Electrochemical Immunoassay with Dual-Signal Amplification for Redox Cycling within a Nanoscale Gap

被引:5
作者
Ito, Kentaro [1 ]
Inoue, Kumi Y. [1 ,3 ]
Ito-Sasaki, Takahiro [1 ]
Ino, Kosuke [2 ]
Shiku, Hitoshi [2 ]
机构
[1] Tohoku Univ, Grad Sch Environm Studies, Sendai, Miyagi 9808579, Japan
[2] Tohoku Univ, Dept Biomol Engn, Grad Sch Engn, Sendai, Miyagi 9808579, Japan
[3] Univ Yamanashi, Fac Engn Grad Fac Interdisciplinary Res, Ctr Basic Educ, Kofu, Yamanashi 4008511, Japan
基金
日本学术振兴会; 日本科学技术振兴机构;
关键词
electrochemical immunosensor; redox cycling; Limulus amebocyte lysate reaction; endotoxin; ENDOTOXIN; SENSITIVITY; SENSOR; FLUCTUATIONS; MOLECULES; STRATEGY; SYSTEM; ARRAYS;
D O I
10.1021/acsanm.1c02837
中图分类号
TB3 [工程材料学];
学科分类号
0805 ; 080502 ;
摘要
Here, we report a highly sensitive and easy-to-use electrochemical immunoassay using a dual-signal amplification strategy of redox cycling within a nanoscale gap and the Limulus amebocyte lysate (LAL) cascade reaction. After the antigen-antibody reaction using an endotoxin-labeled antibody, the LAL reaction was induced by endotoxin as the first signal amplification. Subsequently, p-aminophenol (pAP) liberated from peptideconjugated pAP (Boc-Leu-Gly-Arg-pAP; LGR-pAP), at the last step of the LAL reaction, was detected with redox cycling within a nanoscale gap using a fabricated device as the second signal amplification. First, an electrochemical immunoassay for goat immunoglobulin G (IgG), used as a model analyte, was performed with an endotoxin-labeled antibody using a 96-well plate. The results indicated that the endotoxin-labeled antibody was usable for immunoassays. However, a high background was observed. To decrease the background and increase the efficiency of the reactions, magnetic beads in an endotoxin-free test tube were used for the immunosorbent assay. After optimization of the condition, goat IgG was successfully detected with single-signal amplification of the cascade reaction at concentrations as low as 10 pg/mL (67 fM) using a Au disc electrode. Finally, an immunoassay for goat IgG on magnetic beads using a redox cycling-inducing device was performed. With the dual-signal amplification strategies of redox cycling within a nanoscale gap and LAL reactions, the detection limit was 70 fg/mL (470 aM). Our highly sensitive and easy-to-use immunoassay strategy provides a platform for clinical diagnosis to detect early-stage infections.
引用
收藏
页码:12393 / 12400
页数:8
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