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Journal of Nanomaterials
Volume 2012 (2012), Article ID 582531, 7 pages
Research Article

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

Department of Applied Chemistry, College of Applied Science, Kyung Hee University, Gyeonggi-do, Yongin-si 446-701, Republic of Korea

Received 2 October 2012; Accepted 7 November 2012

Academic Editor: Haiyan Li

Copyright © 2012 Seungah Lee and Seong Ho Kang. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


A highly sensitive immunosensor based on a gold nanopatterned chip was developed for accurate determination of alpha-fetoprotein (AFP) via total 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.