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

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%.