Sensitive electrochemical immunoassay of a biomarker based on biotin-avidin conjugated DNAzyme concatamer with signal tagging

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Abstract
This work reports a new electrochemical immunoassay protocol with signal amplification for the sensitive detection of a biomarker (carcinoembryonic antigen, CEA, used in this case) by coupling hemin/G-quadruplex-constructed DNAzyme concatamer with the biotin-avidin amplification system. Initially, polyclonal rabbit anti-human CEA antibody (pAb1) was immobilized onto a gold nanoparticle-modified glassy carbon electrode, which was used for the capture of target CEA, and biotin-labeled mouse anti-human monoclonal CEA antibody (bio-mAb2) was then used as a secondary antibody for the formation of the sandwiched immunocomplex in the presence of target CEA on the pAb1-modified electrode. The carried biotin could react with streptavidin and a biotin-labeled initiator strand (S0) in sequence. Two auxiliary DNA strands (S1 and S2) were designed for the concatamer reaction with the S0. The DNA initiator stand (S0) could propagate a chain reaction of hybridization events between alternating S1 and S2 to form a nicked double-helix. Upon addition of hemin, the hemin-binding aptamers can be bound to form the hemin/G-quadruplex-based DNAzymes on the S1 probe. Meanwhile, the formed double-helix DNA polymers could also cause the numerous intercalation of electroactive methylene blue. During the electrochemical measurements, the DNAzymes could catalyze the reduction of H2O2 in a detection solution with the aid of the intercalated methylene blue. Under the optimal conditions, the electrochemical immunosensor exhibited a dynamic working range of 1.0 pg mL−1–50 ng mL−1 toward CEA standards with a detection limit of 0.5 pg mL−1. The reproducibility and selectivity of the developed immunoassay were acceptable (CV <10%). In addition, 6 spiked CEA samples with various concentrations were assayed by using the developed immunoassay, and the recovery was 92–120%.

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