Enzyme-free colorimetric determination of EV71 virus using a 3D-MnO₂-PEG nanoflower and 4-MBA-MA-AgNPs

We present a simple colorimetric assay for EV71 virus detection based on the aggregation of 4-mercaptobenzoic acid (4-MBA) and melamine (MA) modified silver nanoparticles (4-MBA-MA-AgNPs) in the presence of Mn²⁺. The EV71-Ab₁ was incubated on a 96-well plate and the EV71-Ab₂ was labeled on the surface of three-dimensional nanoflower-like MnO₂-PEG (3D-MnO₂-PEG). After layer-by-layer immunoreactions, the EV71 virus and the corresponding 3D-MnO₂-PEG-Ab₂ were captured on the plate. With the addition of Vitamin C (Vc), Mn²⁺ was released from the 3D-MnO₂-PEG and then the aggregation of the 4-MBA-MA-AgNPs was induced, allowing a naked-eye detection limit of EV71 virus to be as low as 5 × 10⁴ particles per mL, which is about three orders of magnitude lower than the conventional enzyme-linked immunosorbent assay (ELISA). This enzyme-free immunoassay based on a hybrid 3D-MnO₂ features signal amplification strategies via a simple reduction reaction.