Enhanced colorimetric detection of norovirus using in-situ growth of Ag shell on Au NPs

Norovirus (NoV) is a leading cause of acute gastroenteritis. The low infectious dose and environmental stability of NoV facilitate its effective transmission through a variety of modes such as food, water and person-to-person. The available enzyme-linked immunosorbent assay (ELISA) for NoV detection has low sensitivity due to the low catalytic activity of the peroxidase used, and thus, a reliable ultrasensitive bioassay is needed. In this study, we apply the enhanced peroxidase-like activity of silver ion-incorporated gold nanoparticles (Au/Ag NPs) in a colorimetric bioassay for NoV detection. NoV was captured by anti-NoV genogroup II antibodies, which were immobilized on the surface of a 96-well microtiter plate and formed a sandwich structure among anti-NoV Ab, NoV and Ab-Au NP. Then, Ag ion-containing hydroquinone was added to form Au/Ag core/shell NPs. When H₂O₂/3,3',5,5'-tetramethylbenzidine (TMB) solution was added to the wells, Ag ions were liberated from the surface of Au/Ag NPs and enhanced the oxidation of TMB. These reactions enhanced the oxidation of TMB and developed an intense blue color. The Au/Ag NPs were shown to exhibit higher affinity and catalytic efficiency for H₂O₂ and higher catalytic velocity based on the k_cat of up to 7-fold compared with Au NPs. The bioassay was then optimized to detect clinically isolated NoV using NoV-like particles (NoV-LPs). NoV-LPs were detected with a limit of detection of 10.8 pg/mL, corresponding to 1000- and 100-fold higher sensitivity compared to the gold-immunoassay and horseradish peroxidase-based ELISA, respectively. Clinically isolated NoV GII.4 and NoV GII.3 were detected in the range of 10²-10⁶ copies of viral RNA/mL fecal solution with a detection limit of 13.2 copies/mL fecal solution for NoV GII.4, equivalent to 132 copies of viral RNA/g feces and indicating significantly higher sensitivity compared to commercial immunoassay kits. This bioassay represents a workable detection assay for low concentrations of NoV that is applicable for early-stage diagnosis for public hygiene.