Highly Sensitive Electrochemical Immunoassay Using Signal Amplification of the Coagulation Cascade

Abstract

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-pNA) was used as a substrate for thrombin activation in the last step of the coagulation cascade. During the coagulation cascade, pNA is liberated from FPK-pNA and can be detected electrochemically. Using square wave voltammetry with a glassy carbon electrode, we demonstrated that pNA can be quantified in a solution modeling the coagulation cascade prepared by mixing FPK-pNA and pNA. Characterization of the reactivity of thrombin toward FPK-pNA revealed that thrombin efficiently reacted with FPK-pNA. Subsequent characterization of factor XIa activity of factor XIalabeled 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.

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

“…The calibration plots for goat and human IgG were not linear because of using cascade reactions. In our previous study using LAL or coagulation cascade reaction, ,, the same trend of nonlinear calibration plots was obtained. Performing curve fitting of calibration plots will be realized for practical use in the future.…”

“…Based on thrice the standard deviation of 0 pg/mL human IgG (BG + 3σ), the limit of detection for human IgG with dual-signal amplification is 50 fg/mL (330 amol/L). This result shows that the limit of detection is improved by 100-fold compared to our previous study in which an immunoassay was performed with single-signal amplification of the coagulation cascade reaction . This improvement is mainly attributed to the signal amplification of redox cycling.…”

“…However, to our best knowledge, a multiplex immunoassay with signal amplification of a cascade reaction has not been reported. A multiplex immunoassay has many advantages such as cost and time reduction, particularly for diagnosis requiring detection of multiple marker molecules. , In this study, we developed a dual immunoassay with signal amplification of a cascade reaction and redox cycling within a nanoscale gap, based on our previous studies on immunoassays using LAL and coagulation cascade reactions . Endotoxin and factor XIa were employed as the label molecules for the immunoassays of goat and human IgG, respectively.…”

“…The other immunoassay was based on signal amplification of a coagulation cascade reaction. 10 We performed immunoassays for human IgG using the activated form of factor XI (factor XIa), which induces a blood coagulation cascade reaction, as the label molecule. After the antigen−antibody reaction, the coagulation cascade reaction was induced, allowing detection of p-nitroaniline (pNA) liberated from peptide-conjugated pNA (Z−Phe−Pro−Lys− pNA; FPK−pNA) at its end.…”

“…The liberated p AP was detected at the nanoscale-gap electrode, amplifying the amperometric signals by repeating the redox reaction between two close electrodes, and a detection limit of 470 amol/L was achieved for goat IgG. The other immunoassay was based on signal amplification of a coagulation cascade reaction . We performed immunoassays for human IgG using the activated form of factor XI (factor XIa), which induces a blood coagulation cascade reaction, as the label molecule.…”