Extending the Operational Lifespan of Nucleic Acid-Based Electrochemical Sensors via Protection against Hydrogen Peroxide and Oligonucleotide Displacement.

Nucleic acid-based electrochemical sensors (NBEs) have emerged as a promising approach to continuous molecular monitoring in vivo. NBEs consist of electrically conductive gold surfaces coated with self-assembled monolayers of a mixture of electrode-passivating alkylthiols and functional alkylthiol-modified oligonucleotides (oligos). These oligos also display binding sites for target analytes and are modified with redox reporters capable of transferring electrons to the underlying gold electrode. Although sufficiently robust for continuous, multihour sensing of small molecules and proteins in biological fluids both in vitro and in vivo, the operational lifespan of NBEs under continuous interrogation is only 12 h in biological fluids. To address this issue, we present rigorous experimental evidence using scanning electrochemical microscopy (SECM) showing that applying a negative potential bias during NBE interrogation promotes the localized generation of hydrogen peroxide (HO) at concentrations up to ∼30 μM near the sensor surface, which in turn contributes to progressive degradation of the sensing interface. In addition, we introduce a biofluid mimetic specifically designed to study the competitive displacement of oligonucleotides from NBEs─another critical degradation pathway. Using this mimetic, we identify and validate two mitigation strategies: elongation of monolayer alkyl chains and incorporation of an alginate-based hydrogel. Both approaches significantly reduce HO generation at the sensor surface and suppress competitive displacement, extending in vitro sensor stability by up to 20-fold. Combined, these strategies also improve sensor stability in vivo, as shown by a 95% retention of signaling from sensors implanted in rats' cortexes after 5 h of continuous operation versus only 60% retention for standard sensors.