Nanozyme-linked immunosorbent assay for porcine circovirus type 2 antibody using HAuCl4/H2O2 coloring system

Abstract

It is of great interest but still a huge challenge to construct an enzyme-free method for the early clinical diagnosis of various diseases with naked eyes. In the traditional enzyme linked immunosorbent assay (ELISA), horseradish peroxidase (HRP) as enzyme label and tetramethylbenzidine (TMB) as coloring agent have been widely used for immunoassays. However, the monochromic changes of TMB and the limited catalytic activity of HRP may confine the performance of ELISA. Herein, a new HAuCl4/H2O2 coloring system was proposed for naked-eye detection of porcine circovirus type 2 (PCV2) antibody using nanozyme, Au-Pt/SiO2 as labels. Be similar to the oxidation capacity of H2O2 towards TMB, HAuCl4 can also be reduced into gold nanoparticles (Au NPs) by H2O2. Based on this finding, antibody grafted Au-Pt/SiO2 nanospheres (APS NPs) were prepared and acted as enzyme labels to catalyze the decomposition of H2O2, thus resulting in a color variation from wine red to light purple with the increasing of analyte. The HAuCl4/H2O2 system was applied to the detection of human IgG and porcine circovirus type 2 (PCV2) antibody, which was comparable with Au-Pt/SiO2 labeled TMB/H2O2 system. This method showed the limit of detection (LOD) of 50 pg mL−1 (n = 3, ±4.8%) for HIgG and a 1:107 dilution detection for PCV2 antibody, two orders of magnitude higher in detection sensitivity compared to conventional ELISA, which revealed its great advantage in developing sensitive enzyme-linked immunoassay.