Time-resolve Fluorescence Immunoassay for Determination of Diethylstilbestrol Based on Dual-codified Gold Nanoparticles

Abstract

An ultrasensitive time-resolve fluorescence immunoassay for the determination of diethylstilbestrol (DES) was established based on dual-codified gold nanoparticles (DC-AuNPs). The dual-codified gold nanoparticles was composed of gold nanoparticles (AuNPs) conjugated to anti-DES antibody and SH-dsDNA-biotin. The DES-OVA immobilized on polystyrene microtitration plates and the DES in the standard solution or sample were competitively reacted with the anti-DES antibodies immobilized on DC-AuNPs, and the fluorescence signal was amplified via AuNPs and biotin-streptavidin (B-SA) dual amplification system; followed by the addition of enhancement solution, the Eu3+ was disengaged from the reactive product and formed new chelate with the enhancement solution again, and the fluorescence signal was further enhanced. Under the optimized condition, the method shows a wide linear range from 1.0×10-6 to 10.0 ng/mL with detection limit of 0.4 fg/mL. This method was applied to determine DES in milk sample, with the percent recoveries in the range of 91.9-101.5%.