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Protein-mediated nanoscale biotemplating
Protein-mediated nanoscale biotemplating Shira Lagziel-Simis, Noa Cohen-Hadar, Hila Moscovich-Dagan, Yariv Wine and Amihay Freeman Biomimetics — the concept of taking ideas from nature and implementing them in technology — has found particular use for the development of nanoscale materials. One such approach employs protein-mediated biotemplating for the nanostructuring of inorganic material. Recently, two key advances have been witnessed in this field. Firstly, the number of successfully employed biotemplates, including feasibility demonstrations of using three-dimensional crystalline structures, has been expanded. Secondly, the introduction of site-directed mutations on the protein template, or the display of peptides that exhibit effective biorecognition sequences for inorganic structures, has led to substantial improvements in our ability to control proteinmediated biotemplating. Taken together, these achievements will pave the way for the successful application of proteinmediated biotemplating in the future. Addresses Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel Corresponding author: Freeman, Amihay ([email protected])
Current Opinion in Biotechnology 2006, 17:569–573 This review comes from a themed issue on Chemical biotechnology Edited by Jonathan S Dordick and Amihay Freeman Available online 30th October 2006 0958-1669/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2006.10.005
Introduction The recent trend for structuring inorganic materials and their biocomposites at the nanoscale has led to growing interest in learning from ‘mother nature’ on how to initiate, direct and terminate biomineralization processes to yield nanostructured materials [1,2]. Proteins have a key role in controlling the formation of crystalline materials in vivo, and it was suggested that specific biorecognition processes are involved in the induction of crystal nucleation, growth regulation and growth arrest [3]. As the molecular details of these processes are not yet fully understood, early attempts to practice protein-mediated biotemplating have involved the empirical use of proteinmade patterned physical templates, rather than copying well-characterized natural routes. Here we provide an overview of recent advances in the use of protein-mediated biotemplating and discuss the www.sciencedirect.com
trends involved in its application for the nanostructuring of inorganic material. The range of biotemplates discussed is summarised in Figure 1.
Single-molecule biotemplating One pioneering attempt to use protein biotemplates for the in vitro synthesis of nanosized magnetic particles was based on the use of ferritin. Ferritin comprises a protein shell structure that under normal conditions stores the compound ferrihydrite. By removing ferrihydrite through controlled dialysis, it was possible to obtain an apoferritin empty shell. Reloading the apoferritin shell with iron oxide crystals (Fe3O4 and Fe2O3) in a controlled process led to the synthesis of ‘magnetoferritin’ (Figure 1a). Loading was based on equilibration with Fe2+ under argon, followed by slow exposure to oxygen, and led to the formation of 6 nm crystals within the ferritin shell [4]. In a related study, a different mechanism was used to successfully load apoferritin with cobalt oxide. The apoferritin was first loaded with 200 Co2+ ions per apoferritin shell and then exposed to hydrogen peroxide to yield Co3O4 grains (1.2 nm in diameter) grown on specific sites on the inner surface of the protein shell. The further addition of Co2+ subsequently led to the formation of an inner cobalt oxide layer complementary to the inner side of the apoprotein shell; this was further evolved by the addition of more Co2+ to form a 6 nm cobalt oxide nucleus [5]. An important step in gaining better control of singlemolecule biotemplating was recently described by Douglas and colleagues [6]. In their study, a 24 subunit shell-like heat-shock protein was genetically manipulated to display on its inside a dodecapeptide (derived from screening protein libraries) that exhibited specific affinity for the crystalline edge of CoPt magnetic metallic alloy. Incubation of cobalt and platinum salts with the mutant under reducing conditions led to the synthesis of magnetic nanoparticles (6.5 nm in size) encapsulated within the protein shell.
Protein array mediated biotemplating Biotemplating by bacterial S-layers
Crystalline bacterial cell-surface layers (S-layers) provide an outer cell envelope in many eubacteria and archaebacteria. These layers can be readily isolated and reconstructed in vitro to generate a two-dimensional monomolecular protein array with a pore size in the range 2–6 nm (Figure 1b) [7]. Pioneering works by Mann, Sleytr and coworkers [8,9] employed reconstructed S-layers as templates for the in situ nucleation of ordered Current Opinion in Biotechnology 2006, 17:569–573
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Figure 1
The main configurations of protein biotemplates. (a) The apoferritin protein shell on the outside (blue ovals) is loaded with magnetoferritin (gray) to produce magnetic particles. (b) Bacterial S-layers have been used as templates for the construction of two-dimensional arrays of nanoparticles. Protein molecules are shown as ovals and nanoparticles as spheres. (c) Microtubuli, rhapidosomes, amyloid fibres and actin have all been employed as biotemplates for the construction of nanowires. The deposited metal grains are shown as black spheres. (d) The regularity of viral (e.g. M13) envelopes has been exploited for biotemplating. (e) The three-dimensional array of a protein crystal structure was used to synthesise a nanostructured protein–hydrogel complex. Blue ovals depict the regular arrangement of protein molecules; hydrogel molecules are in gray and fit within the voids.
two-dimensional arrays of cadmium sulfide nanocrystals and gold nanoparticles (5 nm in size). The S-layer template was also used for patterning of nanoparticles by chemically linking the particles to repetitive proteinbinding sites in the S-layer [10]. This approach was recently employed for the synthesis of gold nanoparticle arrays exhibiting different spacings [11], core-shell quantum dots arrays [12] and dendrimer-encapsulated platinum nanoparticle arrays [13]. Biotemplating using a two-dimensional bacterial membrane protein array
Purple membrane — a two-dimensional array comprised of lipids and bacteriorhodopsin — was recently used for the patterning of gold nanoparticles. Bacteriorhodopsin mutants displaying cysteine residues at predetermined sites were used to ‘hook’ the gold nanoparticles. Other bacteriorhodopsin mutants, displaying histidine tags at preselected sites, readily affected nearly homogeneous patterning of silver sulfide nanoparticles (4 nm in size) [14].
Metallization of protein arrays Electroless plating — the deposition of metals on solid surfaces in the absence of electricity — has many applicaCurrent Opinion in Biotechnology 2006, 17:569–573
tions in micro- and nanotechnologies. Electroless methods currently employed by the microelectronics industry offer high-quality ultra-thin films compatible with highresolution patterns such as interconnects smaller than 100 nm [15]. The possibility to adapt the electroless methods used to metallize inorganic templates for use with biotemplates holds great potential, and has stimulated research in this area over the past decade. These metallizations were mostly directed to the surface of the substrate by the adsorption of palladium or platinum ions followed by their chemical reduction. The nucleation sites obtained in this way were then enlarged into continuously deposited metallic films through immersion in plating solution containing the metal ions of choice (e.g. Ag+ or Ni2+) and reducing agents such as NaBH4 or dimethylaminoborane. In recent years, metallization by electroless deposition directed to the surface of protein templates has primarily been used for the preparation of nanowires. The synthesis of nanowires (25–30 nm in diameter) templated using naturally occurring nanosize tubes (e.g. rhapidosomes [16] and microtubuli [17,18]; Figure 1c) was reported. Improved process control of microtubule metallization has recently enabled biotemplating directed to the formation of arrays of metallic www.sciencedirect.com
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silver grains (5.2 nm in size) or to continuous silver deposition [19]. The use of a non-specific ion adsorption mechanism as the crucial initiation step in the metallization of protein arrays was recently substituted by labeling the surface of the array with gold nanoparticles followed by enlargement to reach continuous metal deposition by electroless deposition. Lindquist and colleagues [20] successfully applied this approach for the metallization of genetically modified amyloid fibres with silver or gold, resulting in conducting wires of 100 nm diameter. Patolsky et al. [21] used gold nanoparticle-labeling of actin followed by electroless deposition for the preparation of conducting gold nanowires (80–200 nm in diameter). The authors also demonstrated the synthesis of actin fibres that presented metallized and non-metallized segments, thus enabling ATP-fuelled motility of the segmented actin nanowires on a myosin surface.
Biotemplating using viral envelopes The highly regular protein array of viral envelopes has been used as a biotemplate for the metallization of viruses (Figure 1d). Gold nanoparticles chemically attached to the surface of Chillo Iridescent virus were used as nucleation sites for the electroless deposition of gold to yield gold-coated spherical particles 50–80 nm in diameter [22]. Knez et al. [23] demonstrated the use of the central channel of the tobacco mosaic virus (TMV) as biotemplate for the synthesis of nickel and cobalt nanowires that were a few atoms in diameter. Through the appropriate choice of viral strain and reaction conditions, the authors successfully directed the commonly used ion nucleation/ electroless deposition metallization process into the central channel of the viral protein envelope. Genetically altered TMV displaying cysteine residues on its outer surface was also used for the synthesis of metal clusters of gold, silver and palladium on its surface [24]. Viral capsids have also been used for the synthesis of magnetic virus particles. In a study by Liu et al. [25], adsorption/electroless deposition led to the formation of magnetic cobalt nanoparticles (42 nm in size) encapsulated within a hollow capsid of bacteriophage T7. Moreover, the spherical T7–cobalt hybrids retained their natural outer biorecognition capability [25]. M13 bacteriophage have also been employed for the preparation of magnetic phage bundles in a three-step process. First, a library based on genetically engineered protein 8 was used to select an octapeptide with high-affinity for Co2+. Phages displaying the selected octapeptide were then mixed with Co2+ to generate ‘phage bundles’. Lastly, electroless deposition of CoPt alloy resulted in the production of a highly magnetic fibrilar network [26]. The programmable assembly of M13 phages by Belcher and colleagues [27] has taken the potential inherent in www.sciencedirect.com
the metallization of viruses an important step forward. Before metallization, self-assembly of these viruses into a predetermined linear structure was affected by the display of streptavidin-binding motifs on protein 3 of this phage. The specific binding of gold nanoparticles to gold-binding peptides displayed on the viral envelope (protein 8) then formed nucleation sites and allowed for subsequent electroless deposition, which resulted in the formation of predesigned gold wires (50 nm in diameter). Work in the same laboratory [28] recently expanded these capabilities to synthesize hybrid gold– cobalt oxide wires by the simultaneous expression of a gold-binding peptide motif and a cobalt oxide specific peptide on the envelope protein 8 of M13 bacteriophage. The use of these hybrid nanowires as a highly effective anode in a lithium ion battery was demonstrated. Metallization of viral envelopes was recently practiced by Colvin and coworkers [29] on virus crystals serving as three-dimensional scaffolds for the synthesis of nanostructured composite materials. Crystals of the cowpea mosaic virus were stabilized by glutaraldehyde crosslinking and exposed to Pd2+. Ions adsorbed to proteins of the viral capsid were then reduced to serve as nucleation sites for the electroless deposition of platinum. This process led to the formation of three-dimensional arrays of distinctive metallic grains (10.3 nm in size) within the cavities of the crystal array.
Protein crystal mediated biotemplating Protein crystals, routinely prepared for the elucidation of three-dimensional protein structures by X-ray crystallography, present an ordered and highly accurate threedimensional array of protein molecules. Inherent to the three-dimensional arrangement of the protein molecules in the crystal is a complementary three-dimensional array of voids made of interconnected cavities and exhibiting highly ordered porosity. The permeability of such chemically crosslinked enzyme protein crystals to low molecular weight solutes has been employed both for enzyme-mediated organic synthesis and for sizeexclusion chromatography [30]. This permeability was also explored by Cohen-Hadar et al. [31] who investigated the use of stabilized lysozyme crystals as a biotemplate for the fabrication of a nanostructured protein–synthetic-hydrogel hybrid. The process of ‘filling’ the ordered voids within the protein crystal monomers and their subsequent in situ polymerization into a crosslinked gel was monitored to ensure preservation of the protein crystal template throughout the process. This monitoring was based on a step-by-step comparative analysis of data obtained from X-ray crystallography and fluorescence decay kinetics of an ultra-fast laser-activated dye, impregnated within the crystals. The stabilized protein crystal template retained its three-dimensional structure throughout the process, Current Opinion in Biotechnology 2006, 17:569–573
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demonstrating the feasibility of using stabilized protein crystals as effective biotemplates for the preparation of novel composite materials (Figure 1e).
Conclusions Continuous efforts to realize the potential inherent in nanoscale biotemplating — first initiated in the 1990s — has gradually yielded feasibility demonstrations of fabrication methodologies, characterization means and, in some cases, functionality. On the way to these achievements, a major hurdle was the need to either modify traditional fabrication methodologies already practiced in chemistry and microelectronics, making them compatible with the relatively labile protein biotemplates, or to develop new specific methods to meet this challenge. Two key achievements have been made in proteinmediated biotemplating over the past two years. First, the number of successfully employed biotemplates, including feasibility demonstrations of using threedimensional crystalline structures, has been significantly expanded. Second, substantial improvements have been made in our ability to control protein-mediated biotemplating, through the introduction of site-directed mutations or the display of peptides exhibiting effective biorecognition sequences for inorganic structures on the protein template. A major challenge that remains in the field of proteinmediated biotemplating is the ability to synthesise protein–inorganic-compound functional hybrids that simultaneously provide functionality derived from both the inorganic segment (e.g. electrical conductivity) and from the protein segment (e.g. biocatalysis and biorecognition). Nevertheless, given recent rapid progress in this field, it seems likely that we will see reports of studies addressing this issue in the near future.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest
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Klem MT, Willits D, Solis DJ, Belcher AM, Young M, Douglas T: Biol-inspired synthesis of protein encapsulated CoPt nanoparticles. Adv Func Mater 2005, 15:1489-1494. The authors successfully applied the display of a dodecapeptide on the interior surface of a naturally available protein ‘cage’ to direct ‘filling’ of this cage with CoPt grains through specific biorecognition. 7.
Sleytr UB, Messner P, Pum D, Sara M: Crystalline bacterial cell surface layers (S-layers): from supramolecular cell structure to biomimetics and nanotechnology. Angew Chem Int Ed Engl 1999, 38:1034-1054.
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Shenton W, Pum D, Sleytr UB, Mann S: Synthesis of cadmium sulfide superlattices using self-assembled bacterial S-layers. Nature 1997, 389:585-587.
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Dieluweit S, Pum D, Sleytr UB: Formation of a gold superlattice on an S-layer with square lattice symmetry. Supramolec Sci 1998, 5:15-19.
10. Gyoervary E, Schroedter A, Talapin DV, Weller H, Pum D, Sleytr UB: Formation of nanoparticle arrays on S-layer protein lattice. J Nanosci Nanotech 2004, 4:115-120. 11. Bergkvist M, Mark SS, Yang X, Angert ER, Batt CA: Bionanofabrication of ordered nanoparticle arrays: effect of particle properties and adsorption conditions. J Phys Chem B 2004, 108:8241-8248. 12. Mark SS, Bergvist M, Yang X, Teixeira LM, Bhatnagar P, Angert ER, Batt CA: Bionanofabrication of metallic and semiconductor nanoparticle arrays using S-layer protein lattices with different lateral spacings and geometries. Langmuir 2006, 22:3763-3774. 13. Mark SS, Bergkvist M, Yang X, Angert ER, Batt CA: Self-assembly of dendrimer encapsulated nanoparticle arrays using 2-D microbial S-layer protein biotemplates. Biomacromolecules 2006, 7:1884-1897. 14. Mo X, Krebs MP, Yu MS: Directed synthesis and assembly of nanoparticles using purple membrane. Small 2006, 2:526-529. 15. Shacham-Diamand Y, Ingberg A, Sverdlov Y, Bogush V, Croitoru N, Moscovich H, Freeman A: Electroless processes for micro- and nanoelectronics. Electrochem Acta 2003, 48:2978-2996. 16. Pazirandeh M, Bural S, Campbell JR: Metallized nanotubules derived from bacteria. Biomimetics 1992, 1:41-50. 17. Kirsch R, Mertig M, Pompe W, Wahel R, Sadowski G, Bohm KJ, Unger E: Three dimensional metallization of microtubules. Thin Solid Films 1997, 305:248-253. 18. Mertig M, Kirsch R, Pompe W: Biomolecular approach to nanotube fabrication. App Phys A 1998, 66:S723-S727. 19. Behrens S, Wu J, Habicht W, Unger E: Silver nanoparticle and nanowire formation by microtule templates. Chem Mater 2004, 16:3085-3090. 20. Scheibel T, Parthasarathy R, Sawicki G, Lin XM, Jaeger H, Lindquist SL: Conducting nanowires built by controlled selfassembly of amyloid fibers and selective metal deposition. Proc Natl Acad Sci USA 2003, 100:4527-4532.
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Davis SA, Dujardin E, Mann S: Biomolecular inorganic materials chemistry. Cur Opin Mat Sci 2003, 7:273-281.
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Bauerlein E: Biomineralization of unicellular organisms: an unusual membrane biochemistry for the production of inorganic nano- and microstructures. Angew Chem Int Ed Engl 2003, 42:614-641.
21. Patolsky F, Weizmann Y, Willner I: Actin based metallic nanowires as bio-nanotransporters. Nat Mater 2004, 3:692-695. The authors demonstrate the integration of metallized actin nanowire segments with natural actin segments into a patterned wire. The latter exhibited ATP-fuelled motility on a myosin surface, demonstrating combined conductivity/motility functioning.
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Addadi L, Weiner S, Geva M: On how proteins interact with crystals and their effect on crystal formation. Z Kardiol 2001, 90:SIII92-SIII98.
22. Radloff C, Vaia RA, Brunton J, Bouwer GT, Ward VK: Metal nanoshell assembly on a virus bioscaffold. Nano Lett 2005, 5:1187-1191.
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Meldrum FC, Heywood BR, Mann S: Magnetoferritin: in vitro synthesis of a novel magnetic protein. Science 1992, 257:522-523.
23. Knez M, Bittner AM, Boes F, Wege C, Jeske H, Maiss E, Kern K: Biotemplate synthesis of 3nm nickel and cobalt nanowires. Nano Lett 2003, 3:1079-1082.
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Kim JW, Choi SH, Lillehei PT, Chu SH, King GC, Watt GD: Cobalt oxide hollow nanoparticles derived by bio-templating. Chem Commun 2005:4101-4103.
24. Lee SY, Royston E, Culver JN, Harris MT: Improved metal cluster deposition on a genetically engineered tobacco mosaic virus template. Nanotechnology 2005, 16:S435-S441.
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25. Liu C, Chung SH, Jin Q, Sutton A, Yan F, Hoffman A, Kay BK, Bader SD, Makowski L, Chen L: Magnetic viruses via nano-capsid template. J Magnetism Magnet Mater 2006, 302:47-51. 26. Lee SK, Yun DS, Belcher AM: Cobalt ion mediated self assembly of genetically engineered bacteriophage for biomimetic Co-Pt hybrid material. Biomacromolecules 2006, 7:14-17. The authors successfully demonstrated CoPt grain decoration of the coat protein of M13 phage, directed by displayed affinity octapeptides, combined with self-assembly of the bacteriophage into a fibrous structure. 27. Huang Y, Chiang CY, Lee SW, Gao Y, Belcher AM: Programmable assembly of nanoarchitectures using genetically engineered viruses. Nano Lett 2005, 5:1429-1434. The authors prepared M13 phage equipped with both gold- and strepavidin-binding motifs. This combination was successfully applied to convert these phage into nanowires and to affect their self-assembly into linear — or more complexed — arrays. 28. Nam KT, Kim DW, Yoo PJ, Chiang CY, Meethong N, Hammond PT, Chiang YM, Belcher AM: Virus enabled synthesis and assembly
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of nanowires for lithium ion battery electrodes. Science 2006, 312:885-888. The authors successfully demonstrated the specifically directed preparation of virus-templated gold-cobalt oxide nanowires and their assembly and integration into a functional lithium ion battery. 29. Falkner JC, Turner ME, Bosworth JK, Trentler TJ, Johnson JE, Lin T, Colvin VL: Virus crystals as nanocomposite scaffolds. J Am Chem Soc 2005, 127:5274-5275. This study demonstrates the use of a three-dimensional virus crystal to template the formation of a three-dimensional ordered array of distinct metallic grains. 30. Margolin AL, Navia MA: Protein crystals as novel catalytic materials. Angew Chem Int Ed Engl 2001, 40:2204-2222. 31. Cohen-Hadar N, Wine Y, Nachliel E, Huppert D, Gutman M, Frolow F, Freeman A: Monitoring the stability of crosslinked protein crystals biotemplates: a feasibility study. Biotechnol Bioeng 2006, 94:1005-1011. The authors monitored (step-by-step) the biotemplating of a synthetic hydrogel by a crosslinked protein crystal, demonstrating feasibility and structural stability of the biotemplate throughout the process.
Current Opinion in Biotechnology 2006, 17:569–573
Current Opinion in Biotechnology 2006, 17:569–573 This review comes from a themed issue on Chemical biotechnology Edited by Jonathan S Dordick and Amihay Freeman Available online 30th October 2006 0958-1669/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2006.10.005
Introduction The recent trend for structuring inorganic materials and their biocomposites at the nanoscale has led to growing interest in learning from ‘mother nature’ on how to initiate, direct and terminate biomineralization processes to yield nanostructured materials [1,2]. Proteins have a key role in controlling the formation of crystalline materials in vivo, and it was suggested that specific biorecognition processes are involved in the induction of crystal nucleation, growth regulation and growth arrest [3]. As the molecular details of these processes are not yet fully understood, early attempts to practice protein-mediated biotemplating have involved the empirical use of proteinmade patterned physical templates, rather than copying well-characterized natural routes. Here we provide an overview of recent advances in the use of protein-mediated biotemplating and discuss the www.sciencedirect.com
trends involved in its application for the nanostructuring of inorganic material. The range of biotemplates discussed is summarised in Figure 1.
Single-molecule biotemplating One pioneering attempt to use protein biotemplates for the in vitro synthesis of nanosized magnetic particles was based on the use of ferritin. Ferritin comprises a protein shell structure that under normal conditions stores the compound ferrihydrite. By removing ferrihydrite through controlled dialysis, it was possible to obtain an apoferritin empty shell. Reloading the apoferritin shell with iron oxide crystals (Fe3O4 and Fe2O3) in a controlled process led to the synthesis of ‘magnetoferritin’ (Figure 1a). Loading was based on equilibration with Fe2+ under argon, followed by slow exposure to oxygen, and led to the formation of 6 nm crystals within the ferritin shell [4]. In a related study, a different mechanism was used to successfully load apoferritin with cobalt oxide. The apoferritin was first loaded with 200 Co2+ ions per apoferritin shell and then exposed to hydrogen peroxide to yield Co3O4 grains (1.2 nm in diameter) grown on specific sites on the inner surface of the protein shell. The further addition of Co2+ subsequently led to the formation of an inner cobalt oxide layer complementary to the inner side of the apoprotein shell; this was further evolved by the addition of more Co2+ to form a 6 nm cobalt oxide nucleus [5]. An important step in gaining better control of singlemolecule biotemplating was recently described by Douglas and colleagues [6]. In their study, a 24 subunit shell-like heat-shock protein was genetically manipulated to display on its inside a dodecapeptide (derived from screening protein libraries) that exhibited specific affinity for the crystalline edge of CoPt magnetic metallic alloy. Incubation of cobalt and platinum salts with the mutant under reducing conditions led to the synthesis of magnetic nanoparticles (6.5 nm in size) encapsulated within the protein shell.
Protein array mediated biotemplating Biotemplating by bacterial S-layers
Crystalline bacterial cell-surface layers (S-layers) provide an outer cell envelope in many eubacteria and archaebacteria. These layers can be readily isolated and reconstructed in vitro to generate a two-dimensional monomolecular protein array with a pore size in the range 2–6 nm (Figure 1b) [7]. Pioneering works by Mann, Sleytr and coworkers [8,9] employed reconstructed S-layers as templates for the in situ nucleation of ordered Current Opinion in Biotechnology 2006, 17:569–573
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Figure 1
The main configurations of protein biotemplates. (a) The apoferritin protein shell on the outside (blue ovals) is loaded with magnetoferritin (gray) to produce magnetic particles. (b) Bacterial S-layers have been used as templates for the construction of two-dimensional arrays of nanoparticles. Protein molecules are shown as ovals and nanoparticles as spheres. (c) Microtubuli, rhapidosomes, amyloid fibres and actin have all been employed as biotemplates for the construction of nanowires. The deposited metal grains are shown as black spheres. (d) The regularity of viral (e.g. M13) envelopes has been exploited for biotemplating. (e) The three-dimensional array of a protein crystal structure was used to synthesise a nanostructured protein–hydrogel complex. Blue ovals depict the regular arrangement of protein molecules; hydrogel molecules are in gray and fit within the voids.
two-dimensional arrays of cadmium sulfide nanocrystals and gold nanoparticles (5 nm in size). The S-layer template was also used for patterning of nanoparticles by chemically linking the particles to repetitive proteinbinding sites in the S-layer [10]. This approach was recently employed for the synthesis of gold nanoparticle arrays exhibiting different spacings [11], core-shell quantum dots arrays [12] and dendrimer-encapsulated platinum nanoparticle arrays [13]. Biotemplating using a two-dimensional bacterial membrane protein array
Purple membrane — a two-dimensional array comprised of lipids and bacteriorhodopsin — was recently used for the patterning of gold nanoparticles. Bacteriorhodopsin mutants displaying cysteine residues at predetermined sites were used to ‘hook’ the gold nanoparticles. Other bacteriorhodopsin mutants, displaying histidine tags at preselected sites, readily affected nearly homogeneous patterning of silver sulfide nanoparticles (4 nm in size) [14].
Metallization of protein arrays Electroless plating — the deposition of metals on solid surfaces in the absence of electricity — has many applicaCurrent Opinion in Biotechnology 2006, 17:569–573
tions in micro- and nanotechnologies. Electroless methods currently employed by the microelectronics industry offer high-quality ultra-thin films compatible with highresolution patterns such as interconnects smaller than 100 nm [15]. The possibility to adapt the electroless methods used to metallize inorganic templates for use with biotemplates holds great potential, and has stimulated research in this area over the past decade. These metallizations were mostly directed to the surface of the substrate by the adsorption of palladium or platinum ions followed by their chemical reduction. The nucleation sites obtained in this way were then enlarged into continuously deposited metallic films through immersion in plating solution containing the metal ions of choice (e.g. Ag+ or Ni2+) and reducing agents such as NaBH4 or dimethylaminoborane. In recent years, metallization by electroless deposition directed to the surface of protein templates has primarily been used for the preparation of nanowires. The synthesis of nanowires (25–30 nm in diameter) templated using naturally occurring nanosize tubes (e.g. rhapidosomes [16] and microtubuli [17,18]; Figure 1c) was reported. Improved process control of microtubule metallization has recently enabled biotemplating directed to the formation of arrays of metallic www.sciencedirect.com
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silver grains (5.2 nm in size) or to continuous silver deposition [19]. The use of a non-specific ion adsorption mechanism as the crucial initiation step in the metallization of protein arrays was recently substituted by labeling the surface of the array with gold nanoparticles followed by enlargement to reach continuous metal deposition by electroless deposition. Lindquist and colleagues [20] successfully applied this approach for the metallization of genetically modified amyloid fibres with silver or gold, resulting in conducting wires of 100 nm diameter. Patolsky et al. [21] used gold nanoparticle-labeling of actin followed by electroless deposition for the preparation of conducting gold nanowires (80–200 nm in diameter). The authors also demonstrated the synthesis of actin fibres that presented metallized and non-metallized segments, thus enabling ATP-fuelled motility of the segmented actin nanowires on a myosin surface.
Biotemplating using viral envelopes The highly regular protein array of viral envelopes has been used as a biotemplate for the metallization of viruses (Figure 1d). Gold nanoparticles chemically attached to the surface of Chillo Iridescent virus were used as nucleation sites for the electroless deposition of gold to yield gold-coated spherical particles 50–80 nm in diameter [22]. Knez et al. [23] demonstrated the use of the central channel of the tobacco mosaic virus (TMV) as biotemplate for the synthesis of nickel and cobalt nanowires that were a few atoms in diameter. Through the appropriate choice of viral strain and reaction conditions, the authors successfully directed the commonly used ion nucleation/ electroless deposition metallization process into the central channel of the viral protein envelope. Genetically altered TMV displaying cysteine residues on its outer surface was also used for the synthesis of metal clusters of gold, silver and palladium on its surface [24]. Viral capsids have also been used for the synthesis of magnetic virus particles. In a study by Liu et al. [25], adsorption/electroless deposition led to the formation of magnetic cobalt nanoparticles (42 nm in size) encapsulated within a hollow capsid of bacteriophage T7. Moreover, the spherical T7–cobalt hybrids retained their natural outer biorecognition capability [25]. M13 bacteriophage have also been employed for the preparation of magnetic phage bundles in a three-step process. First, a library based on genetically engineered protein 8 was used to select an octapeptide with high-affinity for Co2+. Phages displaying the selected octapeptide were then mixed with Co2+ to generate ‘phage bundles’. Lastly, electroless deposition of CoPt alloy resulted in the production of a highly magnetic fibrilar network [26]. The programmable assembly of M13 phages by Belcher and colleagues [27] has taken the potential inherent in www.sciencedirect.com
the metallization of viruses an important step forward. Before metallization, self-assembly of these viruses into a predetermined linear structure was affected by the display of streptavidin-binding motifs on protein 3 of this phage. The specific binding of gold nanoparticles to gold-binding peptides displayed on the viral envelope (protein 8) then formed nucleation sites and allowed for subsequent electroless deposition, which resulted in the formation of predesigned gold wires (50 nm in diameter). Work in the same laboratory [28] recently expanded these capabilities to synthesize hybrid gold– cobalt oxide wires by the simultaneous expression of a gold-binding peptide motif and a cobalt oxide specific peptide on the envelope protein 8 of M13 bacteriophage. The use of these hybrid nanowires as a highly effective anode in a lithium ion battery was demonstrated. Metallization of viral envelopes was recently practiced by Colvin and coworkers [29] on virus crystals serving as three-dimensional scaffolds for the synthesis of nanostructured composite materials. Crystals of the cowpea mosaic virus were stabilized by glutaraldehyde crosslinking and exposed to Pd2+. Ions adsorbed to proteins of the viral capsid were then reduced to serve as nucleation sites for the electroless deposition of platinum. This process led to the formation of three-dimensional arrays of distinctive metallic grains (10.3 nm in size) within the cavities of the crystal array.
Protein crystal mediated biotemplating Protein crystals, routinely prepared for the elucidation of three-dimensional protein structures by X-ray crystallography, present an ordered and highly accurate threedimensional array of protein molecules. Inherent to the three-dimensional arrangement of the protein molecules in the crystal is a complementary three-dimensional array of voids made of interconnected cavities and exhibiting highly ordered porosity. The permeability of such chemically crosslinked enzyme protein crystals to low molecular weight solutes has been employed both for enzyme-mediated organic synthesis and for sizeexclusion chromatography [30]. This permeability was also explored by Cohen-Hadar et al. [31] who investigated the use of stabilized lysozyme crystals as a biotemplate for the fabrication of a nanostructured protein–synthetic-hydrogel hybrid. The process of ‘filling’ the ordered voids within the protein crystal monomers and their subsequent in situ polymerization into a crosslinked gel was monitored to ensure preservation of the protein crystal template throughout the process. This monitoring was based on a step-by-step comparative analysis of data obtained from X-ray crystallography and fluorescence decay kinetics of an ultra-fast laser-activated dye, impregnated within the crystals. The stabilized protein crystal template retained its three-dimensional structure throughout the process, Current Opinion in Biotechnology 2006, 17:569–573
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demonstrating the feasibility of using stabilized protein crystals as effective biotemplates for the preparation of novel composite materials (Figure 1e).
Conclusions Continuous efforts to realize the potential inherent in nanoscale biotemplating — first initiated in the 1990s — has gradually yielded feasibility demonstrations of fabrication methodologies, characterization means and, in some cases, functionality. On the way to these achievements, a major hurdle was the need to either modify traditional fabrication methodologies already practiced in chemistry and microelectronics, making them compatible with the relatively labile protein biotemplates, or to develop new specific methods to meet this challenge. Two key achievements have been made in proteinmediated biotemplating over the past two years. First, the number of successfully employed biotemplates, including feasibility demonstrations of using threedimensional crystalline structures, has been significantly expanded. Second, substantial improvements have been made in our ability to control protein-mediated biotemplating, through the introduction of site-directed mutations or the display of peptides exhibiting effective biorecognition sequences for inorganic structures on the protein template. A major challenge that remains in the field of proteinmediated biotemplating is the ability to synthesise protein–inorganic-compound functional hybrids that simultaneously provide functionality derived from both the inorganic segment (e.g. electrical conductivity) and from the protein segment (e.g. biocatalysis and biorecognition). Nevertheless, given recent rapid progress in this field, it seems likely that we will see reports of studies addressing this issue in the near future.
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Current Opinion in Biotechnology 2006, 17:569–573