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Published on: June 28, 2024
Homogeneous immunoassay utilizing fluorescence resonance energy transfer from quantum dots to tyramide dyes deposited
Zihan Xu1, Xiaojun Liu1, Chenghua Zong1
1School of Chemistry and Materials Science, Jiangsu Normal University, Shanghai Road 101, Xuzhou, Jiangsu, China. gai@jsnu.edu.cn.
Insights
This study introduces a novel, highly sensitive homogeneous immunoassay using fluorescence resonance energy transfer (FRET). The method amplifies signals for detecting biomarkers like carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) at sub-femtomolar levels in plasma.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Biotechnology
Background:
- Homogeneous immunoassays require enhanced sensitivity for clinical diagnostics.
- Existing fluorescence resonance energy transfer (FRET)-based assays have limitations in signal amplification.
- Developing sensitive and reliable diagnostic tools is crucial for early disease detection.
Purpose of the Study:
- To develop a highly sensitive, FRET-based homogeneous immunoassay.
- To improve signal amplification in FRET immunoassays by modifying acceptor labeling strategy.
- To validate the assay's performance for quantifying clinically relevant biomarkers.
Main Methods:
- Developed a novel FRET-based homogeneous immunoassay.
- Labeled the entire sandwich-structured immunocomplex with FRET acceptors.
- Quantified carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) in buffer and human plasma.
Main Results:
- Achieved a 10-fold amplification of the FRET signal due to reduced donor-acceptor distance.
- Reached sub-femtomolar limits of detection (LOD) for CEA and AFP in PBS buffer and blank plasma.
- Successfully quantified CEA and AFP in human plasma samples, demonstrating clinical applicability.
Conclusions:
- The developed FRET immunoassay offers superior sensitivity for clinical diagnostics.
- The modified labeling strategy significantly enhances FRET signal amplification.
- This method provides a reliable platform for quantifying biomarkers in biological samples.
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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