Selection of novel ssDNA aptamer for ultra-sensitive colorimetric detection of acetamiprid using ultra-small dot assay

Abstract

An innovative ssDNA aptamer-mediated colorimetric biosensor utilizing an ultra-small dot assay has been developed for the ultra-sensitive detection of acetamiprid, a hazardous insecticide widely used in agriculture. This “Dot assay” leverages ultra-low volumes of less than 20 µL, allowing for rapid visual detection within seconds and addressing the urgent need for accessible and efficient detection methods. Through a non-immobilized graphene oxide-based systematic evolution of ligands by exponential enrichment (SELEX) approach, a specific 79-mer aptamer (Apt-SS5) was identified after 15 enrichment cycles. The Apt-SS5 aptamer demonstrated a remarkable binding affinity for acetamiprid, with a melting temperature (Tm) of 53 °C and a Gibbs free energy (ΔG) of −7.24 kcal/mol at 37 °C. Notably, the binding pocket is formed by nucleotides C-16 to T-32, with critical interactions occurring at C-10, C-16, G-25, A-26, G-27, G-28, and A-29. The optimized colorimetric assay achieved an impressive limit of detection (LOD) of 0.039 ppb and resulted in a significant reduction in assay costs. The target specificity and cross-reactivity of Apt-SS5 were tested against other pesticides, including imidacloprid, thiachloprid, thiamethoxam, chlorpyrifos, pyriproxyfen, and chlorantraniliprole, with no significant color change observed for these non-targets. In contrast, a clear visual color change was noted in the presence of acetamiprid, confirming the aptamer’s high specificity. Additionally, the aptasensor proved effective for testing river and water samples, yielding recovery rates of 84.40 % to 101.66 %. This innovative approach enhances sensitivity and simplifies the detection process, paving the way for rapid screening of pesticide residues in various samples.