A cross-reactive DNA aptamer for neonicotinoid insecticides: Selection and its application in label-free rapid screening and detection of five neonicotinoids in foods

An improved multi-target SELEX yielded an aptamer with binding affinity for 5 NNIs. • Joint characterization clarified enthalpy-driven induced-fit recognition mechanism. • Unlabeled colorimetric sensor realized single/total fast screening of 5 NNIs in food. Neonicotinoid insecticides (NNIs) are a class of fourth-generation insecticides widely used in agriculture. It is crucial to discover bioreceptors with specific binding affinity toward multiple or even all commonly-used NNI compounds, and further develop high-throughput methods for rapid screening/determination of multiple or even all commonly-used NNI compounds in foods. Herein, an improved combined SELEX strategy integrating gradient multi-target parallel selection and serial counter-selection was developed for screening cross-reactive aptamers toward NNIs. One of the obtained cross-reactive aptamers, NNI-03, can recognize five commonly-used NNI compounds, including imidacloprid (IMI), thiacloprid (TCP), imidaclothiz (IMZ), thiamethoxam (TMX) and acetamiprid (ACE), with excellent specificity and binding affinities ( K d values) within comparable orders of magnitude (low-micromolar range). The practicality and reliability of NNI-03 were validated by developing a label-free colorimetric aptasensor for rapid screening/determination of above five NNI compounds, either individually or collectively, with a limit of detection (LOD) of 0.05–0.10 μM, a limit of quantitation (LOQ) of 0.25–0.50 μM and no interference from non‑neonicotinoid pesticides. The LOD and LOQ are all lower than the maximum residue limits (MRL) of these five NNIs in most foods as specified by Chinese national standards (GB-2763-2026). The aptasensor was successfully applied to detect these five NNI compounds, either individually or collectively, in tomato and fresh milk samples. This work not only offers a promising bioreceptor for the construction of diverse high-throughput methods but also directly provides a simple and cost-effective analytical approach, enabling rapid screening/monitoring of individual or co-existing residues of above five NNI compounds in foods. Download: Download high-res image (172KB) Download: Download full-size image