A nanowell-based QCM aptasensor for rapid and sensitive detection of avian influenza virus

In this study, a cost-effective nanowell structure was fabricated and utilized for the development of a nanowell-based quartz crystal microbalance (QCM) aptasensor for rapid, sensitive, and label-free detection of H5N1 avian influenza virus (AIV). A nanoporous gold film with a thickness of 120 nm and a pore size of ∼20 nm was prepared using a metallic corrosion method. Then, the nanoporous gold film was immobilized onto a gold electrode surface using a self-assembled monolayer to form a nanowell-based electrode. A specific H5N1 AIV ssDNA aptamer with a NH₂ conjugated 5′-terminal was used in the fabrication of the QCM aptasensor through covalent bonding. The stepwise assembly of the aptasensor was characterized by means of QCM. The result showed that the binding of target AIV H5N1 onto the immobilized aptamers decreased the sensor's resonant frequency, and the frequency change correlated to the virus titer. We demonstrated that the developed nanowell-based QCM aptasensor could dramatically reduce detection time down to 10 min using a label-free assay. The detection range of 2⁻⁴ to 2⁴ hemagglutination units (HAUs)/50 μl was obtained with a detection limit of 2⁻⁴ HAU/50 μl for AIV H5N1. The binding of target H5N1 virus onto the nanowell-based electrode surface was further confirmed by scanning electron microscopy (SEM). No interference was observed from non-target AIV subtypes of H1N1, H2N2, H7N2 and H5N3. The aptasensor using H5N1 aptamer was validated for the detection of AIV H5N1 in chicken tracheal swab samples. The developed aptasensor could be adopted for detection of other viruses.