Fluorescence Resonance Energy Transfer-Enabled Dual-Emission Nanoplatform for Accurate Alpha-Fetoprotein Sensing

Nanoparticles emerge as alternatives to conventional molecular tags such as fluorophores, owing to their superior optical properties, chemical stability, and long photostability. Herein, we present a fluorescence resonance energy transfer (FRET)-based ratiometric fluorescent probe consisting of nitrogen-doped carbon dots and gold nanoclusters (N-CDs–AuNCs) for the sensitive and selective detection of α-fetoprotein (AFP), a biomarker for early hepatocellular carcinoma. The probe is constructed from a donor–acceptor fluorophore pair, in which blue-emissive N-CDs transfer energy to red-emissive AuNCs, resulting in dual fluorescence at 435 and 710 nm under a single excitation wavelength of 380 nm, enabled by carbodiimide coupling. Blue/red dual-fluorescent nanotags were integrated into a ratiometric fluorescence enzyme-linked immunosorbent assay (RF-ELISA) platform to measure AFP on microplates functionalized with anti-AFP capture antibodies, in combination with a glucose oxidase (GOx)-tagged anti-AFP secondary antibody. After assembly of the sandwich-structured immunocomplex, GOx converted glucose into hydrogen peroxide, thereby quenching the red fluorescence of the AuNCs on the fluorescent nanoprobe. At the same time, the blue emission of the embedded N-CDs remained unchanged, allowing the red/blue fluorescence ratio to serve as a quantitative determination of AFP concentration under optimal conditions. Ratiometric fluorescence at 435:710 nm enabled AFP detection over a concentration range of 0.001 to 45 ng mL–1, with a limit of detection near 1.8 pg mL–1. The method exhibited reliable reproducibility, specificity, and stability. AFP measurements in human serum agreed well with a commercially available ELISA kit.