Sequence-Specific Electrical Purification of Nucleic Acids with Nanoporous Gold Electrodes

Abstract

Nucleic acid-based biosensors have enabled rapid and sensitive detection of pathogenic targets; however, these devices often require purified nucleic acids for analysis since the constituents of complex biological fluids adversely affect sensor performance. This purification step is typically performed outside the device, thereby increasing sample-to-answer time and introducing contaminants. We report a novel approach using a multifunctional matrix, nanoporous gold (npAu), which enables both detection of specific target sequences in a complex biological sample and their subsequent purification. The np-Au electrodes modified with 26-mer DNA probes (via thiolgold chemistry) enabled sensitive detection and capture of complementary DNA targets in the presence of complex media (fetal bovine serum) and other interfering DNA fragments in the range 50 to 1500 base pairs. Upon capture, the non-complementary DNA fragments and serum constituents of varying sizes were washed away. Finally, the surface-bound DNA-DNA hybrids were released by electrochemically cleaving the thiol-gold linkage and the hybrids were iontophoretically eluted from the nanoporous matrix. The optical and electrophoretic characterization of the analytes before and after the detection-purification process revealed that low target DNA concentrations (80 pg/µL) can be successfully detected in complex biological fluids and subsequently released to yield pure hybrids free of polydisperse digested DNA fragments and serum biomolecules. Taken together, this multifunctional platform is expected to enable seamless integration of detection and purification of nucleic acid biomarkers of pathogens and diseases in miniaturized diagnostic devices.

Nanoporous metals by alloy corrosion: Bioanalytical and biomedical applications

“…8 Enzymes such as laccase have also been covalently linked to SAMs formed from lipoic acid activated to produce reactive esters that form an amide linkage to a lysine residue on the enzyme. 9 Thiolated single-stranded DNA oligonucleotides have been directly assembled onto NPG, 10 as have thiolated aptamers. 11 In the case of DNA and aptamer immobilization, subsequent exposure to a short chain alkanethiol such as mercaptohexanol has been applied to cover exposed sites, limit nonspecific adsorption, and promote better orientation of the DNA strand.…”

“…Several groups have reported that NPG films are highly biofouling-resilient and are able to sustain electrochemical function in protein solutions. 10,17 This has been attributed to NPG morphology at a certain pore size (tens of nanometers), which blocks the permeation of large proteins while still allowing the transport of small ions and reporter molecules, effectively serving as a biofouling-resilient electrode (Figure 1b and Figure 2c).…”

Voltage‐Gated Closed‐Loop Control of Small‐Molecule Release from Alumina‐Coated Nanoporous Gold Thin Film Electrodes

“…Np-Au is produced by dissolving silver from a silver-rich gold alloy in heated nitric acid, where gold atoms go under surface diffusion to create a bicontinuous open-pore structure. [14] A wide range of attractive features, including high surface area-tovolume ratio, [15] tunable morphology [16] and surface chemistry, [17] biocompatibility, [18] electrical conductivity, [16a] and compatibility with conventional microfabrication techniques, [19] have led to interest from the sensing and catalysis community. These features, also desirable for precise control of molecular release, enable high small-molecule loading capacity and release kinetics, paved the way for np-Au's use in molecular release applications.…”

Effect of Surface–Molecule Interactions on Molecular Loading Capacity of Nanoporous Gold Thin Films

“…Properties of the drug molecules (e.g., electrical charge, size, chemical reactivity), the materials (e.g., morphology, surface charge), and the liquid medium where the molecules are delivered (e.g., ionic strength, pH) are all crucial factors in dictating drug delivery performance 2,6 . Nanoporous gold (np-Au), with its high effective surface area, well established gold-thiol-mediated surface modifications, and tunable pore morphology, is a promising material for drug delivery applications and studying the influence of physicochemical surface properties on the moleculeloading capacity and release kinetics [8][9][10][11][12][13][14][15] . Our previous studies have focused on the effect of pore morphology and elution medium constituents on the molecular release from np-Au coatings 11,16 .…”