Selection of aptamer targeting levamisole and development of a colorimetric and SERS dual-mode aptasensor based on AuNPs/Cu-TCPP(Fe) nanosheets

Levamisole (LEV) is a veterinary drug that often remains in animal food. Consuming products containing high levels of LEV will cause a series of harmful reactions to human health. This work describes the Capture-SELEX (Capture-systematic evolution of ligands by exponential enrichment) screening strategy of LEV aptamers, using streptavidin modified agarose beads as a solid phase medium to separate target-bound and unbound ssDNA. The affinity and specificity of candidate aptamers were determined by SYBR Green I (SGI) dye and isothermal titration calorimetry (ITC), in which LEV-5 showed good binding affinity and specificity, and the dissociation constant was 66.15 ± 11.86 nM. Circular dichroism (CD) was used to characterize aptamer conformational changes before and after target binding, including increased helicity and enhanced base stacking. To evaluate whether this aptamer can be used for LEV detection, a colorimetric-surface-enhanced Raman spectroscopy (colorimetric-SERS) dual-mode aptasensor was constructed based on the peroxidase-like activity and SERS effect of AuNPs/Cu-TCPP(Fe) nanosheets. The detection limits of this dual-mode aptasensor for LEV were 5 nM and 1.12 nM, respectively. This aptamer-based method was further successfully used to detect LEV in milk, with recoveries ranging from 94.95% to 111.2%, providing a potential application for the detection of harmful substances in food.