Immunoassay detection of tumor-associated autoantibodies using protein G bioconjugated to nanomagnet-silica decorated with Au@Pd nanoparticles

Abstract

A colorimetric immunosensor was developed for the detection of tumor-associated anti-p53 autoantibodies (anti-p53aAbs). The immunosensor platform was prepared by immobilizing human-protein (p53Ag) onto a high binding 96-well plate. The immunoassay was based on the immunometric sandwich protocol, and protein G functionalized nanomagnet-silica nanoparticles decorated with Au@Pd (Fe₃O₄@SiO₂-NH₂-Au@Pd_0.30NPs-protG) was used as the detection nanobioprobe. The Fe₃O₄@SiO₂-NH₂-Au@Pd_0.30NPs-protG exhibited a high binding affinity for the captured anti-p53aAbs and high catalytic performance towards the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB). The oxidation of TMB resulted in significant color change and a UV-vis absorption signal. The detection was achieved by measuring the changes in UV-Vis absorption as the concentrations of anti-p53aAbs changed. The apparent binding affinity (K_D) between the p53aAbs and Fe₃O₄@SiO₂-NH₂-Au@Pd_0.30NPs-protG was 35.2 ng mL^-1. The plot of change in the absorption intensity against the logarithm of anti-p53aAbs was linear within 1.0-500.0 ng mL^-1 with a correlation coefficient (R²) of 0.98. The detection limit (LoD) using 3σ was calculated to be 15 pg mL^-1, which is lower than the conventional HRP-label based colorimetric immunoassay. The real sample detection was investigated using the serum recovery method. The recovery of the anti-p53aAbs ranges from 98.5% to 105.7%, demonstrating its potential for practical applications.

Keywords: Cancer biomarker; Colorimetric immunosensor; Nanomagnet bioconjugate; Nanozyme; anti-p53 autoantibodies.