Dual-Fluorescence Ratiometric Aptasensor Using Structure-Switching Aptamer Modified With 2-Aminopurine for Detecting Ochratoxin A

Structure-switching aptamers (SSAs) generate measurable signals through target-induced conformational changes, serving as sensitive molecular recognition elements. We developed a dual-fluorescence SSA for ochratoxin A (OTA) detection by inserting 2-aminopurine (2AP) into the OTA aptamer and hybridizing a complementary DNA (cDNA) strand at the 5' end. OTA binding induced the aptamer-cDNA duplex to form a G4, enhancing 2AP fluorescence at 370 nm. Together with the intrinsic fluorescence of OTA at 450 nm, this enabled ratiometric dual-fluorescence measurements without external quenchers or fluorophores. To elucidate the molecular mechanism, all-atom molecular dynamics (MD) simulations and spectroscopic experiments assessed the effects of cDNA hybridization sites and strand lengths on sensing performance. Simulations further examined OTA binding at various 2AP insertion sites. Apt@2AP-cDNA1, with cDNA hybridized to bases 1-12, exhibited a binding pattern closely resembling the original aptamer. Umbrella sampling simulations revealed similar binding affinities: 46.3 kJ/mol for Apt@2AP-cDNA1 and 43.2 kJ/mol for the original aptamer. Experimentally, Apt@2AP-cDNA1 converted to G-quadruplexes more efficiently than other designs. A ratiometric dual-fluorescence aptasensor using Apt@2AP-cDNA1 was developed, with a linear detection range of 10-800 nM, a detection limit of 5.9 nM. The dual-fluorescence ratiometric aptasensor has good selectivity and was proven effective for detecting OTA in beer samples.