A novel electrochemical immunoassay based on diazotization-coupled functionalized bioconjugates as trace labels for ultrasensitive detection of carcinoembryonic antigen

Abstract In this article, a novel sandwich-type electrochemical immunosensor based on the signal amplification strategy of diazotization-coupling concept for ultrasensitive detection of carcinoembryonic antigen (CEA) was reported. It operates through physisorption of monoclonal anti-CEA on 4-aminothiophenol (4Atp) functionalized gold electrode interface as the detection platform. Diazo-4Atp-coupled-thionine (Thi)-conjugated gold nanoparticles (GNPs) were prepared for immobilization of horseradish peroxidase (HRP) and secondary anti-CEA to form core–shell bioconjugates that were used as electrochemical signal amplification reagent. The sensitivity of the immunosensor was greatly amplified by a dual amplification: one is that a large number of thionine and HRP was introduced on the electrode surface through sandwich immunoreaction, the other is that HRP as enhancer could catalyze the oxidation reaction of thionine by H 2 O 2 , which results in great enhancement of the reduction peak current. Thus, the bioconjugates-based assay provided an amplification approach for detecting CEA at trace levels and led to a detection limit as low as 0.7 pg/mL (at a three times signal-to-noise ratio) that is well-below the threshold value of 2.5 ng/mL for clinical diagnosis. The assay was evaluated for clinical serum samples with various CEA concentrations and received in excellent accordance with the results obtained from the referenced enzyme-linked immunosorbent assay (ELISA).