Abstract
There is an urgent need for homogeneous immunoassays that offer sufficient sensitivity for routine clinical practice. In this study, we have developed a highly sensitive, fluorescence resonance energy transfer (FRET)-based homogeneous immunoassay. Unlike previous FRET-based homogeneous immunoassays, where acceptors were attached to antibody molecules located far from the donor, we employed acceptors to label the entire sandwich-structured immunocomplex, including two antibodies and one antigen. As a result, the FRET signal was amplified by a factor of 10, owing to the reduced distance between the donor and acceptors. We validated our method by quantifying carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) in PBS buffer and blank plasma. The limits of detection (LOD) for CEA and AFP in both PBS buffer and blank plasma were comparable, reaching sub-femtomolar levels. Furthermore, we successfully quantified CEA and AFP in three human plasma samples, thereby confirming the reliability of our method for clinical applications.
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