Gan Ning, Jia Liyong, Zheng Lei
The State Key Laboratory Base of Novel Functional Materials and Preparation Science, Faculty of Material Science and Chemical Engineering, Ningbo University, Ningbo 315211, Zhejiang, China; E-Mail:
Int J Mol Sci. 2011;12(11):7410-23. doi: 10.3390/ijms12117410. Epub 2011 Oct 28.
A novel magnetic nanoparticle-based electrochemical immunoassay of carcinoembryonic antigen (CEA) was designed as a model using CEA antibody-functionalized magnetic beads [DNA/Fe(3)O(4)/ZrO(2); Fe(3)O(4) (core)/ZrO(2) (shell) nano particles (ZMPs)] as immunosensing probes. To design the immunoassay, the CEA antibody and O-phenylenediamine (OPD) were initially immobilized on a chitosan/nano gold composite membrane on a glassy carbon electrode (GCE/CS-nano Au), which was used for CEA recognition. Then, horseradish peroxidase (HRP)-labeled anti-CEA antibodies (HRP-CEA Ab(2)) were bound to the surface of the synthesized magnetic ZMP nanoparticles as signal tag. Thus, the sandwich-type immune complex could be formed between secondary antibody (Ab(2)) modified DNA/ZMPs nanochains tagged by HRP and GCE/CS-nano Au. Unlike conventional nanoparticle-based electrochemical immunoassays, the recognition elements of this immunoassay included both electron mediators and enzyme labels, which obviously simplifies the electrochemical measurement process. The sandwich-type immunoassay format was used for online formation of the immunocomplex of CEA captured in the detection cell with an external magnet. The electrochemical signals derived from HRP during the reduction of H(2)O(2) with OPD as electron mediator were measured. The method displayed a high sensitivity for CEA detection in the range of 0.008-200 ng/mL, with a detection limit of 5 pg/mL (estimated at a signal-to-noise ratio of 3). The precision, reproducibility, and stability of the immunoassay were good. The use of the assay was evaluated with clinical serum samples, and the results were in excellent accordance with those obtained using the standard enzyme-linked immunosorbent assay (ELISA) method. Thus, the magnetic nanoparticle-based assay format is a promising approach for clinical applications, and it could be further developed for the detection of other biomarkers in cancer diagnosis.
设计了一种基于新型磁性纳米粒子的癌胚抗原（CEA）电化学免疫分析法，以CEA抗体功能化的磁珠[DNA/Fe(3)O(4)/ZrO(2)；Fe(3)O(4)（核）/ZrO(2)（壳）纳米粒子（ZMPs）]作为免疫传感探针作为模型。为设计该免疫分析法，首先将CEA抗体和邻苯二胺（OPD）固定在玻碳电极（GCE/CS-纳米金）上的壳聚糖/纳米金复合膜上，用于CEA识别。然后，将辣根过氧化物酶（HRP）标记的抗CEA抗体（HRP-CEA Ab(2)）作为信号标签结合到合成的磁性ZMP纳米粒子表面。因此，在由HRP标记的二抗（Ab(2)）修饰的DNA/ZMPs纳米链与GCE/CS-纳米金之间可形成夹心型免疫复合物。与传统的基于纳米粒子的电化学免疫分析法不同，该免疫分析法的识别元件包括电子媒介体和酶标记物，这明显简化了电化学测量过程。夹心型免疫分析法用于在检测池中通过外部磁铁在线形成捕获的CEA免疫复合物。测量了以OPD作为电子媒介体在H(2)O(2)还原过程中来自HRP的电化学信号。该方法在0.008 - 200 ng/mL范围内对CEA检测显示出高灵敏度，检测限为5 pg/mL（估计信噪比为3）。该免疫分析法的精密度、重现性和稳定性良好。用临床血清样本评估了该分析法的实用性，结果与使用标准酶联免疫吸附测定（ELISA）方法获得的结果高度一致。因此基于磁性纳米粒子的分析法形式是临床应用的一种有前景的方法，并且可进一步开发用于癌症诊断中其他生物标志物的检测。
