Highly sensitive electrochemical biosensor based on redox - active monolayer for detection of anti-hemagglutinin antibodies against swine-origin influenza virus H1N1 in sera of vaccinated mice.

In this work, we report an electrochemical biosensor for the detection of anti-hemagglutinin antibodies against the swine virus H1N1 present in mice sera immunized with mixture of His6-H1 HA in monomeric and oligomeric form. The oriented immobilization of the recombinant His-tagged hemagglutinin (His6-H1 HA) consists of: (i) formation of a mixed layer of 4-mercaptobutanol (MBT) and the thiol derivative of dipyrromethene (DPM); (ii) complexation of Cu (II) by DPM; (iii) immobilization of His6-H1 HA via coordination bonds between Cu (II) sites from DPM-Cu (II) complex and imidazole nitrogen atoms of a histidine tag; (iv) filling free spaces with bovine serum albumin. The interactions between recombinant His6- H1 HA covalently attached to the electrode surface and the anti-hemagglutinin H1 antibodies present in mice sera were explored with Osteryoung square-wave voltammetry. This analytical device was able to detect the antibodies present in vaccinated mice sera diluted from 1 × 109 to 1 × 108 fold. The unprecedented sensitivity of described biosensor is much better than widely use ELISA test and other analytical methods for determination of antibodies against the influenza A viruses. It has been proved that redox active DPM-Cu (II) monolayer is a universal platform suitable for stable and oriented immobilization of any His-tagged sensing elements. Thus, this universal layer could be a base of numerous analytical devices suitable for detection of antibodies against different viruses.