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

Abstract

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.

Interplay of Effective Surface Area, Mass Transport, and Electrochemical Features in Nanoporous Nucleic Acid Sensors

“…On the contrary, the relative probe current displayed the opposite of the expected trend (Figure A), that is, initially a constant value followed by a rapid decrease for the samples with the largest EF (27.9 and 36.1). The amount of DNA desorbed from the samples with different EFs (Figure B) was in agreement with the amount of DNA recovered from each sample (∼10–50 ng/mL), as determined by our previous studies using the same grafting solution concentration (0.5 μM) . In those studies, we observed that the surface-bound and unbound DNA amounts strongly depended on the grafting solution concentration, where the recovered DNA probe approached 5000 ng/mL for grafting solution concentrations of 8 μM.…”

“…Only a higher concentration gradient between the bulk and the pores, attained by high grafting solution concentrations, can drive DNA into the deeper pores. Figure C is a composite plot of electrochemical and fluorometric quantification of the DNA probe layer that illustrates the significant deviation from the expected linear relationship (shown with a trendline) between the two readouts. Taken together, the severely reduced efficiency in grafting the electrochemically active surface at higher EFs likely plays a role in the impaired sensor performance.…”

“…The amount of DNA desorbed from the samples with different EFs (Figure 4B) was in agreement with the amount of DNA recovered from each sample (∼10−50 ng/mL), as determined by our previous studies using the same grafting solution concentration (0.5 μM). 32 In those studies, we observed that the surface-bound and unbound DNA amounts strongly depended on the grafting solution concentration, where the recovered DNA probe approached 5000 ng/mL for grafting solution concentrations of 8 μM. These results suggest that there is significant hindrance in permeation of the DNA probe into the pores.…”

“…Eight different types of electrodes (4 thicknesses each of unannealed and annealed np-Au, all n = 3) were used. Note that, for the entire study, four identical sets of such electrodes were prepared using previously described grafting protocols that consisted of incubating the working electrode overnight (∼15 h) with 0.5 μM MB-tagged DNA probe solution prepared in 25 mM PB and 50 mM MgCl 2 . , To obtain a well-ordered DNA probe monolayer, electrodes were backfilled with mercaptohexanol (MCH), as routinely used. − Finally, the DNA probe-functionalized electrode was assembled in the electrochemical cell for electrochemical characterization (Figure S1).…”

Chemically-Gated and Sustained Molecular Transport through Nanoporous Gold Thin Films in Biofouling Conditions

“…In addition to the tunable morphology, np-Au exhibits other desirable features such as high surface area to volume ratio [ 36 ], electrical conductivity [ 37 ], biocompatibility [ 38 ], biofouling resilience [ 39 ], compatibility with conventional microfabrication processes [ 40 , 41 ], and facile surface functionalization via gold-thiol chemistry [ 42 ]. These benefits make np-Au an attractive material system for drug delivery [ 43 ], short nucleic acid detection [ 44 ], and catalysis [ 45 , 46 ]. Here, we report a computational and experimental study of in-plane molecular transport in np-Au thin films integrated into a microfluidic model of a reservoir distal to the pharmaceutical delivery site.…”