Using the GOLD-SELEX approach to generate highly specific and sensitive DNA aptamers against carbendazim

Carbendazim (CBZ) is a fungicide that is widely used in agriculture for controlling fungal diseases. However, its excessive presence in food, feed, agricultural soils, and water bodies poses severe environmental, human, and animal health risks due to its toxicity. Among the various available detection techniques, aptamers have gained prominence as highly sensitive, cost-effective, and reliable tools for on-site detection. This study employed gold nanoparticles (GNPs) combined with the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, specifically using the GOLD-SELEX approach, to identify a highly specific and sensitive aptamer for CBZ detection in food and feed products. The process involved ten rounds of SELEX, incorporating counter-SELEX against commonly used and structurally related pesticides, such as Mancozeb, Chlorpyrifos, Glyphosate, Monocrotophos, Atrazine, Thiamethoxam, and Methyl Parathion, to enhance specificity. A graphene oxide (GO)-based fluorescence assay was utilized to monitor aptamer enrichment throughout the SELEX process. In the final SELEX round, PCR amplification was performed using unmodified primers, and the amplified sequences were cloned using a blunt-end cloning kit from Takara. High-throughput sequencing (HT-SELEX) was then conducted to analyze the abundance and motifs of the enriched aptamers. The identified aptamer sequences will undergo further characterization to determine their dissociation constant (kD) and assess their sensitivity and specificity toward CBZ. Ultimately, the most effective aptamer will be utilized to develop a rapid, cost-effective, and user-friendly diagnostic kit for detecting carbendazim in food and environmental samples.