Key Laboratory of Medical Diagnostics of Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, PR China.
Biosens Bioelectron. 2012 Mar 15;33(1):216-21. doi: 10.1016/j.bios.2012.01.004. Epub 2012 Jan 16.
A novel electrochemical immunoassay system for the detection of human epididymis-specific protein 4 (HE4) was developed. A chitosan-titanium carbide (TiC) nanocomposition film was first electrodeposited onto a tin-doped indium oxide (ITO) electrode at a constant potential. Gold (Au) nanoparticles were then electrodeposited on the surface of the chitosan-TiC film by cyclic voltammetry (CV). The capture antibody (anti-HE4) was adsorbed onto the Au and TiC nanoparticles. After a specific sandwich immunoreaction among the capture antibody, HE4, and biotinylated secondary antibody, biotinylated primer DNA was immobilized on the secondary antibody by biotin-streptavidin system. Appropriate amounts of circular template DNA and biotinylated primer DNA were used for rolling circle amplification (RCA) under optimal conditions. The RCA products provided a large number of sites to link DNA detection probes. Doxorubicin hydrochloride intercalated the CG-GC steps between the RCA products and the DNA detection probes, which was monitored by differential pulse voltammetry (DPV) based on the current signal of doxorubicin hydrochloride. With the above-mentioned amplification factors, the current responded to HE4 linearly in the concentration range of 3-300 pM under optimal detection conditions, with a detection limit of 0.06 pM. Stepwise changes in the microscopic features of the surfaces and electrochemical properties upon the formation of each layer were confirmed by scanning electron microscopy (SEM), atomic force microscopy (AFM), and electrochemical impedance spectroscopy (EIS). This system was successfully employed for the detection of HE4 with good accuracy and renewable ability.
一种新型电化学免疫分析系统用于检测人附睾蛋白 4（HE4）。首先在恒定电位下将壳聚糖-碳化钛（TiC）纳米复合材料薄膜电沉积在掺锡氧化铟（ITO）电极上。然后通过循环伏安法（CV）在壳聚糖-TiC 薄膜表面电沉积金（Au）纳米粒子。捕获抗体（抗-HE4）吸附在 Au 和 TiC 纳米粒子上。在捕获抗体、HE4 和生物素化二级抗体之间发生特异性三明治免疫反应后，生物素化引物 DNA 通过生物素-链霉亲和素系统固定在二级抗体上。在最佳条件下，适量的圆形模板 DNA 和生物素化引物 DNA 用于滚环扩增（RCA）。RCA 产物提供了大量的连接 DNA 检测探针的位点。盐酸多柔比星嵌入 RCA 产物和 DNA 检测探针之间的 CG-GC 步骤，这可以通过基于盐酸多柔比星电流信号的差分脉冲伏安法（DPV）进行监测。在最佳检测条件下，电流在 3-300 pM 的浓度范围内对 HE4 呈线性响应，检测限为 0.06 pM。通过扫描电子显微镜（SEM）、原子力显微镜（AFM）和电化学阻抗谱（EIS）证实了每一层形成时表面微观特征和电化学性质的逐步变化。该系统成功用于 HE4 的检测，具有良好的准确性和可再生能力。
