Abstract
In the present study, we fabricated a label-free avian influenza (AIV H5N1) detection biosensor composed of a multi-functional DNA 3 way-Junction (3 W J) on a hollow Au spike-like nanoparticle (hAuSN) using a localized surface plasmon resonance (LSPR) method. To construct the multi-functional DNA (MF-DNA) as a bioprobe, the 3 W J was introduced. The proposed AIV detection bioprobe should contain three functionalities: target recognition, signal amplification, and connection to substrate. To achieve this goal, each piece of the DNA 3 W J was tailored to a hemagglutinin (HA) binding aptamer, FAM dye and thiol group, respectively. The assembly of each DNA 3 W J functional fragment was then confirmed by TBM-Native PAGE. Moreover, the hAuSN was immobilized on the indium-tin-oxide (ITO) substrate for LSPR measurement. The DNA 3 W J was immobilized onto the hAuSN electrode through the thiol-group of DNA 3 W J. The fabricated DNA 3 W J/hAuSN heterolayer on the ITO substrate was investigated by field emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM). LSPR experiments were conducted to confirm HA protein binding to the DNA 3 W J/ hAuSN -modified electrode. The proposed biosensor can detected the HA protein in PBS buffer (LOD: 1 pM) as well as in the diluted chicken serum (LOD: 1 pM). The present study details a label-free, simple fabrication method consisted of DNA 3 W J/ hAuSN heterolayer that uses easy-to-tailor elements to detect not only AIV but also various viruses detection platform easily.