Structure-switching electrochemical aptamer-based (E-AB) sensors are a class of chemical sensors that provides specific target recognition and high sensitivity for the detection of proteins and small molecules. Intermittent pulse amperometry (IPA) is a recently introduced electrochemical technique that allows rapid interrogation of E-AB sensor response. Here, we use IPA to study kinetics of small molecule binding to surface-bound aptamers. To pursue this study, the E-AB sensor platform is combined with a flow injection analysis system that allows the determination of the observed binding rate (kobs), association rate (kon), and dissociation rate (koff) constants for the binding of ATP and aminoglycoside tobramycinto their corresponding electrode-bound DNA aptamer. These kinetic parameters further allow for the calculation of the thermodynamic binding affinity (KD,surface). The flow injection system comprises of a two-position six valve injector and is controlled automatically to manage the injection of buffer and target. A continuous 5 s injection of the target analyte to the electrode surface results in a rapid, single-exponential rise in current that corresponds to the target association phase followed by a steady-state region indicative of reaching equilibrium. Finally, the buffer is reintroduced to the sensor surface where the aptamer-target complex will dissociate regenerating the sensor surface. Employing IPA for fast E-AB sensor interrogation facilitates the determination of rapid binding kinetics of small molecules-surface-bound aptamers, which is a difficult task to be achieved via conventional techniques such as surface plasmon resonance. The high temporal resolution gained with IPA provides a fundamental foundation to study real-time monitoring of biological processes like exocytosis.

Interrogation of small molecule to surface-bound aptamer binding kinetics with electrochemical, aptamer based sensors using intermittent pulse amperometry

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