Nanowires as Electrochemical DNA Sensors

The construction of gold nanowires using templated metal deposition [23] (Figure 6.2) provides an easy route to a nanostructured material without the need for expensive or difficult nanofabrication. Both track-etched polycarbonate [23,24] and alumina membranes [24] can be used in conjunction with electroless or electrodeposition of gold and other metals, but most electrochemical studies have focused on the polycarbonate membranes with chemically plated gold, given that there are commercial sources for the template and the plating process is quite straightforward. Early studies of electrochemical processes at nanowires revealed that the minimal background currents achieved with this type of electrode allowed very low levels of redox-active species to be detected [23].

Our work has explored the use of nanowire electrodes as a platform for electrochemical biosensing applications [11,20]. Although electrochemical biosensing holds promise as a means to achieve

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Au deposition

Removal of top Au surface layer

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FIGURE 6.2 Generation of gold nanowire electrodes using templated electrodeposition. Gold nanowires can be generated in track-etched polycarbonate membranes by electroless deposition in solution. After the removal of one face plane of Au, flat nanodisks are present at the surface of the membrane. Subsequent oxygen plasma etching exposes three-dimensional wires that can be used as an ensemble as an electrode.

cost-effective and accurate detection of nucleic acids and protein-based biomarkers [25,26], the difficulty inherent in achieving sufficient sensitivity and specificity for assays based on this type of readout has limited implementation in useful devices. One of the challenges arises from the poor performance of immobilized probe molecules and a lowered ability to complex with target analytes. Nanostructured materials offer a novel way to overcome this difficulty, as the features of nanostructures often match those of biomolecules, which should in theory permit better display of probes and more efficient capture of target analytes.

Our initial studies of electrochemical biosensing at nanowire electrodes were aimed towards nucleic acids detection, an important technology for the diagnosis of genetic and infectious diseases (Figure 6.1). In order to generate an appropriate nanowire-based ensemble for electrochemical biosensing, we imagined that the most effective architecture would feature templated nanowires with exposed tips. Wires generated within polycarbonate membranes can be exposed to an oxygen plasma, which selectively etches away polycarbonate while leaving the wires intact (Figure 6.3). Sealing of the polycarbonate membrane around the Nanowires is achieved by heat treatment, and is a crucial step that significantly reduces double-layer charging currents. Thiolated probe DNA sequences form robust monolayers on the nanowires (Figure 6.1), thus providing an ideal configuration for electrochemical detection of target sequences with an appropriate redox reporter strategy.

The nanowire electrodes were tested as a DNA detection system using an electrocatalytic DNA detection method developed in our laboratory [27]. This label-free system reports on the binding of a target DNA sequence to an immobilized probe oligonucleotide using a catalytic reaction between

FIGURE 6.3 Exposure of gold nanowires by oxygen plasma etching. The length of gold nanowires synthesized within a polycarbonate template can be controlled using oxygen plasma etching. After 1 min of etching (A), ~80 nm of the nanowires protrude from the membrane. After 5 min (B), ~300 nm is exposed. Subsequent heat treatment then causes shrinking of the membrane and sealing of the pores around the wires.

FIGURE 6.3 Exposure of gold nanowires by oxygen plasma etching. The length of gold nanowires synthesized within a polycarbonate template can be controlled using oxygen plasma etching. After 1 min of etching (A), ~80 nm of the nanowires protrude from the membrane. After 5 min (B), ~300 nm is exposed. Subsequent heat treatment then causes shrinking of the membrane and sealing of the pores around the wires.

two transition-metal ions, Ru(NH3)|+ and Fe(CN)|_. The Ru(III) electron acceptor is reduced at the electrode surface and then reoxidized by excess Fe(III), making the electrochemical process catalytic. The increased concentration of anionic phosphates at the electrode surface that accompanies DNA hybridization increases the local concentration ofRu(NH3)|+, and therefore produces large changes in the electrocatalytic signal. This approach works with sequences of varied composition [27] and is thus widely applicable to any target gene of interest.

The utility of nanowire electrodes for nucleic acid analysis was tested using oligonucleotide sequences that correspond to a portion of the 23S rRNA gene from Helicobacter pylori (a pathogen implicated in gastric ulcers and cancer) [11]. When hybridization of sequences from this pathogen was monitored electrocataytically, very low detection limits were established—5 attomoles of DNA could be sensed at a three dimensional nanowire electrode (Figure 6.4). The analysis was performed on an electrode with an exposed geometric area of 0.07 cm2, indicating that zeptomole detection limits could easily be achieved with a modest decrease in the size of the aperture used in the electrochemical analysis. Previous studies that used the Ru(III)/Fe(III) electrocatalysis assay to detect the same DNA sequences using macroscopic gold electrodes achieved femtomole sensitivity. An attomole-level detection limit compares favorably with recently reported electrochemical methods for the direct detection of oligonucleotides [28-32]. The achievement of this unprecedented detection limit with nanoscale electrodes, generated by a simple and lithography-free method, may facilitate the development of miniaturized devices for biomolecular sensing.

The increase in sensitivity observed can be attributed to properties of the nanowire-based electrodes imposed by their nanoscale geometries. Foremost, hybridization efficiencies are enhanced with the nanowire electrodes [32]. Both the kinetics of complexation between an immobilized probe sequence and an incoming target sequence, and the overall extent of hybridization are enhanced. This fact likely reflects that the structure of the monolayer displayed on the curved nanowire surface promotes access

FIGURE 6.4 Evaluation of DNA detection limit at a three-dimensional nanowire electrode using cyclic voltam-metry. In the experiments shown, electrodes were treated with thiolated single-stranded DNA, and exposed to a target complementary sequence. In the scans shown, 0, 1 pM, 1 nM, 1 mM, and 20 mM target DNA are introduced. From these experiments, a detection limit of 5 attomoles could be determined; this represents a significant enhancement in sensitivity over what can be achieved with bulk materials. (Reprinted from Gasparac, R. et al., J. Am. Chem. Soc., 126, 12750, 2004. With permission.)

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FIGURE 6.4 Evaluation of DNA detection limit at a three-dimensional nanowire electrode using cyclic voltam-metry. In the experiments shown, electrodes were treated with thiolated single-stranded DNA, and exposed to a target complementary sequence. In the scans shown, 0, 1 pM, 1 nM, 1 mM, and 20 mM target DNA are introduced. From these experiments, a detection limit of 5 attomoles could be determined; this represents a significant enhancement in sensitivity over what can be achieved with bulk materials. (Reprinted from Gasparac, R. et al., J. Am. Chem. Soc., 126, 12750, 2004. With permission.)

and binding by the incoming sequence. DNA monolayers deposited on bulk gold surfaces at the high densities required for electrochemical sensing are known to possess many inaccessible sites because of the steric and electrostatic crowding that occurs [27]. On a three-dimensional nanoarchitecture, however, the monolayer can still display a high density of probe strands without as much crowding because the curved surface provided by the nanowire will enforce offset of adjacent strands. For example, a 10 nm nanowire will display DNA strands composed of 4.5 nm (15 base) strands on a curved surface with a spacing of nm. The immobilized molecules would adopt a splayed orientation, leaving a significant fraction of their termini remaining unshielded by adjacent strands, hence promoting penetration of a target sequence into the monolayer. It is the matching of the size of this type of nanoscale object and the size of the DNA that makes this structural advantage possible, hence highlighting why nanostructured materials exhibit a significant advantage for biosensing at solid interfaces.

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