrence of redox cycling combined with a low background current yields an electrochemical immunosensor with a very low detection limit (100 fg/mL). Mouse IgG could be detected at concentrations ranging from 100 fg/mL to 100 microg/mL (i.e., 9 orders of magnitude) in a single assay.