A time-resolved fluorescence immunoassay for the ultrasensitive determination of diethylstilbestrol based on the double-codified gold nanoparticles

• An ultrasensitive method for determination of diethylstilbestrol was established.
• Gold nanoparticle-based signal amplification platform for TRFIA was constructed.
• The detection limit for diethylstilbestrol is 0.4 fg mL−1.

An ultrasensitive and selective method is presented for the determination of diethylstilbestrol (DES) using time-resolved fluorescence immunoassay (TRFIA) based on double-codified gold nanoparticles (DC-AuNPs). In this system, the DC-AuNPs, that are gold nanoparticles (AuNPs) modified with anti-DES antibody and SH-dsDNA-biotin, was regarded as signal amplifier. A competitive immunoreaction was performed on polystyrene microtitration plates, where the DES compete with the immobilized DES-ovalbumin on polystyrene microtitration plates to bind to anti-DES antibodies on DC-AuNPs, and the europium(III)-labeled streptavidin was added to link to the SH-dsDNA-biotin as a tracer. Fluorescence signal was amplified via the AuNPs and the biotin–streptavidin double amplification systems. Under the optimized condition, DES can be quantified by TRFIA. The linear range and the limit of detection of DES were 1.0 × 10−6–10 ng mL−1 and 0.4 fg mL−1, respectively. This method was applied to determine DES in beef sample, with the recoveries ranging from 88% to 105%.

Schematic diagram of the DC-AuNPs preparation (A) and the amplification assay of competitive time-resolved fluorescence immunoassay for DES (B).Figure optionsDownload as PowerPoint slide