Selection of ssDNA aptamer using GO-SELEX and development of DNA nanostructure-based electrochemical aptasensor for penicillin

Penicillin is among the most used antibiotics for the prevention and treatment of infections in livestock. However, residual penicillin present in animal food products can cause an allergic immune response or induce several human diseases. Herein, we identified aptamer sequences highly specific to penicillin G (PenG) through GO-SELEX screening of the ssDNA library. The target-binding affinity and the dissociation constants of the aptamer sequences were determined by an electrochemical sensing platform. The effectiveness of the sensing platform was enhanced by modifying the working electrode surface using gold nanoparticles and electrochemically reduced graphene oxide and further functionalized with tetrahedral DNA nanostructures (TDNs). The TDN functionalization allowed to precisely control the surface density of the biosensing interface and orientation of attached aptamers with high target accessibility. The specificity of the selected aptamer sequence was confirmed by testing against different antibiotics. The results show that the designed aptasensor was fast, sensitive, and specific in detecting and quantifying PenG in a wide concentration range from 0.2 nM to 1 mM with an ultralow limit of detection of 0.05 nM. Additionally, the aptasensor exhibited great potential for application in a real sample when tested with penG-spiked milk samples with recovery rates in the range of 98–109%.