Abstract
A novel magnetic nanoparticle-based electrochemical immunoassay of carcinoembryonic antigen (CEA) was designed as a model using CEA antibody-functionalized magnetic beads [DNA/Fe3O4/ZrO2; Fe3O4 (core)/ZrO2 (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 Ab2) 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 (Ab2) 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 H2O2 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.
Highlights
Carcinoembryonic antigen (CEA) is a protein found in many types of cells, but it is associated with tumors and the developing fetus
CEA is very important in tumor screening and diagnosis, and the increasing demand for its early and ultrasensitive detection has driven the development of novel detection technologies
The ZMP nanocomposites were characterized by X-ray diffraction (XRD) and transmission electron microscopy (TEM)
Summary
Carcinoembryonic antigen (CEA) is a protein found in many types of cells, but it is associated with tumors and the developing fetus. CEA is very important in tumor screening and diagnosis, and the increasing demand for its early and ultrasensitive detection has driven the development of novel detection technologies. Among the various analytical techniques available, electrochemical biosensing, electrochemical immunosensor (ECI), has gained considerable interest and has been used extensively to determine the serum CEA level because of its intrinsic advantages such as its portability, low cost, high sensitivity, and high specific molecular recognition of immunoreactions [2,3,4,5]. The sandwich-type ECI for CEA has recently been developed [6]. The signal generated from the enzyme-labeled antibody can catalyze a specific substrate, whose signal increases with an increase in the antigen concentration. The concentration of antigen can be determined by this method [7,8,9,10,11]
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