Signal-Amplifiable Porous Nanostructures for Detection of Infectious Viruses

Avian influenza virus (AIV) is the highly contagious and fatal viral disease that occurs among wild birds worldwide, mainly caused by influenza A virus. The proliferation of high pathogenicity AIVs, which requires massive killing, has a significant impact on the poultry industry and causes serious social and economic damages, therefore diagnosing AIV at early stage and prevent its spread is important. In addition to the large amount of enzyme required for signal amplification, it is important that the antibody is located on the outside of the particle to increase the accessibility to the antigen. The purpose of this study was to develop a size-selective biomolecule immobilization strategy that facilitates optimal porous silica nanoparticles (PSNPs) and conjugation system, which enables larger molecules (such as antibodies) to be conjugated to the outside of the nanoparticles, and smaller molecules (such as HRP) to be incorporated into its internal pores.<br/>The rationally designed nanoprobe has been successfully adapted for chemiluminescent lateral flow stick immunoassay (CL-LFA). The detection limit of the CL-LFA using the signal-amplifiable nanoprobe for the nucleoprotein of the H3N2 virus was 5 pM. Sensitivity tests for low pathogenicity avian influenza H9N2, H1N1, and high pathogenicity avian influenza H5N9 viruses were conducted, and the detection limits of CL-LFA were found to be 10^3.5 50% egg infective dose (EID₅₀)/mL, 10^2.5 EID₅₀/mL, and 10⁴ EID₅₀/mL, respectively, which is 20 to 100 times lower than that of a commercial AIV rapid test kit. Additionally, CL-LFA showed high sensitivity and specificity against 37 clinical samples. The signal-amplifiable probe used in this study may be a potential diagnostic probe with ultrahigh sensitivity for applications in the field of clinical diagnosis, which requires sensitive antigen detection as evidenced by enhanced signaling capacity and sensitivity of the LFAs.