A label-free ratiometric immunoassay using bioinspired nanochannels and a smart modified electrode

Labeling with redox reporter is often required in developing electrochemical bioassay for most proteins or nucleic acid biomarkers. Herein, a label-free ratiometric immunosensing platform is firstly developed by integrating the antibody-conjugated nanochannels with a smart modified electrode. The electrode modifier is the composite of C₆₀, tetraoctylammonium bromide (TOA⁺) and Prussian blue (PB). Cyclic voltammograms of the ultimate C₆₀-TOA⁺/PB modified electrode exhibited two pairs of peaks at 0.15 V and -0.13 V, ascribing to the redox of PB and C₆₀, respectively. With the addition of K₃[Fe(CN)₆] in the electrolyte solution, the peaks of PB decreased due to the adsorption of [Fe(CN)₆]^3- while the peaks of C₆₀ increased because of the formation of the ternary complex (TC) C₆₀-TOA⁺-[Fe(CN)₆]^3-. As a result, the peak current ratio I_PB/I_TC decreased gradually with the increment of the concentration of [Fe(CN)₆]^3-. For the nanochannels-based immunosensing platform, the steric hindrance of the bioconjugated nanochannels varied with the loading amount of the target CA125, and thus [Fe(CN)₆]^3- passing through the channels was quantitatively affected. And the higher CA125 level was, the less [Fe(CN)₆]^3- concentration was. And thus, the ratio I_PB/I_TC monitored at the C₆₀-TOA⁺/PB modified electrode increased with the increase of the concentration of CA125. The ratiometric immunoassay featured a linear calibration range from 1.0 U mL^-1 to 100 U mL^-1 with a low detection limit of 0.86 U mL^-1. In addition, the ratiometric immunosensing platform demonstrated good specificity and stability as well as acceptable accuracy in overcoming the effect of electrode passivation which was an inherent problem of electroanalysis.