MINIREVIEW Use of biomolecular templates for the fabrication of metal nanowires Ehud Gazit Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel Bionanotechnology – the use of biological tools for nanotechnology Many functional biological assemblies represent genu- ine nanotechnological systems and devices [1,2]. These nano-objects are formed by the process of self- assembly, facilitated by molecular recognition events between building blocks, resulting in the formation of functional devices. Even the simplest living organism contains functional complex elements such as motors, pumps, and cables, all functioning at the nano-scale [3]. Much research is being devoted to the use of nanotechnology tools for the advancement of biology (nanobiotechnology) [4]. This is directly related to the use of nanotechnology to address biological and med- ical needs (Fig. 1). However, another very interesting research direction involves the use of ordered biologi- cal building blocks for the fabrication of various non- biological nanostructures [5]. In recent years there has been increasing interest in the utilization of biological tools for nanotechnological applications that are not related to biology such as micro-electronics and nano- electronics, micro-fluidics and nano-fluidics, and micro-electromechanical and nano-electromechanical systems. This general field could be referred to as ‘bio- nanotechnology’, the use of biology (or biological tools and scaffolds) for nanotechnology. The present review will focus on bionanotechnological applications for the formation of metal and other inorganic wires. As will be discussed next, biology may actually provide unique tools for such fabrication at the nano-scale (Fig. 1). The biological building blocks include proteins, pep- tides, nucleic acids (DNA and RNA), bacteriophages (viruses that infect bacteria), and plant viruses. These biologically templated nanostructures may have appli- cations in diverse fields that are very remote, such as electronics, telecommunication, and materials engineer- ing. In this minireview, I will limit the discussion to the scheme in which the biological assemblies define the 1D nature of the nanowire. However, it is worth Keywords bionanotechnology; electroless deposition; fibrils; molecular recognition; self-assembly Correspondence E. Gazit, Department of Molecular, Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel Fax: +972 3 640 5448 Tel: +972 3 640 9030 E-mail: ehudg@post.tau.ac.il (Received 6 October 2006, accepted 3 November 2006) doi:10.1111/j.1742-4658.2006.05605.x The nano-scale spatial organization of metallic and other inorganic materi- als into 1D objects is a key task in nanotechnology. Nano-scale fibers and tubes are very useful templates for such organization because of their inherent 1D organization. Fibrillar biological molecules and biomolecular assemblies are excellent physical supports on which to organize the inor- ganic material. Furthermore, these biological assemblies can facilitate high- order organization and specific orientation of inorganic structures by their utilization of highly specific biological recognition properties. In this mini- review, I will describe the use of biomolecules and biomolecular assemblies, including DNA, proteins, peptides, and even viral particles, which are excellent templates for 1D organization of inorganic materials into wires. This ranges from simple attempts at electroless deposition on inert biologi- cal templates to the advanced use of structural motifs and specific protein– DNA interactions for nano-bio-lithography as well as the fabrication of multilayer organic and inorganic composites. The potential technological applications of these hybrid biological–inorganic assemblies will be dis- cussed. FEBS Journal 274 (2007) 317–322 ª 2006 The Author Journal compilation ª 2006 FEBS 317 mentioning that other research directions involve bio- logical modifications of nonbiological 1D objects such as carbon nanotubes [6,7]. Use of DNA as a template for nanowire formation DNA molecules are very intriguing building blocks for nanotechnological applications. Interestingly, more than two decades ago, Seeman [8,9] showed that specific recognition between complementary DNA single-strands allowed them to be engineered to form well-ordered structures at the nano-scale. The inherent addressing capabilities, facilitated by specific inter- actions between complementary single strands, are manifested in specific recognition and self-assembly processes. The formation of 2D arrays as well as 3D nanocubes could be achieved by clever design of the building blocks [8,9]. DNA is also a very interesting biomolecule for nanotechnological applications from the material sci- ence point of view. The diameter of ssDNA is less than 1 nm, and that of dsDNA is 2 nm (Fig. 2). Further- more, DNA molecules are chemically very robust and their frequent use in molecular biology applications has significantly reduced the cost of large-scale chem- ical DNA synthesis. Consequently, large amounts of native and modified DNA molecules (for example, by biotinylation or thiolation) can be rapidly synthesized at a relatively low cost. One of the early applications of DNA for the forma- tion of nanowires, in 1998, involved the metallization of dsDNA between two electrodes to form conductive silver nanowire [10]. More specifically, the researchers used complementary ssDNA to bridge a 12–lm gap between two gold electrodes. The dsDNA formed was then coated with silver by a deposition and enhance- ment process to form 12–lm long, 100nm-wide con- ductive silver wires. Other seminal work paved the way to form a gold nanowire based on the use of a DNA template [11]. This was achieved by the intercalation of functionalized gold nanoparticles into dsDNA, fol- lowed by covalent photochemical attachment of the intercalator [11]. The use of metal-coated DNA mole- cules was also demonstrated for DNA-assisted wiring of gold electrodes on silicone wafers [12] and for the specific metallization of a Y-shaped DNA that incor- porated a central biotin moiety [13]. These patterned and directed metallization schemes hold promise for novel applications in the design and manufacture of nanoelectronic devices in the future [12,13]. Although lithography methods are constantly being improved, template-assisted nanowire formation may be very use- ful for making interconnections between lithographic- ally defined elements [14]. Other research into much higher resolution pattern- ing involves specific recognition between proteins and defined DNA sequences by a process termed ‘mole- cular lithography’ [15] (Fig. 3). In this case RecA, a sequence-specific DNA-binding protein, was allowed ygolonhcetonaNygoloiB ygolonhcetonanoiB serutcurtsonande lbmessa-fleS slairetamderipsni-oiB scinortcelera l u celom- o iB seilbmessa-oib f o n oit a z il l ate M ygolonhcetoibonaN pihc-A-nO-lleC s c i t so n g aidyar r a-naN ygoloibnistodm u tnauQ - on an nog n i re e nign eeu ss i T setalpmet Fig. 1. Interplay between biology and nano- technology. Nanobiotechnology involves the use of nanotechnological tools for various biological and medical applications. Bionanotechnology is the use of biological and bio-inspired molecules and assemblies for technological uses. Biological templates for nanowire fabrication E. Gazit 318 FEBS Journal 274 (2007) 317–322 ª 2006 The Author Journal compilation ª 2006 FEBS to bind a specific region on a DNA template before the metallization process, thus serving as the equival- ent of a ‘resist’ (Fig. 3). As the metallization process proceeded, only noncovered parts of the DNA mole- cule were coated, thus achieving nano-scale patterned metallization of the DNA molecule [15]. RecA–DNA interaction was also used to attach a genetically engin- eered RecA containing a surface-associated cysteine which allowed specific metal–thiol interactions [16]. Other DNA–protein complexes used for the formation of ordered metallic assemblies at the nano-scale have involved the strepavidin protein array of a 2D array of biotinylated DNA, followed by metallization of the array [17]. Use of the naturally occurring amyloid fibrils for metal coating Another use of DNA is to utilize protein and peptide fi- bers [18–20]. Such nano-scale fibrils are formed by the assembly of various building blocks and could be pro- duced in large amounts by over-expression. Unlike tnemaliFnitcA mn7 lirbiFdiolymA mn01-7 ebutonanTNDA mn02 AND mn2 egahpsuotnemaliF m n 6 Fig. 2. Molecular dimensions of 1D biological molecules and biomolecular assemblies for nanotechnological use. The biological molecules and assemblies are schematically presented to provide an approximate indication of their dimensions. The DNA structure is formed by bio- molecular assembly of double helix. All other structures are formed by self-assembly of the large number of nano building blocks. Photo Lithography Molecular Lithography Photoresist SiO 2 Wafer Mask UV radiation Photoresist removal Etching DNA Recognition sequences DNA-binding proteins Metallization Protein removal Fig. 3. Use of DNA-binding proteins for ‘molecular lithography’. In photolithography, a photoresist layer is deposited on the silicone oxide surface. The use of a mask allows differential treatment of the photoresist and the etching of specific parts of the layer. In molecular litho- graphy, the specific DNA sequence is the equivalent of a mask, and the DNA-binding protein serves as the resist. E. Gazit Biological templates for nanowire fabrication FEBS Journal 274 (2007) 317–322 ª 2006 The Author Journal compilation ª 2006 FEBS 319 DNA structures, these are supramolecular assemblies formed by the recognition and association of numerous building blocks to create ordered structures. Compared with DNA, protein allows much more chemical and bio- logical flexibility as well as providing building blocks with heterogeneity. As discussed above in the case of DNA-based structures, genetically engineered DNA- binding protein is used to achieve such variability. The first reported attempt to use naturally occur- ring fibers to make conductive nanowires involved the use of amyloid fibrils as template [20]. Amyloid fibrils are naturally occurring fibrillar assemblies with a diameter of 7–10 nm and a length that can reach several microns (Fig. 2). These assemblies are usually associated with human disorders [18–20]. Yet the for- mation of typical amyloid fibrils is observed in cases involving bacterial biofilms and in yeast ‘prion-pro- teins.’ In a pioneering study, yeast-derived amyloid fibrils were found to be a useful protein template for the formation of conductive metal wires [18]. Over- expressed yeast amyloid proteins were genetically engineered to contain a cysteine residue (as described above for the RecA-mediated DNA metallization) [18]. This additional thiol group served as a nuclea- tion site for the metallization of the fibrils. The researchers were able to demonstrate the formation of conductive nanowires by directly measuring the cur- rent carried by the modified fibrils across a nano-scale gap between electrodes. The novel concept of the use of amyloid fibrils for nanowire formation may actually be utilized to make wires by coating amyloid fibrils formed by simpler building blocks. As it has been demonstrated that typ- ical amyloid fibrils can be formed by peptides as short as pentapeptides and tetrapeptides [21,22], and as the molecular structure of amyloid assemblies has been revealed by high-resolution methods [23–25], simpler peptide building blocks could be used for future appli- cations of amyloid fibrils for bionanotechnology. Simpler building blocks could be synthesized in large quantities by solid-phase techniques, as previously des- cribed for DNA oligomers. Use of cytoskeletal elements for the assembly of nanowires Another interesting use of naturally occurring fibers for metal deposition is the use of cytoskeletal elements. Various nano-scale fibers comprise part of the eukary- otic cell skeleton including actin and tubulin as well as intermediate filaments. Such fibers are ubiquitous in the biological world, and homologous proteins, such as the FtsZ protein, can also be found in bacteria. The first use of cytoskeletal proteins for nanotech- nology was the utilization of actin filaments as tem- plates for nanowire formation [26]. Briefly, 7-nm actin filaments were formed by self-assembly of the actin protein, providing mechanical support for the cell (Fig. 2). Preformed actin fibrils were covalently modi- fied by the attachment of gold nanoparticles using an amine-reactive agent (N-hydroxysuccinimide). This was followed by disassembly using dialysis, repolymeriza- tion of fibers, and an enhancement process, resulting in the formation of a continuous gold nanowire. The use of cytoskeletal elements adds another dimension to the biological template of nanowires, as these elements can be translated at the nano-scale using biological nanomotors. The myosin nanomotor can bind actin fibers and use ATP hydrolysis to gener- ate force and can ‘walk’ along the filament. Thus, fur- ther study of cytoskeletal modification may lead to various nano-electromechanical system applications in which mechanics, in addition to electrical conductivity, is provided by the biological–inorganic complex. Use of peptide nanostructures to form conductive nanowires Another key research direction for the fabrication of biological fibrils involves the use of peptide and hybrid–peptide building blocks for the assembly of bio-inspired fibrillar assemblies. Such bio-inspired assemblies were also used for the fabrication of metal- lic nanowires. The simple peptide and peptide–hybrid building blocks could be synthesized in large amounts and readily modified. Various classes of peptide nanotube had already been used for the formation of 1D metal assemblies. Glycylglycine bolaamphiphile peptide nanotubes are examples of such bio-inspired peptide nanostructures [27]. The functionalization of these peptide nanotubes with histidine-rich peptide motifs enabled the forma- tion of copper coating on the nanotube surface [27]. Other studies utilized aromatic dipeptide nanotubes (Fig. 2). The preferential entrance of metal ions into the lumen of aromatic dipeptide nanotubes allowed the reduction of silver ions, with the formation of silver-filled nanotubes [28]. After the peptide coat is removed, silver nanowires 20 nm in diameter are formed [28]. Another study used aromatic dipeptide nanotubes to assemble platinum nanoparticles [29]. In a follow-up study, silver-filled peptide nanotubes were further coated with gold to achieve trilayer coaxial nanocables [30]. Peptide–amphiphile nanofibers form part of another class of peptide-based nanostructures. These fibers are Biological templates for nanowire fabrication E. Gazit 320 FEBS Journal 274 (2007) 317–322 ª 2006 The Author Journal compilation ª 2006 FEBS formed by the self-assembly of hydrophilic peptide building blocks that are conjugated to a hydrophobic aliphatic tail [31,32]. Amphiphile nanofibers were shown to form 1D arrays of gold nanoparticles on the surface of modified peptide fibers [31]. Such peptide– amphiphile nanofibers were also modified using the paramagnetic gadolinium(III) metal ion, forming inor- ganically modified peptide fibers that could be used for magnetic resonance imaging [32]. Use of bacteriophages and viruses for nanowire assembly Earlier in this minireview, I discussed the use of DNA molecules or peptide and protein assemblies. Another research direction in this organic–inorganic template- assisted fabrication process is the use of much more complex assemblies such as bacteriophages and viruses. These viruses are self-assembled structures at the nano- scale (Fig. 2). Viral structures are also very attractive assemblies for fabricating 1D metallic objects. Both viruses and bacteriophages have been used for this purpose. One of the first studies in bionanotechnology was the metallization of tobacco mosaic virus particles [33,34]. This nano-scale biological entity is very effective as a seamless template for the fabrication of various inor- ganic materials. In the last few years, several protocols for the deposition of various metals on the tobacco mosaic virus surface have been developed [33,34]. Filamentous bacteriophages can provide an even bet- ter molecular system for the formation of well-ordered 1D inorganic assemblies [35–38]. This is based on the ability of bacteriophages to express various protein motifs, including single-chain antibodies, on their sur- face, a technique known as ‘phage display’. These are proteins and peptides expressed on 6-nm elongated fibrillar structures (Fig. 2). This technique, which is widely used for selecting various peptide-binding motifs, was later used for selecting peptide motifs that can bind various inorganic metallic and semiconductive nanoparticles [35–38]. This property was later used for fabricating various metal and semiconductive nano- wires by utilizing the bacteriophages. The bacterio- phages used are engineered to express motifs that interact with specific metal and semiconductive parti- cles. These phages can then be aligned in such a way that macroscopic metal or semiconductive wires are formed. The application of these wires was recently demonstrated for the fabrication of electrodes for thin lithium-ion batteries [38]. The binding of gold to the viruses followed by reduction of the cobalt ions resul- ted in composite wires that contained both cobalt oxide and gold, which serve as superb electrodes for batteries. These wires have very good specific capacity, allowing the production of batteries with high-energy density. 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One of the early applications of DNA for the forma- tion of nanowires, in 1998, involved the metallization of dsDNA between two electrodes to form conductive silver. des- cribed for DNA oligomers. Use of cytoskeletal elements for the assembly of nanowires Another interesting use of naturally occurring fibers for metal deposition