A novel ratiometric aptasensor based on dual-emission fluorescent signals and the conformation of G-quadruplex for OTA detection

Ochratoxin A (OTA) is known as an important mycotoxin that is widely distributed in food products. The increasing requirement for sensitive and convenient OTA detection has promoted the development of aptasensors based on nanomaterials and elegant design strategy for aptamer probes. Ratiometric fluorescence aptasensors greatly reduce the probability of generating false-positive signals, compared to one-signal detection methods. In this work, an efficient ratiometric aptasensor was constructed, based on the dual-emission fluorescence of N-methyl mesoporphyrin IX (NMM) and copper nanoclusters (CuNCs). The key binding region of aptamer was obtained by theoretical results of molecular docking, and experiments of optimizing the length of complementary DNA (cDNA) verified the conjecture. With the addition of OTA, CuNCs-cDNA dissociated from the aptamer sequence, and the fluorescence of CuNCs (λem=510 nm) decreased. Meanwhile, the aptamer contained G-quadruplex structure and enhanced the fluorescence of NMM (λem=610 nm). In this way, fluorescence intensity at the emission wavelength of 610 nm and 510 nm were monitored and I610/I510 was set as the signal output of this aptasensor. Stability tests indicated that the established aptasensor can reduce the interference of pH and storage time. The limit of detection (LOD) was as low as 0.045 ng/mL, with a wide linear range of 0.05–100 ng/mL. Moreover, specificity tests provided excellent selectivity towards other common mycotoxins, and the applicability was confirmed by performing real sample analysis, indicating that the NMM/CuNCs ratiometric biosensor for OTA detection has practical application prospects in food samples.