ELISA type fluorescence turn-on immunoassay based on Fe3+ induced fluorescence enhancement

ELISA type fluorescence immunoassay for the sensitive detection of protein biomarker tumor necrosis factor α (TNF-α) was proposed. The immuno-reaction was taken place in the wells of the 96 well plate using Fe3O4 nanoparticles as label. The signal amplification strategy was based on the released Fe3+ from Fe3O4 nanoparticles after acid treatment to trigger the fluorescence from nonfluorescent fluorophore. The fluorophore selected was rhodamine 6G derivative N-(rhodamine-6G)lactam-ethylenediamine (Rh6G-LEDA). Such signal turn-on strategy has advantages of low background current and less susceptibility to fluorescence quench. The immunoassay displays high sensitivity, wide linear range and good selectivity for TNF-α detection. The developed immunoassay combines the high throughput of ELISA type assay and the low cost as well as simplicity of nanoparticle label, which could find wide applications for clinical screening purposes.