Highly Sensitive\nElectrochemical Immunoassay Using\nSignal Amplification of the Coagulation Cascade

Abstract

Here, we report a highly sensitive immunoassay for human\nimmunoglobulin\nG (IgG) that uses signal amplification of the coagulation cascade.\nZ-Phe-Pro-Lys-<i>p</i>-nitroaniline (FPK-<i>p</i>NA) was used as a substrate for thrombin activation in the last step\nof the coagulation cascade. During the coagulation cascade, <i>p</i>NA is liberated from FPK-<i>p</i>NA and can be\ndetected electrochemically. Using square wave voltammetry with a glassy\ncarbon electrode, we demonstrated that <i>p</i>NA can be\nquantified in a solution modeling the coagulation cascade prepared\nby mixing FPK-<i>p</i>NA and <i>p</i>NA. Characterization\nof the reactivity of thrombin toward FPK-<i>p</i>NA revealed\nthat thrombin efficiently reacted with FPK-<i>p</i>NA. Subsequent\ncharacterization of factor XIa activity of factor XIa-labeled antibody\nrevealed that factor XIa was not inactivated during labeling. Finally,\na coagulation cascade-based immunoassay for human IgG was performed\nusing a factor XIa-labeled antibody on magnetic beads. The limit of\ndetection for human IgG was 5.0 pg/mL (33 fM) indicating that the\ncoagulation cascade can amplify the immunoassay sensitivity compared\nto immunoassay using a thrombin-labeled antibody as a condition without\na coagulation cascade. Coagulation cascade-based immunoassay was also\nhighly selective. In the near future, we will report a highly sensitive\nimmunoassay for the simultaneous detection of multiple analytes using\na coagulation cascade-based immunoassay and <i>Limulus</i> amebocyte lysate reaction-based immunoassay we previously reported.