An electrochemical immunosensor based on a 4,4′-thiobisbenzenethiol self-assembled monolayer for the detection of hemagglutinin from avian influenza virus H5N1

Abstract

An electrochemical immunosensor for the detection of hemagglutinin from avian influenza virus H5N1 is presented in this paper. The following steps lead up to the construction of immunosensor: (i) modification of gold electrodes with 4,4′-thiobisbenzenethiol, (ii) modification of self-assembled monolayer of 4,4′-thiobisbenzenethiol with gold colloidal nanoparticles, (iii) immobilization of single chain variable fragments of antibodies (scFv) against hemagglutinin H5 via Ssingle bondAu covalent bonds, (iv) blocking of the remaining free space with bovine serum albumin. The interactions between the scFv and hemagglutinin variants have been explored with electrochemical impedance spectroscopy in the presence of [Fe(CN)6]3−/4− as an electroactive marker. The immunosensor was able to detect two different His-tagged variants of recombinant hemagglutinin from H5N1 viruses: the short fragment (17–340 residues) of A/swan/Poland/305-135V08/2006 and the long (17–530 residues) of A/Bar-headed Goose/Qinghai/12/2005. The strongest response has been observed for the long variant with a detection limit of 0.6 pg/mL and a dynamic range from 4.0 to 20.0 pg/mL. The recombinant hemagglutinin (17–527 residues) from A/chicken/Netherlands/1/03 (H7N7), used as the negative control generated a weak response. This confirms the selectivity of the immunsensor proposed. A miniaturized version of the immunosensor, based on screen-printed gold electrodes, was tested with the same set of recombinant hemagglutinins and it achieved a linear range from 1 to 8 pg/mL with a detection limit of 0.9 pg/mL for the long fragment of hemagglutinin.