Precise Spectral Overlap-Based Donor–Acceptor Pair for a Sensitive Traffic Light-Typed Bimodal Multiplexed Lateral Flow Immunoassay

Bimodal-type multiplexed immunoassays with complementary mode-based correlation analysis are gaining increasing attention for enhancing the practicability of the lateral flow immunoassay (LFIA). Nonetheless, the restriction in visually indistinguishable multitargets induced by a single fluorescent color and difficulty in single acceptor ineffectual fluorescence quenching due to the various spectra of multiple different donors impede the further execution of colorimetric–fluorescence bimodal-type multiplexed LFIAs. Herein, the precise spectral overlap-based donor–acceptor pair construction strategy is proposed by regulating the size of the nanocore, coating it with an appropriate nanoshell, and selecting a suitable fluorescence donor with distinct colors. By in situ coating Prussian blue nanoparticles (PBNPs) on AuNPs with a tunable size and absorption spectrum, the resultant APNPs demonstrate efficient fluorescence quenching ability, higher colloidal stability, remarkable colorimetric intensity, and an enhanced antibody coupling efficiency, all of which facilitate highly sensitive bimodal-type LFIA analysis. Following integration with competitive-type immunoreaction, this precise spectral overlap-supported spatial separation traffic light-typed colorimetric–fluorescence dual-response assay (coined as the STCFD assay) with the limits of detection of 0.013 and 0.152 ng mL–1 for ractopamine and clenbuterol, respectively, was proposed. This work illustrates the superiority of the rational design of a precise spectral overlap-based donor–acceptor pair, hinting at the enormous potential of the STCFD assay in the point-of-care field.