Abstract
There has been a need for rapid detection of Avian Influenza virus (AIV) H5N1 due to it being a potential pandemic threat. Most of the current methods, including culture isolation and PCR, are very sensitive and specific but require specialized laboratories and trained personnel in order to complete the tests and are time-consuming. The goal of this study was to design a biosensor that would be able to rapidly detect AIV H5N1 using aptamers as biosensing material and a quartz crystal microbalance (QCM) for transducing method. Specific DNA aptamers against AIV H5N1 were immobilized, through biotin and streptavidin conjugation, onto the gold surface of QCM sensor to capture the target virus. Magnetic nanobeads (150 nm in diameter) were then added as amplifiers considering its large surface/volume ratio which allows for faster movement and a higher target molecule binding rate. The result showed that the captured AIV caused frequency change, and more change was observed when the AIV concentration increased. The nanobead amplification was effective at the lower concentrations of AIV, however, it was not significant when the AIV concentration was 1 HA or higher. The detection limit of the aptasensor was 1 HAU with a detection time of 1 h. The capture of the target virus on to the surface of QCM sensor and the binding of magnetic nanobeads with the virus was confirmed with electron microscopy. Aptamers have unlimited shelf life and are temperature stable which allows this aptasensor to give much more consistent results specifically for in field applications.
Highlights
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Inactivated avian influenza virus (AIV) H5N1 with titers in the range of 0.01-4 Hemagluttination unit/50 μl (HAU)/50 μl in PBS were added to the flow cell for capturing by the aptamer immobilized on the surface of Quartz crystal microbalance (QCM) sensor and allowed to incubate for 30 min
Because we focused on the change in frequency before and after the addition of AIV H5N1, the liquid portion of the equation could be cancelled out due to both measurements being taken in a PBS solution
Summary
Influenza A viruses are a genus of virus in the family Orthomyxoviridae consisting of a single stranded negative sense RNA genome segmented into 8 fragments (Lee & Saif, 2009). The lipoprotein envelope of Orthomyxoviridae viruses can occur in either spherical or helical forms with the shape being dependent on its surface proteins (Jin et al, 1997). The natural host of influenza A particles are wild birds, primarily water fowl. These can carry and shed virus even while showing no outward symptoms. The cleavage site of high pathogenic H5N1 contains a large number of basic amino acid residues allowing it to be cleaved by a variety of host proteases. This allows the virus to be able to infect more of the body outside the respiratory tract (Steinhaeur, 1999). It is estimated that many mild cases of H5N1 go unreported which would cause the reported mortality rate of 60% in humans to be lower (Thorson et al, 2006)
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