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 (k_obs), association rate (k_on), and dissociation rate (k_off) 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 (K_D,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.

Sanduni Abeykoon

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