Four different film thicknesses of np-Au were sputtered using Lesker Labline Sputter System. For each of the sets, piranha-cleaned glass coverslips were sputter-coated with 160 nm-thick Cr layer to serve as an adhesive layer between the glass substrate and 80 nm-thick seed layer of Au. Ag and Au were co-sputtered on top of the Au-seed layer to obtain 36% Au and 64% Ag (atomic %) alloy 1. Alloy co-sputtering time was varied to allow for fabrication of varying film thicknesses (t = 5, 10, 20 and 30 min). All metal depositions were performed in argon ambient at a pressure of 10 mTorr without breaking the vacuum between deposition of different metal layers. To obtain unannealed np-Au morphology, the alloy samples were dealloyed in 70% nitric acid at 55 °C for 15 min, followed by copious rinsing under deionized water (DI) water stream. This process is a chemical corrosion process, in which nitric acid dissolves less noble metal (Ag) from the AuAg alloy as gold atoms go under surface diffusion to assemble the bi-continuous porous structure with interconnected nanochannels. To obtain coarser, yet self-similar morphologies, thermal annealing was used. Final product of the fabrication process were 8 sets of varying properties thin films (4 thicknesses and 2 morphologiesunannealed and annealed np-Au).

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.