Electrochemical Immunoassay with Dual-Signal Amplification for Redox Cycling within a Nanoscale Gap

Here, we report a highly sensitive and easy-to-use electrochemical immunoassay using a dual-signal amplification strategy of redox cycling within a nanoscale gap and the Limulus amebocyte lysate (LAL) cascade reaction. After the antigen-antibody reaction using an endotoxin-labeled antibody, the LAL reaction was induced by endotoxin as the first signal amplification. Subsequently, p-aminophenol (pAP) liberated from peptide-conjugated pAP (Boc-Leu-Gly-Arg-pAP; LGR-pAP), at the last step of the LAL reaction, was detected with redox cycling within a nanoscale gap using a fabricated device as the second signal amplification. First, an electrochemical immunoassay for goat immunoglobulin G (IgG), used as a model analyte, was performed with an endotoxin-labeled antibody using a 96-well plate. The results indicated that the endotoxin-labeled antibody was usable for immunoassays. However, a high background was observed. To decrease the background and increase the efficiency of the reactions, magnetic beads in an endotoxin-free test tube were used for the immunosorbent assay. After optimization of the condition, goat IgG was successfully detected with single-signal amplification of the cascade reaction at concentrations as low as 10 pg/mL (67 fM) using a Au disc electrode. Finally, an immunoassay for goat IgG on magnetic beads using a redox cycling-inducing device was performed. With the dual-signal amplification strategies of redox cycling within a nanoscale gap and LAL reactions, the detection limit was 70 fg/mL (470 aM). Our highly sensitive and easy-to-use immunoassay strategy provides a platform for clinical diagnosis to detect early-stage infections.