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Updated: Jan 14, 2026
Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
Published on: December 11, 2021
Precise regulation of donor-acceptor pairing maximizes energy transfer efficiency for fluorescence and colorimetric
Jincheng Xiong1, Boyan Sun2, Sihan Wang3
1National Key Laboratory of Veterinary Public Health Security, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China; Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, China Agricultural University, Beijing, 100193, China; Guangdong Provincial Key Laboratory of Advanced Biomaterials, Shenzhen Key Laboratory of Smart Healthcare Engineering, Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China; Institute of Quality Standard and Monitoring Technology for Agro-products of Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, China.
Abstract:
Traditional enzyme-linked immunoassays are constrained by time-consuming multi-step procedures, limited detection sensitivity, and instability of enzymatic activity, restricting their performance in complex sample matrices. To address these challenges, we propose a precisely regulated donor-acceptor pairing strategy that maximizes energy transfer efficiency to develop an ultrasensitive dual-mode immunoassay. Eight emissive gold nanoclusters were systematically screened as fluorescence donors, while the oxidized product of 3,3',5,5'-tetramethylbenzidine served as the energy acceptor. The results reveal that effective donor-acceptor pairing requires not only spectral overlap, but also consideration of physicochemical compatibility, system adaptability, and interference resistance. Among the candidates, glutathione-stabilized Au/Ag bimetallic nanoclusters demonstrated good photostability, strong luminescence, high spectral overlap, and minimal background interference, making them ideal transducers for energy transfer-based signal modulation. Furthermore, a pH-mediated antibody immobilization strategy was developed to achieve oriented assembly of antibodies onto mesoporous core-shell palladium@platinum nanozymes, resulting in bifunctional immunoprobes with high catalytic activity and target specificity. As a proof-of-concept, chloramphenicol detection was demonstrated, fluorescence modality achieved a broad linear range of 0.004-10 ng/mL, with visual and calculated detection limits of 4 pg/mL and 1.1 pg/mL, respectively. The assay was completed within 1 h and exhibited satisfactory recoveries of 80.3-112.0 % in real samples. This work provides new insights into the rational regulation of donor-acceptor systems and provides a generalizable framework for constructing ultrasensitive immunoassays through integrating multiple enhancement strategies.
