On-wire lithography: synthesis, encoding and biological applications 

The next step in the maturing field of nanotechnology is to develop ways to introduce unusual architectural changes to simple building blocks. For nanowires, on-wire lithography (OWL) has emerged as a powerful way of synthesizing a segmented structure and subsequently introducing architectural changes through post-chemical treatment. In the OWL protocol presented here, multisegmented nanowires are grown and a support layer is deposited on one side of each nanostructure. After selective chemical etching of sacrificial segments, structures with gaps as small as 2 nm and disks as thin as 20 nm can be created. These nanostructures are highly tailorable and can be used in electrical transport, Raman enhancement and energy conversion. Such nanostructures can be functionalized with many types of adsorbates, enabling the use of OWL-generated structures as bioactive probes for diagnostic assays and molecular transport junctions. The process takes 13–36 h depending on the type of adsorbate used to functionalize the nanostructures.

INTRODUCTION
Over the past few decades, nanowires and nanorods (the former typically have a smaller diameter and larger aspect ratio, but the terms are often used interchangeably) have become a major  research area because of their unusual properties and potential utility in a variety of technologies. Similar to their zero-dimensional counterparts (e.g., nanoparticles and quantum dots), they can now be synthesized and fabricated by many methods including solution-phase synthetic methods, vapor–liquid–solid growth  processes and nanolithographic techniques. These first-generation nanowires/rods have provided important fundamental insight into many important scientific problems. However, the development of wire structures with greater architectural complexity (including multiple segments made of different chemical compositions, branched structures, surface coatings and in-wire doping) has led to materials for novel applications, has helped develop physical models of electron and optical transport and has created new synthetic challenges . In addition to control over the diameter, length and composition of such structures (factors that dramatically influence their physical properties), introducing positive and negative features (e.g., disks shapes or gaps) along the long-wire axes would produce structures with even greater flexibility. In this regard, development of methods for nanowire fabrication and manipulation that are analogous to many powerful types of two-dimensional nanolithographies (e.g., electron beam lithography , nanoimprint lithography and dip-pen nanolithography) could dramatically increase the scope and utility of such structures. For example, controlling the substructure of one-dimensional nanomaterials will lead to materials with additional functionalities (e.g., plasmonic signatures), which may prove useful in fields such as biodiagnostics, data encoding and light manipulation. This is especially true from a biodetection standpoint where appropriately functionalized plasmonic and electronic materials have been shown to be powerful sensing , detection  and even therapeutic agents. Recently, our group has developed an approach to synthesize nanowires and subsequently to introduce positive and negative architectural features along the long axis of the wire with a high degree of precision and reproducibility20 .

This method, termed onwire lithography (OWL), is based on the selective electrodeposition and etching of multicomponent nanowires and allows one to control feature composition and size from the sub-5 nm to many micrometer length scale (Fig. 1). It also allows one to make structures that would be difficult, if not impossible, to fabricate through any other technique . In addition, structures produced through OWL are dispersible in a wide range of common solvents, which allows for a host of applications not possible with substrate immobilized nanostructures (e.g., dispersible barcodes  that can be solution processed and drop cast onto any substrate or device of choice for covert tracking or tagging). OWL has been used to fabricate catalytic nanomechanical systems, high-throughput devices for the study of molecular electronics , structures that facilitate light harvesting and energy transfer  and novel systems for probing the physical underpinnings of the well-known surface-enhanced Raman spectroscopy (SERS) phenomenon .

The OWL technique has even been used to prepare unusual nanogap structures called ‘nanodisk codes’ and electrical ‘nanotraps’. The nanodisk codes consist of pairs of disks oriented along the long wire axis with gaps between the disks forming hotspots for Raman spectroscopy enhancement . These structures, when functionalized with the appropriate dyes, can be utilized in a novel encoding system where information is stored on the basis of the number and position of the disk codes and the types of dyes they have on their surfaces. They have been used as dispersible taggants and biological labels for high-sensitivity Raman-based molecular diagnostic assays. The electrical nanotrap consists of a nanowire with a nanometer-scale gap, which can be used to localize charged materials such as oligonucleotides with an appropriate electric field and simultaneously to enhance the Raman spectroscopic signal of materials that enter the gap . This type of  nanostructure is interesting for addressing and probing small quantities of materials that flow near the gap and has been demonstrated in the context of nucleic acids.

Figure 1| On-wire lithography protocol. A silver backing is evaporated onto an alumina template
(i). A sacrificial silver layer is electrochemically
deposited to ensure a clean connection to the
evaporated backing (ii). Multicomponent nanowires
are grown by electrochemical deposition (iii). The silver backing and alumina template are dissolved and the rods are dispersed onto a glass slide (iv). PECVD or PVD is used to deposit a backing material on one-half of each nanorod (v). The nanorods are sonicated off the surface (vi). The sacrificial segments are then etched (vii). The rods may be functionalized with small molecules and identified by CRM (viiia). Alternatively, the rods can be functionalized with DNA or other biomolecules (viiib). In the case of nucleic acids, the DNA is subsequently suspended in buffer and stabilized with surfactant in the presence of salt (ix). Target DNA strands are hybridized to these rods and identified by CRM (x) (stars represent chromophores generating signal). CRM, confocal Raman spectroscopy. A clear protocol to describe how to make such systems, encompassing the OWL process itself and subsequent functionalization and characterization methods, will be an important step toward aiding the development of more advanced systems. Thus, in this protocol, we outline the necessary steps to design, synthesize, functionalize and eventually characterize these systems in a variety of formats including those necessary for SERS studies and biodetection assays. However, the procedures are general and can be readily adapted for a variety of different studies or applications, which should allow OWL to become a powerful tool for many nanotechnology-based investigations.

Applications and limitations Nanomaterials made by OWL allow for a variety of intriguing applications in nanotechnology. For example, gap-based nanoelectronics, SERS and biosensing modalities have all been realized , and further applications in these and related fields e.g., metal-enhanced fluorescence) can be envisioned. This technology will enable the development of more robust, rapid, reliable and sensitive mobile detection schemes for biomolecules. It is also envisioned that the spatially specific ability to enhance spectroscopic information in real time will allow for the study of biological processes. As just one example, we can create a hybrid nanostructure that contains a diode junction and three gold nanodisk pairs. The diode section can be used for electrical measurement of biomolecule binding, and the gold disk pairs can serve as Raman hotspots for spectroscopic measurements. Using this structure, it should be possible to show the parallel electrical and spectroscopic measurement of telomerase binding onto surface-immobilized oligonucleotide receptors and subsequent elongation of the oligonucleotide strands.

The OWL technique has a demonstrated resolution of B2 nm and is also capable of producing features that are spaced micrometers apart . In addition (as noted above), a wide variety of metals can be electroplated in the nanowire deposition step. The main challenge of using these metals with OWL is combining several metals into one structure. In this case, care must be taken to choose metals with lattice parameters that are similar enough to ensure adhesion of the two components and to develop etching procedures that are highly selective, even in the presence of multiple metals. In addition, using smaller diameter nanowires (e.g., sub nm pores as a template) may lead to difficulties in redispersing nanostructures during sonication steps after plasma-enhanced chemical vapor deposition (PECVD) or physical vapor deposition (PVD) is performed .

Finally, it should be possible to adapt the biodetection strategy on the basis of OWL-generated structures to analytes other than DNA, such as proteins, viruses, metal ions and certain smallmolecule analytes. Importantly, in principle, any conventional metal-ligand-binding chemistry can be used to attach chemical or biological species of interest to the OWL structures for the applications listed above. Experimental design OWL device fabrication. In the OWL process, anodic aluminum oxide (AAO) membranes (either purchased from commercial vendors, such as Whatman Inc. (part of GE Healthcare) and Synkra Technologies Inc., or fabricated in the lab ) are used as templates to electrochemically deposit nanowires. Cylindrical, aligned, nonintersecting pores permeate the templates and serve as discrete regions for nanowire growth. AAO films can be purchased or prepared with pores ranging in diameter from 500 to 5 nm (Whatman Inc. (part of GE Healthcare) produces templates with B300 nm pores. Synkera Technologies Inc. produces templates with a variety of pore sizes ranging from 13 to 150 nm) . Although the quality of lab-synthesized templates tends to be better with more uniform pores and narrower pore size distributions (leading to nanowires with more well-defined plasmonic features), for many applications, commercially available AAO templates are often satisfactory . For Whatman (GE Healthcare) templates, better results are achieved if the template is oriented such that Ag is deposited on the side without the polymer support ring. This is because this side has a smoother surface and more uniform pore distribution that makes evaporated Ag films more adherent (see below) and also generates more regular-shaped nanorods.

(Matthew J Banholzer, Lidong Qin, Jill E Millstone, Kyle D Osberg & Chad A Mirkin, Department of Chemistry and International Institute for Nanotechnology, Northwestern University, Evanston, Illinois, USA.)