Interrogation of Small Molecules to Surface-Bound Aptamer Binding Kinetics with Electrochemical Aptamer-Based Sensors Using Intermittent Pulse Amperometry

Structure-switching electrochemical-aptamer-based (E-AB) sensors are a group of chemical sensors that allow specific target recognition of biological molecules with high sensitivity. In this article, intermittent pulse amperometry (IPA) is utilized to perform millisecond-resolved interrogation of E-AB sensors to monitor binding kinetics of small-molecule target analyte binding to surface-tethered DNA aptamers. To achieve this goal, the E-AB sensor platform is coupled with a flow injection analysis system that permits the determination of observed binding rate (<i>k</i>_obs), association rate (<i>k</i>_on), and dissociation rate (<i>k</i>_off) constants for the binding of aminoglycoside tobramycin to electrode-bound DNA aptamer. IPA, a technique with high temporal resolution (milliseconds), enables the assessment of fast binding kinetics between small molecules and surface-bound aptamers, which cannot be obtained via traditional interrogation techniques used for this class of sensors, such as square wave voltammetry (SWV). In addition, we compare IPA to surface plasmon resonance (SPR), a common technique, to demonstrate that the IPA signal is specific to the target-induced conformational change of the surface-bound aptamers, while SPR suffers from signaling from nonspecific interactions. Using IPA, we were able to determine the binding affinity for the tobramycin aptamer using both the kinetic analysis and equilibrium measurements to be 41 ± 11 μM and 46 ± 6 μM, respectively, which are comparable values and therefore validate the use of IPA for determining fast binding kinetic parameters.