High-Sensitivity Amperometric Dual Immunoassay Using Two Cascade Reactions with Signal Amplification of Redox Cycling in Nanoscale Gap

Here, we report a high-sensitivity dual immunoassay using Lumulus amebocyte lysate (LAL) and blood coagulation cascade reactions with redox cycling in a nanoscale-gap electrode. Endotoxin and factor XIa were used as the label molecules for the immunoassay of two types of analytes to induce the LAL and coagulation cascade reactions, respectively, when each corresponding analyte existed in the sample solution. In addition to the signal amplification by the cascade reactions, we employed redox cycling in a nanoscale gap to achieve a highly sensitive assay. The nanoscale-gap electrode amplifies the amperometric signals from p- aminophenol liberated from artificial substrates in the final steps of the cascade reactions. First, the cross reaction between the LAL and coagulation cascade reactions was investigated. The results indicated that these cascade reactions did not efficiently proceed in a single solution owing to the cross reaction. Therefore, we selected to induce two cascade reactions in different solutions by bisecting the beads after the immunocomplex formation on the beads. The cross reactions of factor XIa with the LAL cascade reaction and of endotoxin with the coagulation cascade reaction were investigated. The effects of these cross reactions were revealed to be negligible by bisecting the beads before inducing the cascade reactions. Finally, a dual immunoassay for goat and human immunoglobulin G was performed, for which the limits of detection were 70 pg/mL (470 fmol/L) and 1.0 ng/mL (6.6 pmol/L), respectively. Thus, our dual immunoassay provides a sensitive platform for clinical diagnosis requiring detection of multiple analytes.