Precise regulation of donor-acceptor pairing maximizes energy transfer efficiency for fluorescence and colorimetric dual-mode immunoassays

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.