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

Abstract

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 paminophenol 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.

Highly Sensitive Electrochemical Endotoxin Sensor Based on Redox Cycling Using an Interdigitated Array Electrode Device

“…In the future, further characterization of the Au flat plate as a quasi-reference electrode may result in a highly portable device. The IDAE device has a robust structure compared to the redox cycling device in a nanoscale gap [ 19 , 22 , 23 ]. The redox cycling device in a nanoscale gap can break as a result of a small impact due to the broken spatial structure, which is not the case with the IDAE device.…”

“…Additionally, an IDAE device with a 10 μm electrode distance is easier to produce than a device for redox cycling in a nanoscale gap because devices with a small electrode distance are difficult to manufacture, which is an important factor when considering mass production. In our previous study involving a device for redox cycling in a nanoscale gap [ 19 , 22 , 23 ], the shape of amperograms was influenced by the adsorption and desorption of a redox molecule to the electrode surface [ 30 ]. A LAL reagent without a chromogenic substrate can be effectively used to obtain 1.0 EU/L of the limit of detection with 30 min of the LAL reaction by using the IDAE device.…”

“…This cycling of redox reaction amplifies the electrochemical signal [ 20 , 21 ]. The IDAE device has the advantages of simplicity and robustness compared the device for redox cycling in a nanoscale gap used in our previous study [ 19 , 22 , 23 , 24 ]. This difference is attributed to the fragile nanometer-sized spatial structure of the device for redox cycling in a nanoscale gap, which is not present in the IDAE device.…”

Progress in Electrochemical Immunosensors with Alkaline Phosphatase as the Signal Label

“…In this process, a small number of signal molecules can produce a significantly enhanced electrochemical signal. Nowadays, electrochemical immunosensors based on the signal amplification of enzymes plus electrochemical–chemical (EC) or electrochemical–chemical–chemical (ECC) redox cycling arouse wide interest due to their excellent sensitivity and selectivity [ 112 , 113 , 114 , 115 , 116 , 117 ].…”