Redox Reporter ‐ Ligand Competition to Support Signaling in the Cocaine‐Binding Electrochemical Aptamer‐Based Biosensor

Abstract

Electrochemical aptamer‐based (E‐AB) biosensors have demonstrated capabilities in monitoring molecules directly in undiluted complex matrices and in the body with the hopes of addressing personalized medicine challenges. This sensing platform relies on an electrode‐bound, redox‐reporter‐modified aptamer. The electrochemical signal is thought to originate from the aptamer undergoing a binding‐induced conformational change capable of moving the redox reporter closer to the electrode surface. While this is the generally accepted mechanism, it is notable that there is limited evidence demonstrating conformational change or distance‐dependent change in electron transfer rates in E‐AB sensors. In response, we investigate here the signal transduction of the well‐studied cocaine‐binding aptamer with different analytical methods and found that this sensor relies on a redox‐reporter ‐ ligand competition mechanism rather than a ligand‐induced structure formation mechanism. Our results show that the covalently bound redox reporter, methylene blue, binds at or near the ligand binding site on the aptamer resulting in a folded conformation of the cocaine‐binding aptamer. Addition of ligand then competes with the redox reporter for binding, altering its electron transfer rate. While we show this for the cocaine‐binding aptamer, given the prevalence of methylene blue in E‐AB sensors, a similar competition‐based may occur in other systems.

Kinetic FRET Assay to Measure Binding-Induced Conformational Changes of Nucleic Acids

“…Our results are comparable with the results of Reinstein et al (2013) 25 from NMR, ITC, and SAXS measurements, the latter showing differences in the pairwise density distribution functions (PDDF) plot between the free and quinine-bound MN19 aptamer forms. Other methods, such as 1 H NMR spectra, 45 EPR spectra, 46 and aptamer-based electrochemical biosensors, 47 have also confirmed a conformational change upon analyte binding to the aptamer.…”

Codeposition Enhances the Performance of Electrochemical Aptamer-Based Sensors

“…Presumably, this means that the bindinginduced signal change associated with the phenylalanine sensor is likely generated by target-induced displacement of the redox reporter from the target binding pocket. 33 That this aptamer is always folded may account for the inability of codeposition and target-assisted deposition to improve its gain. Specifically, unfolded aptamers expose their nucleobases, which are then free to bind the surface of our gold electrodes nonspecifically.…”

“…Upon addition of the denaturant urea, the ellipticity of the phenylalanine aptamer also falls much more significantly than that of the other aptamers (Figure S6), again suggesting that, unlike the other aptamers we have employed, it is folded in the absence of a target. Presumably, this means that the binding-induced signal change associated with the phenylalanine sensor is likely generated by target-induced displacement of the redox reporter from the target binding pocket . That this aptamer is always folded may account for the inability of codeposition and target-assisted deposition to improve its gain.…”

Long-TermIn VivoMolecular Monitoring Using Aptamer-Graphene Microtransistors

“…Generally, EAB sensors consist of single-stranded DNA or RNA aptamers functionalized on the surface of a gold working electrode 17 . Redox reporters, like methylene blue or ferrocene, are typically functionalized to one end of the aptamers, allowing for charge transfer upon the conformational change of aptamers caused by the binding of targets 19 . More specifically, the binding and dissociation of target analytes to aptamers cause the conformational rearrangement 4 of aptamers, which changes the electron transfer rate between redox reporters and the working electrode and results in a measurable current change.…”