Single Electrode Genosensor for Simultaneous Determination of Sequences Encoding Hemagglutinin and Neuraminidase of Avian Influenza Virus Type H5N1

The duo-genosensor consisting of two different oligonucleotide probes immobilized covalently on the surface of one gold electrode via Au-S bond formation was used for simultaneous determination of two different oligonucleotide targets. One of the probes, decorated on its S'-end with ferrocene (SH-ssDNA-Fc), is complementary to the cDNA representing a sequence encoding part of HS hemagglutinin from H5N1 virus. The second probe, decorated on its S'-end with methylene blue (SH-ssDNA-MB), is complementary to cDNA representing the fragment of N1 neuraminidase from the same virus. The presence of both probes on the surface of gold electrodes was confirmed with Osteryoung square-wave voltammetry (OSWV). The changes in redox activity of both redox active complexes before and after the hybridization process were used as analytical signal. The peak at +400 +/- 2 mV was observed in the presence of 40 nM ssDNA used as a target for SH-ssDNA-Fc probe. This peak increased with the increase of concentration of target ssDNA. It indicates the signal on mode of analytical signal generation. The peak at -250 +/- 4 mV, characteristic for SH-ssDNA-MB probe, was decreasing with the increase of the concentration of the complementary ssDNA target starting from 8 to 100 nM. This indicates the generation of electrochemical signal according to the signal off mode. The proposed duo-genosensor is capable of simultaneous, specific, and good sensitivity probing for the sequences derived from genes encoding two main markers of the influenza virus, hemagglutinin and neuraminidase.