Study of dual binding specificity of aptamer to ochratoxin A and norfloxacin and the development of fluorescent aptasensor in milk detection

Target specificity, one of aptamer characteristics that determine recognition efficiency of biosensors, is generally considered to be an intrinsic property of aptamer. However, a high-affinity aptamer may have additional target binding specificity, little is known about the specificity of aptamer binding to multiple targets, which may result in false-positive results that hinder the accuracy of detection. Herein, an aptamer OBA3 with dual target ochratoxin A (OTA) and norfloxacin (NOR) was used as an example to explore the binding specificity mechanism and developed rapid fluorescent aptasensing methods. The nucleotide 15th T of aptamer OBA3 was demonstrated to be critical for specificity and affinity binding of target OTA via site-saturation mutagenesis. Substituting the 15th T base for C base could directly improve recognition specificity of aptamer for NOR and remove the binding affinity for OTA. The combination of π-π stacking interactions, salt bridges and hydrogen bonds between loop pocket of aptamer and quinolone skeleton, piperazinyl group may contributes to the fluoroquinolone antibiotics (NOR and difloxacin)-aptamer recognition interaction. Based on this understanding, a dual-aptamer fluorescent biosensor was fabricated for simultaneous detection of OTA and NOR, which has a linear detection range of 50–6000 nM with a detection limit of 31 nM for OTA and NOR. Combined with T15C biosensor for eliminating interference of OTA, the assay was applied to milk samples with satisfactory recovery (94.06–100.93%), which can achieve detection of OTA and NOR individually within 40 min.