Redox reporter - ligand competition to support signaling in the cocaine-binding electrochemical aptamer-based biosensor.

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.