Re-engineering Electrochemical Aptamer-Based Biosensors to Tune Their Useful Dynamic Range via Distal-Site Mutation and Allosteric Inhibition.

Electrochemical aptamer-based (E-AB) sensors, exploiting binding-induced changes in biomolecular conformation, are rapid, specific, and selective and perform well even in a complex matrix, such as directly in whole blood and even in vivo. However, like all sensors employing biomolecular recognitions, E-AB sensors suffer from an inherent limitation of single-site binding, i.e., its fixed dose-response curve. To circumvent this, we employ here distal-site mutation and allosteric inhibition to rationally tune the dynamic range of E-AB sensors, achieving sets of sensors with a significantly varied target affinity (∼3 orders of magnitude). Using their combination, we recreate several approaches to narrow (down to 5-fold) or extend (up to 2000-fold) the dynamic range of biological receptors. The thermodynamic consequences of aptamer-surface interactions are estimated via the free-energy difference in solution-phase and surface-bound biosensors employing the same aptamer as a recognition element, revealing that an allostery strategy provides a more predictable and efficient means to finely control the target affinity and dynamic range. Such an ability to rationally modulate the affinity of biomolecule receptors would open the door to applications including cancer therapy, bioelectronics, and many other fields employing biomolecule recognition.