Highly Sensitive Electrochemical Immunoassay Using Signal Amplification of the Coagulation Cascade.

Here, we report a highly sensitive immunoassay for human immunoglobulin G (IgG) that uses signal amplification of the coagulation cascade. Z-Phe-Pro-Lys- p -nitroaniline (FPK- p NA) was used as a substrate for thrombin activation in the last step of the coagulation cascade. During the coagulation cascade, p NA is liberated from FPK- p NA and can be detected electrochemically. Using square wave voltammetry with a glassy carbon electrode, we demonstrated that p NA can be quantified in a solution modeling the coagulation cascade prepared by mixing FPK- p NA and p NA. Characterization of the reactivity of thrombin toward FPK- p NA revealed that thrombin efficiently reacted with FPK- p NA. Subsequent characterization of factor XIa activity of factor XIa-labeled antibody revealed that factor XIa was not inactivated during labeling. Finally, a coagulation cascade-based immunoassay for human IgG was performed using a factor XIa-labeled antibody on magnetic beads. The limit of detection for human IgG was 5.0 pg/mL (33 fM) indicating that the coagulation cascade can amplify the immunoassay sensitivity compared to immunoassay using a thrombin-labeled antibody as a condition without a coagulation cascade. Coagulation cascade-based immunoassay was also highly selective. In the near future, we will report a highly sensitive immunoassay for the simultaneous detection of multiple analytes using a coagulation cascade-based immunoassay and Limulus amebocyte lysate reaction-based immunoassay we previously reported.