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 C 60 , tetraoctylammonium bromide (TOA + ) and Prussian blue (PB). Cyclic voltammograms of the ultimate C 60 -TOA + /PB modified electrode exhibited two pairs of peaks at 0.15 V and −0.13 V, ascribing to the redox of PB and C 60 , respectively. With the addition of K 3 [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 C 60 increased because of the formation of the ternary complex (TC) C 60 -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 C 60 -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.