Single-Molecule Sandwich Immunoassay for Quantification of Alpha-Fetoprotein Based on Evanescent Field-Enhanced Fluorescence Imaging

Abstract

A highly sensitive immunosensor based on a gold nanopatterned chip was developed for accurate determination of alpha-fetoprotein (AFP)viatotal internal refection fluorescence microscopy (TIRFM). The surface of the gold nanopatterned chips was modified with dithiobis(succinimidyl propionate) and protein A/G for immobilization of the AFP antibody. The immunoassay created a sandwich of antigen between the AFP antibody on the chip that was modified with protein A/G, and the secondary antibody, a monoclonal anti-human-AFP labeled with biotin (biotin-labeled anti-AFP). AFP concentration was determined based on evanescent field fluorescence signal, which was generated by interaction between biotin-labeled anti-AFP and a streptavidin-labeled fluorescence dye. AFP concentration could be measured in a wide dynamic linear range of 720 zM–10 nM with a detection limit of 720 zM. A significant enhanced sensitivity (~40,000-fold) was achieved with the AFP-nanoarray chip compared to conventional chemiluminescence immunosensors. The immunoassay exhibited a wide detection range and high sensitivity and accuracy, qualities valuable for clinical assay of AFP.