Yu Fengli, Du Ping, Lei Xi, Zhang Shusheng
Key Laboratory of Eco-chemical Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China.
Talanta. 2009 Jun 15;78(4-5):1395-400. doi: 10.1016/j.talanta.2009.02.031. Epub 2009 Feb 24.
A new voltammetric enzyme-linked immunoassay system using the electrochemical substrate 2,3-diaminopyridine (DAP) and horseradish peroxidase (HRP) system has been developed. DAP is oxidized with H2O2 catalyzed by HRP, and the resulting electroactive product produces a sensitive voltammetric peak at potential of -0.72V (vs. saturated calomel electrode (SCE)) in Britton-Robinson (BR) buffer solutions. The enzyme-catalyzed reaction conditions and voltammetric detection conditions have been investigated in detail. Under the selected optimum conditions, the linear range for detection of free HRP is from 6.0 x 10(-11) to 1.0 x 10(-8) g mL(-1) with a detection limit of 1.0 x 10(-12) g mL(-1). The new voltammetric detection system has been successfully applied for the assay of prostate specific antigen (PSA) in human serum ranging from 0.4 to 100 ng mL(-1) with a detection limit of 0.1 ng mL(-1), which is five times lower than that of traditional o-phenylenediamine (OPD) spectrophotometric enzyme-linked immunosorbent assay (ELISA) method. The proposed N-heterocyclic electrochemical detection system of DAP-H2O2-HRP has provided a new and much improved immunoassay method.
一种使用电化学底物2,3 - 二氨基吡啶（DAP）和辣根过氧化物酶（HRP）系统的新型伏安酶联免疫分析系统已被开发出来。DAP在HRP催化下被H₂O₂氧化，生成的电活性产物在Britton - Robinson（BR）缓冲溶液中于 - 0.72V（相对于饱和甘汞电极（SCE））的电位处产生一个灵敏的伏安峰。对酶催化反应条件和伏安检测条件进行了详细研究。在选定的最佳条件下，游离HRP的检测线性范围为6.0×10⁻¹¹至1.0×10⁻⁸ g mL⁻¹，检测限为1.0×10⁻¹² g mL⁻¹。该新型伏安检测系统已成功应用于人血清中前列腺特异性抗原（PSA）的检测，检测范围为0.4至100 ng mL⁻¹，检测限为0.1 ng mL⁻¹，比传统的邻苯二胺（OPD）分光光度酶联免疫吸附测定（ELISA）方法低五倍。所提出的DAP - H₂O₂ - HRP的N - 杂环电化学检测系统提供了一种新的且有显著改进的免疫分析方法。
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