Construction of a Self-Enhanced Fluorescent Aptasensor Based on Lanthanide Ligand Self-Assembly and Catalytic Hairpin Assembly for Highly-Sensitive Detection of Anatoxin-a
Alina Bari, Guiying Jin, Yinbo Ban, Fuqiang Zhang, Guangfeng Wang
Abstract
Anatoxin-a (ATX-a) is a potent neurotoxin produced by cyanobacteria, with growing evidence unequivocally linking it to acute aquatic toxicity, neurological impairment, and increased mortality in wildlife and livestock, highlighting its significant threat to ecosystem stability and public health. Here, we report a novel fluorescent aptasensor system (FAS) that integrates novel DNA-functionalized Eu nanoparticles (DNA-Eu NPs) with a catalytic hairpin assembly (CHA) strategy for ultrasensitive ATX-a detection. We used single-stranded DNA C 16 T 20 A 16 (ssDNA) oligomers rich in cytosine, thymine, and adenine as effective antenna ligands to sensitize the luminescence of Eu. The Black Hole Quencher (BHQ) labeled hairpin DNA (H 3 ) hybridizes with the exposed DNA sequence on the surface of NPs through complementary base pairing, promoting the fluorescence energy transfer between the quenching group BHQ and DNA-Eu NPs, leading to the formation of DNA-Eu-H 3 and providing stable “on–off” fluorescence. In the presence of ATX-a, it specifically binds to the aptamer (B) to release trigger DNA (T) from duplex DNA (B-T). T initiates a CHA-driven cascade amplification between H 1, H 2, and DNA-Eu-H 3, resulting in fluorescence recovery and signal amplification of DNA-Eu NPs. This FAS exhibits good stability, excellent specificity, and high sensitivity, with a detection limit (LOD) of 0.30 pg/mL and a linear range of 0.001–500 ng/mL. This strategy provides an innovative, highly sensitive, label-free assay and is expected to be a powerful tool for environmental toxin analysis.