Enzyme-catalyzed Ag Growth on Au Nanoparticle-assembled Structure for Highly Sensitive Colorimetric Immunoassay

We have developed a sensitive colorimetric immunoassay with broad dynamic range using enzyme-catalyzed Ag growth on gold nanoparticle (NP)-assembled silica (SiO₂@Au@Ag). To reduce Ag⁺ ion content and promote Ag growth on the assembled Au NPs, alkaline phosphatase (AP)-based enzymatic amplification was incorporated, which considerably increased the colorimetric read-out. As a model study, sandwich enzyme-linked immunosorbent assay (ELISA) was used to quantify target IgG. The immune complexes capture the Ab-IgG-AP-labeled detection Ab and trigger the enzyme-catalyzed reaction to convert 2-phospho-L-ascorbic acid to ascorbic acid in the presence of the target IgG. Ascorbic acid reduced Ag⁺ to Ag, which formed Ag shells on the surface of SiO₂@Au and enhanced the absorbance of the SiO₂@Au@Ag solution. Plasmonic immunoassay showed a significant linear relationship between absorbance and the logarithm of IgG concentration in the range of ca. 7 × 10^-13 M to 7 × 10^-11 M. The detection limit was at 1.4 × 10^-13 M, which is several hundred folds higher than that of any conventional colorimetric immunoassay. Thus, our novel approach of signal-amplification can be used for highly sensitive in vitro diagnostics and detection of target proteins with the naked eye without using any sophisticated instrument.