Anomalous Trends in Nucleic Acid-Based Electrochemical Biosensors with Nanoporous Gold Electrodes

Molecular diagnostics have significantly advanced the early detection of diseases, where electrochemical sensing of biomarkers has shown considerable promise. For a nucleic acid-based electrochemical sensor with signal-off behavior, the performance is evaluated by percent signal suppression (% ss), which indicates the change in current after hybridization. The % ss is generally due to more redox molecules (e.g., methylene blue) associating with the probe DNA bases in the single-strand form than the double-strand form upon hybridization with the target nucleic acid. Nanostructured electrodes generally enhance electrochemical sensor performance via several mechanisms, including increased number of capture probes per electrode volume and unique nanoscale transport phenomena. Here, we employ nanoporous gold (np-Au) as a model electrode material to study the influence of probe immobilization solution concentration on sensor performance and the underlying mechanisms. Unlike planar gold (pl-Au) electrodes, where % ss reaches a steady state with increasing concentration of the grafting solution, the % ss displays peak performance at certain grafting solution concentrations followed by rapid deterioration and reversal of the % ss polarity, suggesting an unexpected case of increased charge transfer upon hybridization. Fluorometric assessments of electrochemically desorbed nucleic acids for different electrode morphologies reveal that a significant amount of DNA molecules (unhybridized and hybridized) remain within the nanopores posthybridization. Analysis of electrochemical signals (e.g., square wave voltammogram shape) suggests that the large unbound nucleic acid concentration may be altering the modes of methylene blue interaction with the nucleic acids and charge transfer to the electrode surfaces.