Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University , Wuhan, Hubei 430079, P. R. China.
ARC Centre of Excellence in Nanoscale Biophotonics (CNBP), Macquarie University , North Ryde, New South Wales 2109, Australia.
Anal Chem. 2016 Oct 4;88(19):9614-9621. doi: 10.1021/acs.analchem.6b02353. Epub 2016 Sep 21.
In this study, a label-free electrochemical immunosensor was developed for detection of cytokine tumor necrosis factor-alpha (TNF-α). First, AuNPs loaded reduced graphene oxides nanocomposites (RGO-ph-AuNP) were prepared, and then, a mixed layer of 4-carbxyphenyl and 4-aminophenyl phosphorylcholine (PPC) was modified to the surface of AuNPs for the subsequent modification of anti-TNF-α capture antibody (Ab) to form the capture surface (Au-RGO-ph-AuNP-ph-PPC(-ph-COOH)) for the analyte TNF-α with the antifouling property. For reporting the presence of analyte, the anti-TNF-α detection antibody (Ab) was modified to the graphene oxides which have been modified with the 4-ferrocenylaniline through diazonium chemistry to form Ab-GO-ph-Fc. Then, a sandwich assay was formed on gold surfaces for the quantitative detection of TNF-α based on the electrochemical signal of ferrocene. X-ray photoelectron spectra (XPS), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), UV-vis, and electrochemistry were used for characterization of the stepwise fabrications on the interface. The prepared electrochemical immunosensor was successfully used for the detection of TNF-α over the range of 0.1-150 pg mL. The lowest detection limit of this immunosensor is 0.1 pg mL TNF-α in 50 mM phosphate buffer at pH 7.0. The fabricated immunosensor provided high selectivity and stability and can be used to detect TNF-α secreted by live BV-2 cells with comparable accuracy to enzyme-linked immunosorbent assay (ELISA) but with lower limit of detection.
在这项研究中，开发了一种用于检测细胞因子肿瘤坏死因子-α（TNF-α）的无标记电化学免疫传感器。首先，制备了负载金纳米粒子的还原氧化石墨烯纳米复合材料（RGO-ph-AuNP），然后，将混合层的 4-羧基苯基和 4-氨基苯基膦酸酯（PPC）修饰到 AuNPs 的表面，以便随后修饰抗 TNF-α捕获抗体（Ab）以形成具有抗污染性的分析物 TNF-α的捕获表面（Au-RGO-ph-AuNP-ph-PPC（-ph-COOH））。为了报告分析物的存在，将抗 TNF-α检测抗体（Ab）修饰到通过重氮化学修饰的 4-二茂铁苯胺修饰的氧化石墨烯上，以形成 Ab-GO-ph-Fc。然后，在金表面上形成三明治测定法，基于铁的电化学信号对 TNF-α进行定量检测。X 射线光电子能谱（XPS）、透射电子显微镜（TEM）、傅里叶变换红外光谱（FT-IR）、紫外-可见光谱和电化学用于对界面上的逐步构建进行表征。所制备的电化学免疫传感器成功用于在 0.1-150 pg mL 范围内检测 TNF-α。该免疫传感器的最低检测限为在 pH 7.0 的 50 mM 磷酸盐缓冲液中为 0.1 pg mL TNF-α。所制造的免疫传感器提供了高选择性和稳定性，并且可以与酶联免疫吸附测定（ELISA）相当的准确性用于检测活 BV-2 细胞分泌的 TNF-α，但检测限更低。
