Dual-Signaling Electrochemical Ratiometric Method for Competitive Immunoassay of CYFRA21-1 Based on Urchin-like Fe3O4@PDA-Ag and Ni3Si2O5(OH)4-Au Absorbed Methylene Blue Nanotubes

Abstract

A novel ratiometric electrochemical (EC) sensing platform was established for sensitive immunoassay of target cytokeratin 19 fragment 21-1 (CYFRA21-1) biomarker by combining competitive immunoreaction and multisignal output. This immunosensor utilized Ag nanoparticles (NPs)-functionalized urchin-like Fe₃O₄@polydopamine (u-Fe₃O₄@PDA-Ag) as a matrix to immobilize CYFRA21-1 antigens and methylene blue (MB)-absorbed Ni₃Si₂O₅(OH)₄-Au nanotubes (NTs) to label the anti-CYFRA21-1 (Ab). During the competitive immunoreaction, square wave voltammetric (SWV) current changes of Ag NPs from u-Fe₃O₄@PDA-Ag indicator and MB from Ni₃Si₂O₅(OH)₄-Au/MB indicator are relevant to the dosage of CYFRA21-1-acquired Ni₃Si₂O₅(OH)₄-Au/MB/Ab. More importantly, numerous CYFRA21-1 loaded stably on u-Fe₃O₄@PDA-Ag exhibited strong competitive capacity toward the target-CYFRA21-1 to combine Ni₃Si₂O₅(OH)₄-Au/MB/Ab, causing sensitive changes in the ratio of two measured SWV currents. Prominently, “ΔI = ΔI_MB + |ΔI_Ag NPs|” (ΔI_MB and |ΔI_Ag NPs| represents the change values of the oxidation peak currents of MB and Ag NPs, respectively) could be regarded as significantly amplifying the signal response and ultimately improving the sensitivity of CYFRA21-1 detection, from which we derived a wide dynamic range from 500 fg/mL to 50 ng/mL and a low detection limit of 0.39 pg/mL (S/N = 3). This work may exert a profound impact on monitoring other biomarkers in early diagnosis of diseases.