Expanding the Monolayer Scope for Nucleic Acid-Based Electrochemical Sensors Beyond Thiols on Gold: Alkylphosphonic Acids on ITO.

Electrochemical biosensors are a powerful and rapidly evolving molecular monitoring technology. Evidenced by the success of the continuous glucose monitor in managing Type 1 Diabetes, these sensors are capable of precise, accurate measurements in unprocessed biological environments. Nucleic acid-based electrochemical sensors (NBEs) are a specific type of biosensor that employs the target binding and conformational dynamics of nucleic acids for signal transduction. Currently, the vast majority of NBEs are fabricated via self-assembly of alkylthiols on Au electrodes. However, this architecture is limited in scope, as Au electrodes are not universally deployable for all potential NBE applications. Here, to expand the repertoire of materials on which NBEs can be made, we describe the multistep procedure for creating sensing monolayers of alkylphosphonic acids on a conductive oxide surface. Using such monolayers on indium tin oxide (ITO)-coated glass slides, we couple redox reporter-modified nucleic acids and demonstrate signaling of procaine-binding NBE sensors in buffer and human serum. We investigate the operational stability of these NBE sensors to reveal faster signal loss relative to benchmark thiol-on-gold sensing layers, a result that arises due to poor stability of the underlying ITO. Finally, we discuss future directions to continue expansion of NBE sensor materials and applications.