Rapid detection of avian influenza H5N1 virus using impedance measurement of immuno-reaction coupled with RBC amplification

Avian influenza virus (AIV) subtype H5N1 was first discovered in the 1990's and has been reported in 46 countries for animal cases and 15 countries for human infection with 501 people infected and 297 human deaths since 2003. Since its emergence it has become a likely source of a global pandemic and economic loss. Controlling influenza outbreaks relies on rapid detection and identification followed by eradication of infected animals, quarantine of a 2 mile radius, and vaccination and observation of animals outside the quarantine zone. Currently accepted gold standard methods of influenza detection, viral culture with immunological tests and RT-PCR, are time consuming, expensive, and require special training and laboratory facilities. A rapid, sensitive, and specific screening method is needed for in-field or bedside testing of AI virus to effectively implement quarantines and medications. Therefore, the objective of this study was to improve the specificity and sensitivity of an impedance biosensor that has been developed for the detection of AIV H5. Three major components of the developed biosensor are immunomagnetic nanoparticles for separation of AI virus, a microfluidic chip for sample control, and an interdigitated microelectrode for impedance measurement. In this study, polyclonal antibody against N1 subtype was immobilized on the surface of the microelectrode to specifically bind AIV H5N 1 to generate more specific impedance signal, and chicken red blood cells (RBC) were used as biolabels to attach to AIV H5N 1 captured on the microelectrode to amplify impedance signal. The impedance of the nanoparticle-virus-RBC complex was measured and compared to the negative control. RBC amplification was shown to increase the impedance signal change by more than 100% compared to the protocol without RBC biolabels and was necessary for forming a linear calibration curve for the biosensor. The use of a second antibody against N1 offered much great specificity and reliability than the previous biosensor protocol. The results of this study showed that the biosensor was able to detect AIV H5N I at concentrations down to 103 EID50/ml in less than 2 hours.Keywords: Impedance biosensor Avian influenza H5N1 magnetic nanoparticles...