Impedimetric detection of double-tagged PCR products using novel amplification procedures based on gold nanoparticles and Protein G

Double-tagged DNA coming from PCR amplification of a Salmonella spp. sample was detected by an electrochemical impedimetric genosensor based on avidin bulk-modified graphite-epoxy biocomposite (Av-GEB). The double-tagging PCR strategy provided the amplicon with both biotin and digoxigenin (DIG) moieties. The immobilization of the double-tagged DNA was based on its biotin moiety, while the DIG label was used for signal amplification. Impedance spectra were recorded to detect the change in interfacial charge transfer resistance (R(ct)), experimented by the redox marker ferri-/ferro-cyanide after the avidin-biotin fixation of the sample DNA onto the electrode surface. A further step in the genosensing strategy was the amplification of impedimetric signal by the use of an enhancing procedure. The latter was based on the reaction of the DIG moiety belonging to the amplicon with an anti-DIG antibody from mouse. Two different secondary enhancing steps based both on gold nanoparticle-labelled anti-mouse IgG or on Protein G were performed and compared for improving assay sensitivity.