Immobilization Strategies for Enhancing Sensitivity of Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (E-AB) sensors are a versatile sensing platform that can achieve rapid and robust target detection in complex matrices. However, the limited sensitivity of these sensors has impeded their translation from proof-of-concept to commercial products. Surface-bound aptamers must be sufficiently spaced to bind targets and subsequently fold for signal transduction. We hypothesized that electrodes fabricated using conventional methods result in sensing surfaces where only a fraction of aptamers are appropriately spaced to actively respond to the target. As an alternative, we presented a novel aptamer immobilization approach that favors sufficient spacing between aptamers at the microscale to achieve optimal target binding, folding, and signal transduction. We first demonstrated that immobilizing aptamers in their target-bound, folded state on gold electrode surfaces yields an aptamer monolayer that supports greater sensitivity and higher signal-to-noise ratio than traditionally prepared E-AB sensors. We also showed that performing aptamer immobilization under low ionic strength conditions rather than conventional high ionic strength buffer greatly improves E-AB sensor performance. We successfully tested our approach with three different small-molecule-binding aptamers, demonstrating its generalizability. On the basis of these results, we believe our electrode fabrication approach will accelerate development of high-performance sensors with the sensitivity required for real-world analytical applications.