Synergistic dual-mechanism fluorescence quenching immunochromatographic assay based on Fe-polydopamine submicrobeads for sensitive detection of enrofloxacin

• Fe-PDAN was prepared and used as a highly efficient quencher and immunoprobe; • The fluorescence quenching ability of Fe-PDAN was superior to PDAN; • FRET and IFE co-dominated the quenching mechanism of Fe-PDAN-based FQ-ICA; • FQ-ICA sensitivity with Fe-PDAN improved 5.7-fold more than that with PDAN. A fluorescence quenching immunochromatographic assay (FQ-ICA) was developed in recent years to improve detection sensitivity. However, inefficient fluorescence quenchers and unclear fluorescence quenching mechanisms limit the development of FQ-ICA. Here, Fe-polydopamine submicrobeads (Fe-PDAN) were prepared by integrating Fe(III) and PDAN as a quencher quenching the fluorescence of quantum dot beads (QB) to establish a highly sensitive FQ-ICA. Fe-PDAN not only absorbs the excitation and emission light of QB due to the strong inner filter effect, but also accepts part of the released energy of QB by fluorescence resonance energy transfer, resulting in efficient fluorescence quenching and ensuring ultralow fluorescence background signal. With the synergistic effect of fluorescence resonance energy transfer and inner filter effect, the fluorescence quenching constant of Fe-PDAN was 14.9-fold higher than that of PDAN on the nitrocellulose membrane, indicating the stronger quenching ability of Fe-PDAN. The fluorescence lifetime of QB (25.2 ns) in FQ-ICA decayed by 51.9% after Fe-PDAN quenching (12.1 ns). Enrofloxacin, an abused antibiotic, was specifically recognized by antigen-antibody immunoaffinity reaction and detected by monoclonal antibodies conjugated to Fe-PDAN based on the competitive inhibition principle. The FQ-ICA sensitivity based on Fe-PDAN (limit of detection, LOD=0.016 ng/mL) improved 5.7-fold more than that based on PDAN (LOD=0.091 ng/mL). The proposed Fe-PDAN can act as a dual-mechanism quencher to enable highly sensitive FQ-ICA, thereby leading to the innovative design of high-efficiency quenchers in the development of sensitive fluorescence quenching biosensors.