Introduction: Rapid detection of biomolecular markers is essential for early diagnosis and management of diseases as well as for fundamental biological studies. Electrochemical sensors have been powerful tools for addressing this need, where nanostructured electrodes have shown significant promise in enhancing the performance of these sensors. In order to develop electrochemical biosensors with rationally-designed nanostructures, it is imperative to have a fundamental understanding of the structure-property relationships that dictate sensor performance. In this study, microfabrication-compatible nanoporous gold (np-Au) thin films with tunable pore morphology are used as a model electrode to investigate the effect of electrode morphology on sensor performance. Specifically, by modifying np-Au pore morphology (i.e., pore size, effective surface area) and employing electrochemical characterization, we studied the effect of morphology on (i) grafting density of 26mer DNA probes, (ii) limit of detection in hybridization to a DNA target, and (iii) dynamic range in detecting different target concentrations¹. We subsequently assessed the performance of np-Au electrodes for nucleic acid detection in complex biological media, with the hypothesis that the optimal pore geometry should allow permeation of nucleic acids into the porous electrode while sieving out biofouling molecules.

Results and Discussion: Np-Au samples were fabricated by sputtering gold/silver alloy on glass coverslips and subsequent etching of silver to self-assemble the np-Au films (un-annealed np-Au). To obtain samples with different pore morphology (annealed np-Au), the films were thermally-treated at 225 °C. The np-Au sensor displayed a 100-fold shift in dynamic range of detection towards lower concentrations of DNA (10 nm – 200 nM) as compared to a planar gold electrode. Annealed np-Au films showed improved accessibility and a further 10-fold shift in dynamic range (Figure 1). The hybridization on np-Au films can be viewed as a combination of two events: (i) target DNA entering the pores and (ii) target DNA binding with probe upon entering the pore. Geometric accessibility determines the efficiency of a target entering the porous structure. Once inside the porous structure, the probability of a target binding to the probe depends on probe density. Target molecule encounters a significantly higher number of probes within a pore compared to a planar surface. These two factors together (i.e., enhanced accessibility and higher probe grafting density in porous films) led to the detection of lower concentrations of target and resulted in a shift in dynamic range of detection with morphology (Figure 1). 

A major obstacle to nucleic acid detection in biological fluids is electrode fouling by constituents that obscure the transport of analytes to the active electrode surfaces. Np-Au electrodes circumvent this by enabling DNA detection in physiologically relevant complex media (bovine serum albumin and fetal bovine serum (FBS))². In contrast, sensor performance was compromised for planar gold electrodes in the same conditions. Un-annealed np-Au electrodes produced a signal suppression (hybridization efficiency) of ~38% with a dynamic range of 25 nM to 200 nM (Figure 2) in FBS. Hybridization efficiency decreased by 10% for np-Au with coarser pores (annealed np-Au) revealing a pore size dependence of sensor performance in biofouling conditions. This nanostructure dependent functionality in complex media suggests that pores with the optimal size act as sieves for  blocking the biomolecules from inhibiting the surfaces within the porous volume while allowing the transport of nucleic acids analytes and redox molecules

Conclusions: In summary, we demonstrated that np-Au electrodes exhibit high electrochemical sensing performance in the presence of common biofouling media and allow for sensitive electrochemical detection of nucleic acids. The un-annealed np-Au electrodes exhibited the highest signal suppression followed by annealed np-Au and planar Au electrodes in presence of FBS. This indicates that pore size plays a significant role in dictating the extent of biomolecule adsorption and transport. This novel technology for detecting target DNA concentrations in tunable dynamic ranges, coupled with sustained functionality in complex biological media, should assist in the development of robust point-of-care diagnostic platforms.

References:

1. Daggumati et al. “Effect of nanoporous gold thin film morphology on electrochemical DNA sensing,” Anal. Chem., 2015, 87 (16), pp 8149–8156

2. Daggumati et al. “Biofouling resilient nanoporous gold electrodes for DNA sensing,” Anal. Chem., 2015, 87 (17), pp 8618–8622