A novel exonuclease III-aided amplification assay for lysozyme based on graphene oxide platform

Based on exonuclease III (Exo III) aided amplification and graphene oxide (GO) platform for fluorescence quenching, a novel, turn-on fluorescent aptasensor for lysozyme (Lys) protein was constructed. The system contains a hairpin probe (HP) and a signal probe (SP) labeled with carboxyfluorescein (FAM) at its 5′ end. HP, which consists of the aptamer sequence of Lys, is partially complementary to SP. Lys could bind with the aptamer region of the HP and facilitate the opening of the hairpin structure of HP, exposing a single-stranded sequence to hybridize with SP. This triggered the Exo III aided amplification and caused the degradation of SP, which liberated the free fluorophore labels. Upon the addition of GO, the released fluorophore could not be adsorbed and no fluorescence quenching occured, while the intact SPs could be adsorbed on GO surface with the fluorescence substantially quenched. The results revealed that the proposed method displayed fluorescence responses in a linear correlation to the concentrations of Lys within the range from 0.125 μg/ml to 1 μg/ml and the detection limit is 0.08 μg/ml. Besides such sensitivity, the proposed strategy is also low-cost and simple due to its homogeneous and fluorescence-based detection format.