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

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 = ΔIMB + |ΔIAg NPs|” (ΔIMB and |ΔIAg 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.