Intrinsic “Turn-On” Aptasensor Detection of Ochratoxin A Using Energy-Transfer Fluorescence

Abstract

Ochratoxin A (OTA) is an intrinsically fluorescent phenolic mycotoxin that contaminates a wide range of food products and is a serious health threat to animals and humans. An OTA binding aptamer (OTABA) that folds into an antiparallel G-quadruplex (GQ) in the absence and presence of target OTA has been incorporated into a vast variety of aptasensor platforms for OTA detection. The development of a simple, aptamer-based approach would allow for detection of the toxin without the use of complex analytical instrumentation, which has been the gold standard for OTA detection thus far. However, to date, none of the aptasensor platforms have utilized the natural fluorescence of the phenolic toxin for detection. Herein, we report that OTA binding to OTABA involves π-stacking interactions that lead to GQ-to-toxin energy transfer (ET), which affords a “turn-on” fluorescence self-signaling platform in which the emission of the aptamer–target complex is enhanced in comparison to the free toxin alone. Selective excitation of the GQ–OTA complex at 256 nm leads to a 4-fold enhancement in OTA fluorescence. The GQ–OTA ET detection platform boasts a limit of detection ∼2 ng/mL, which is comparable to a previously demonstrated fluorescence resonance energy transfer immunoassay platform for OTA detection, and displays excellent OTA selectivity and recovery from red wine samples.

On-Strand Knoevenagel Insertion of a Hemicyanine Molecular Rotor Loop Residue for Turn-On Fluorescence Detection of Pb-Induced G-Quadruplex Rigidity

“…The fluorescence turn-on response was further increased to ∼10-fold using energy-transfer (ET) fluorescence (Figure A). ET fluorescence between DNA (donor) and a fluorescent chromophore (acceptor) occurs when acceptor binding involves π-stacking interactions, which permits energy of UV absorbance by DNA base pairs to be efficiently transferred to the ground-state fluorescent probe to cause excitation to furnish a turn-on emission response. , ET fluorescence within GQ structures provides ET efficiencies that are higher than duplex-to-probe examples , because the central metal ion promotes polarization enhancement (Figure B) of donor G-residues to furnish greater enol character (like O 6 -methylG, which is more emissive than its keto counterpart). Interestingly, the excitation wavelength for the Pb 2+ -bound TBA sample displayed a noticeable shift to the red compared to the K + -bound sample for a maximum wavelength change at 310 nm (Figure B).…”

Lighting Up the Thrombin-Binding Aptamer G-Quadruplex with an Internal Cyanine-Indole-Quinolinium Nucleobase Surrogate. Direct Fluorescent Intensity Readout for Thrombin Binding without Topology Switching

“…For the internally modified 4QI–TBA 16 sample (solid red trace), λ ex /λ em was observed at 510/581 nm, and the internal dye displayed an impressive 40.6-fold increase in intensity compared to free 4QI and 10.9-fold greater than label-free 4QI/TBA 16 . The excitation spectrum also displayed a prominent ET band at 256 nm, confirming strong π-stacking interactions between the internal 4QI surrogate and the GQ structure. , This observation suggested that favorable π-stacking interactions between 4QI and DNA base pairs accounted for the relatively small Δ T m values for duplex and GQ structures produced by 4QI–TBA 16 compared to the native strand (Table ).…”

Recent Progress and Development of G-Quadruplex-Based Luminescent Assays for Ochratoxin A Detection

“…A simple detection method based on the unique antiparallel G4 structure formed by OTA was reported (Armstrong-Price et al, 2020 ). The “turn-on” fluorescence self-signal (λ ex /λ em = 256/425 nm) is generated by π-stacking interactions that lead to G4-to-toxin energy transfer (ET).…”