Protein- and Peptide-Directed Syntheses of Inorganic Materials

Chem. Rev. 2008, 108, 4935–4978

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Protein- and Peptide-Directed Syntheses of Inorganic Materials Matthew B. Dickerson,† Kenneth H. Sandhage,‡,§ and Rajesh R. Naik*,† Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson Air Force Base, Ohio 45433-7702; School of Materials Science and Engineering, Georgia Institute of Technology, 771 Ferst Drive, Atlanta, Georgia 30332-0245; and School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta, Georgia 30332-0245 Received March 18, 2008

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Contents 1. Introduction 2. Biosilicification 2.1. Introduction to Biosilicification 2.2. Diatom Silica-Associated Biomolecules 2.2.1. Long-Chain Polyamines 2.2.2. Silica Precipitating Proteins: Silaffins 2.2.3. Diatom Silica-Regulation Proteins 2.3. Silica-Associated Proteins in Sponges 2.3.1. Silica Precipiting Proteins: The Silicateins 2.3.2. Silica-Precipiting LCPAs 3. Sources of Biomolecules for the Biomimetic Synthesis of Materials 3.1. Biomolecules Isolated or Derived from Biomineralizing Organisms 3.2. Biomineralizing Protein Analogs 3.3. Peptides Identified Through Biopanning 3.3.1. Screening of Peptide Libraries 3.3.2. Application of Biopanning to Functional Inorganic Materials 4. Oxides Produced Under the Influence of Proteins and Peptides 4.1. Silicon Dioxide 4.1.1. Introduction 4.1.2. Silica Formation in the Presence of Biomolecules and Biomolecule Analogues 4.1.3. Exploiting Environmental Influences in Biomimetic Silicification 4.1.4. Peptide Sequence Programmed Silica Architecture 4.1.5. Synergistic Co-Self-Assembly of Silica/ Template Structures 4.1.6. Summary of the Bioenabled Synthesis of Silica 4.2. Titanium Dioxide 4.3. Germanium Oxide 4.4. Gallium Oxide and Zinc Oxide 4.5. Mixed-Valence Oxides of Iron and Cobalt (Fe3O4 and Co3O4) 4.6. Multicomponent Oxides: BaTiO3, BaTiOF4, CaMoO4, and (Fe,Co)3O4

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* To whom correspondence should be addressed. E-mail: Rajesh.Naik@ wpafb.af.mil. † Air Force Research Laboratory, Wright-Patterson Air Force Base. ‡ School of Materials Science and Engineering, Georgia Institute of Technology. § School of Chemistry and Biochemistry, Georgia Institute of Technology.

5. Protein- and Peptide-Directed Syntheses of Non-Oxide Semiconductors 5.1. Introduction 5.2. Biogenic and Biologically-Derived Production of Semiconductor Nanoparticles 5.3. Syntheses of Sulfide Semiconductors with Zinc Fingerlike Peptides and Nanotubes 5.4. Syntheses of Sulfide Semiconductors with Amphiphilic Peptides 5.5. Apoferritin-Templated Semiconductor Nanoparticles 5.6. Semiconductor Nanoparticles from Transport Proteins and Enzymes 5.7. Oriented QDs from Genetically-Modified Phage 5.8. Biomimetic Synthesis of an Elemental Semiconductor 5.9. Summary of the Bio-Enabled Synthesis of Semiconductor Nanocrystals 6. Biomimetic Syntheses of Metallic Nanoparticles, Nanorods, and Nanowires 6.1. Application and Chemical Syntheses of Metal Nanostructures 6.2. Biogenic and Biologically-Derived Production of Metal Nanoparticles 6.2.1. Production of Metal Nanoparticles by Bacteria, Fungi, and Plants 6.2.2. Cell-Free Extracts for in vitro Gold Nanoprism Synthesis 6.3. Amino Acids Influencing the Biomimetic Syntheses of Metal Nanoparticles 6.4. Peptide-Induced Syntheses of Gold Nanoparticles and Nanotubes 6.5. Protein-Templated Production of Gold Nanoparticles and Nanowires 6.5.1. BSA-Au Nanoparticles 6.5.2. Silk- and Enzyme-Induced Au Nanoparticles 6.5.3. Au Nanoparticles and Nanowires Templated by Protein Assemblies 6.6. Bioenabled Syntheses of Silver Nanostructures 6.6.1. Peptide- and Protein-Mediated Syntheses of Ag Nanoparticles and Nanoplatelets 6.6.2. Bioenabled Formation of Silver Nanotubes and Nanowires 6.7. Polypeptide-Synthesized Pd and Pt Nanostructures 6.7.1. Protein-Templated Pt and Pd

10.1021/cr8002328 CCC: $71.00  2008 American Chemical Society Published on Web 10/31/2008

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6.7.2. Peptide-Templated Pt Nanostructures 6.8. Protein- and Peptide-Mediated Syntheses of Transition Metals 6.8.1. Copper 6.8.2. Nickel and Cobalt 6.9. Protein- and Peptide-Mediated Syntheses of Bimetallic Nanoparticles 7. Chimeric/Fusion Proteins and Peptides 7.1. Mineralizing/Size-Constraining Chimeras 7.2. Chimeric Biomolecules for Serial Materials Synthesis 7.3. Binding/Mineralizing Fusion Proteins/Peptides 7.4. Biopolymer/Biomineralizing Fusion Proteins 7.5. Chimeric Phage and Cell-Surface Proteins 8. Promising Developments and Applications of Protein- and Peptide-Templated Materials 8.1. Entrapment of Biological and Nanoscale Materials by Biomimetic Syntheses 8.2. Virus Capsid-Templated Energy-Storage Materials 9. Summary and Outlook 10. Acknowledgments 11. References

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1. Introduction The course of evolution on our planet has resulted in the appearance, diversification, and proliferation of organisms capable of producing complex structures from hard inorganic materials,viaprocessesknowncollectivelyasbiomineralization.1-4 For example, magnetotactic bacteria produce nanoparticles of Fe3O4 or Fe3S4 that have well-defined sizes and shapes that enable these microorganisms to utilize magnetic fields for alignment and migration.2,3,5,6 Fish grow structures, known as otoliths or “ear stones”, within the inner ear that assist in inertial sensing.3,7 These otoliths are composed of the aragonite polymorph of calcium carbonate.3,7 Remarkably, mollusks produce shells that contain a single distinct crystalline form of calcium carbonate, such as aragonite, or may contain segregated layers of calcite and aragonite.1,8 In addition to the crystalline forms of calcium carbonate, an amorphous phase of this mineral is synthesized by sea urchins to produce spines (spicules).9 Marine sponges produce silica spicules that have been demonstrated to possess light-guiding characteristics and may reach lengths up to 3 m.3,10-14 The diatoms, a type of unicellular eukaryotic algae, produce intricately detailed silica cell walls, known as frustules, that are organized over multiple length scales.2,3,15 In each of the examples listed above, specialized biomolecules have been found, or are thought, to play a major role in directing theformationoftheseoftencomplexinorganicstructures.2,3,5-9,12 The use of peptides and proteins to direct the “bottom up” syntheses of inorganic materials in Vitro is thus a logical outgrowth of the discoveries made in biomineralization. The use of proteins and peptides to direct the in Vitro syntheses of inorganic materials is attractive for a number of reasons; a few of these are highlighted here and detailed throughout the course of this review. The first of these potential benefits is the production of materials under reaction conditions that are much milder than those used in traditional materials-processing techniques. Select peptides and proteins can facilitate the syntheses of inorganic materials at or near room temperature, in aqueous solutions, and at or near neutral pH. The reduction of energy input and avoidance of non-

Matthew B. Dickerson was born in Columbus, Ohio. He received a Bachelor’s degree in Materials Science and Engineering from The Ohio State University in 2000. In 2002, he received a M.S. degree in Materials Science and Engineering from The Ohio State University, studying the processing of high-temperature composite materials under the guidance of Professors K. H. Sandhage and R. L. Snyder. Matthew received his Ph.D. in 2008 from the Georgia Institute of Technology, with a minor in biochemistry. Working with advisors Professor K. H. Sandhage and Professor N. Kro¨ger, he studied the biomimetic syntheses of inorganic materials. Matthew is currently a National Research Council postdoctoral research associate working at the Air Force Research Laboratory in the research group led by R. R. Naik.

Kenneth H. Sandhage was born in West Lafayette, IN, in 1959. He obtained a Ph.D. in the field of Ceramics from the Massachusetts Institute of Technology in 1987. After working as a Senior Scientist on process/ product research and development at Corning Glass Works and American Superconductor Corporation, he joined the faculty in the Department of Materials Science and Engineering at The Ohio State University (Columbus, OH) as an Assistant Professor in 1991. In 1999-2000, he was a Humboldt Fellow in the Advanced Ceramics Group of Prof. Dr. Nils Claussen at the Technical University of HamburgsHarburg. In 2003, he joined the School of Materials Science and Engineering at the Georgia Institute of Technology as a Full Professor, where he is currently the B. Mifflin Hood Professor (2005) and an Adjunct Professor (2007) in the School of Chemistry and Biochemistry. His research interests include biologically enabled (biomolecular, bioclastic) and/or shape-preserving reaction-based routes to advanced inorganic and composite materials for sensor, electromagnetic, optical, biomedical, refractory, and structural applications.

natural solvents (e.g., organic solvents) required to produce materials in the presence of proteins and peptides makes bioenabled syntheses inherently “green” processing. The second major advantage in exploiting biomolecules for materials synthesis is the exquisite control that proteins and peptides can impose on the size, shape, chemistry, and crystal structure of the inorganic product. This is significant because these characteristics often impact or determine the properties of the synthesized material. Third, peptides and proteins offer

Protein- and Peptide-Directed Syntheses of Inorganic Materials

Rajesh R. Naik was born in Udupi, India, in 1968. He obtained his B.S. in Microbiology from the University of Bombay, India, in 1990 and a Ph.D. in the field of Molecular Biology from Carnegie-Mellon University, Pittsburgh, PA, in 1998. He later worked as a Howard Hughes Research Fellow at the Center for Advanced Biotechnology and Medicine at Rutgers University. In 1999, he joined the Materials and Manufacturing Directorate at the Air Force Research Laboratory, Dayton, OH, as a Research Scientist. Currently, he is the research lead for the biotechnology program and a technical advisor to the Nanostructured and Biological Materials Branch. His research interests include biologically enabled routes to advanced inorganic and composite materials, biomimetic sensors for chembio detection, and the interface between biological and nanomaterials.

the potential to produce materials with highly specific or multiple functions. For example, proteins and peptides may direct the formation of enzymatically active composites or generate materials that specifically recognize substrates and self-assemble. Additionally, the large diversity of natural and synthetic proteins provides high probability that proteins that recognize, interact with, and direct the formation of many inorganic materials are likely to exist; they need only be discovered or synthesized. This review is focused on providing a summary of existing work on the protein- and peptide-directed syntheses of ceramic oxides, semiconductors, and metals. The application of protein- and peptide-directed materials syntheses for entrapping and stabilizing enzymes, as well as for generating materials for battery electrodes, are also discussed within this review. Readers seeking information on the biological or biomimetic syntheses of carbonate and phosphate materials are directed to books edited by Baeuerlein, as well as articles by Aizenberg, Addadi, and Weiner.2,3,8,9,16 While this review provides some comparative examples of the use of synthetic polymers to produce inorganic materials, it is not intended to provide a comprehensive account of this subject; readers are directed to a review by Xu and colleagues for further information on this topic.17

2. Biosilicification 2.1. Introduction to Biosilicification To provide perspective for the biomimetic studies reviewed in this manuscript, prior work on the identification of biomolecules involved in diatom and sponge silica formation are summarized in this section. A more detailed treatment of the biomolecules and biosilicification processes of diatoms and sponges may be found in the excellent reviews (including those presented in this issue of Chem. ReV.) and book chapters recently authored by Kro¨ger, Sumper, Hildebrand, Mu¨ller, Morse, and their associated colleagues.2,3,18-22 Although a number of nonsilica-forming organisms, such as

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Figure 1. SEM images of cleaned diatom silica cell walls (frustules) from several different diatom species, depicting a variety of shapes and patterns created by these organisms. Images of the overall form of the frustule are presented in the top two rows, and higher-magnification images of frustule details are presented in the bottom row. Reprinted with permission from ref 27 (Kro¨ger, N. Prescribing Diatom Morphology: Toward Genetic Engineering of Biological Nanomaterials. Curr. Opin. Chem. Biol. 2007, 11, 662-669). Copyright 2007 Elsevier.

the mollusks, have also inspired biomimetic materials syntheses and design, the biomolecules associated with silica formation by diatoms and sponges are most pertinent to the research covered in this review article.

2.2. Diatom Silica-Associated Biomolecules One of the most prominent sources of inspiration for the application of biological approaches and molecules to inorganic materials synthesis has been the diatom. Diatoms are a major group of unicellular eukaryotic algae that produce cell walls composed of hydrated, amorphous silica.2,3,18,19 The relative importance of these algae to biomimetics is often attributed to their ability to produce silica under relatively mild intracellular conditions.23,24 While the biogenesis of silica does contrast quite heavily to the conditions surrounding the commercial production of this oxide, diatoms are not the only organisms endowed with biosilicification activity. Indeed, a wide range of terrestrial plants including rice, cucumbers, grasses, and ferns produce silica.25,26 However, the silica architectures created by the diatoms are exceptional, in (i) their intricacy and organization over multiple length scales (i.e., from the nanoscale to the microscale) and (ii) the genetic control exerted over the formation of these hierarchical structures.2,3,15,18,19 The former of these points is clearly evident in the scanning electron microscope (SEM) images of cleaned diatom cell walls presented in Figure 1.27 Evidence for genetic influence over the production of diatom silica cell walls (termed frustules) comes from observations that the patterning of these structures is species-specific and is precisely replicated in diatom progeny.2,3,18,19 During the replication and division of diatom cells, new components of the frustule must be produced for the daughter cells.2,3,18,19 The synthesis and patterning of these new silica components occurs within a specialized, membrane-bound


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LCPAs through the establishment of hydrogen bond and ionic interactions among individual LCPAs.2,18 Silicic acid species may absorb in and/or onto the LCPA aggregates, forming a “liquid precipitate”, which may then polymerize into silica.2,18 LCPAs may also induce the precipitation of silica by serving as flocculating agents.2,18 The cationic polyamines may counteract the negative surface charges that repel polysilicic acid particles in solution, allowing silica embryos to nucleate and precipitate from solution as silica particles.2,18 Sumper has recently theorized that the silica structures generated by controlled LCPA aggregation may extend beyond simple spherical morphologies to the intricate designs of the diatom valve.30 Specifically, Sumper hypothesizes that the honeycomblike patterns of silica present on the valves of the Coscinodiscus genera of diatoms may be formed by silicic acid condensation around the periphery of closely packed arrays of LCPA droplets.30

2.2.2. Silica Precipitating Proteins: Silaffins

Figure 2. (A) Heavily post-translationally modified structure of silaffin-1A1. The peptide backbone of the silaffin is shown in one letter amino acid code and modifications to the lysine and serine residues of silaffin-1A1 are depicted in color. (B) Generalized schematic of the chemical structure of LCPAs where the putrescine basis is denoted in red. Reprinted with permission from ref 18 (Sumper, M.; Kro¨ger, N. J. Mater Chem. 2004, 14, 2059). Copyright 2004 Royal Society of Chemistry.

compartmentknownasthesilicadepositionvesicle(SDV).2,3,18,19 While isolation of a functional SDV would allow for detailed study of the processes, chemistries, and biomolecules involved in diatom silica biogenesis and patterning, there are currently no methods available that allow for the isolation of the SDV or its specialized components.2 Nonetheless, important insights into diatom biosilicification have been made by isolating and characterizing specific biomolecules entrapped within the amorphous silica of the frustule.2,3,18,19,28 Two categories of silica-associated biomolecules have been isolated from the diatom frustules: long-chain polyamines (LCPAs) and proteins (silaffins and silacidins).2,3,18,19,28

2.2.1. Long-Chain Polyamines Long-chain polyamines have been identified in every species of diatom investigated to date.29-32 These LCPAs consist of repeating units of N-methylpropylamine attached to putrescine or a putrescine derivative (Figure 2).29-32 The structure of the LCPAs varies in length (i.e., number of propylamine repeat units), position of secondary amine functionalities, degree of methylation, and incorporation of quaternary ammonium moieties.29-32 While the structures of the diatom LCPAs vary, recent research suggests that diatoms produce species-specific sets of these polyamines.29,32 Long-chain polyamines liberated from diatom frustules have been observed to induce the formation of silica precipitates under in Vitro conditions thought to mimic the environment of the SDV.29 The ability of LCPAs to produce silica was found to depend on the ability of these polyamines to self-assemble into aggregates (in Vitro) in solution.33 LCPA aggregation and aggregate size were found be to directly correlated with the presence and concentration of multivalent anions such as phosphate, sulfate, or citrate ions.33 These negatively charged ions may act to cross-link

In addition to LCPAs, proteins have been recovered from the silica of diatom frustules and are believed to play an important role in the biogenesis of these inorganic cell wall structures.28,29,31,34-37 These frustule-isolated proteins may be categorized into two general classes according to their observed behavior in Vitro: (i) those possessing intrinsic silica precipitation activity and (ii) those that do not initiate the precipitation of silica but, instead, act to regulate or modify the silica formation behavior of other biomolecules. Proteins capable of precipitating silica, silaffins-1A1, -1A2, -1B (the term “silaffin” is derived from the silica affinity of these proteins), have been isolated from the frustules of the diatom Cylindrotheca fusiformis.34,36 Silaffins-1A1, -1A2, -1B are highly homologous peptides that are enriched in serine, lysine, and glycine residues and are generated through the enzymatic cleavage of a single protopolypeptide (i.e., sil1p).34 The lysine and serine residues of these peptides are highly modified (Figure 2).34,36 Post-translational lysine modifications were found by Kro¨ger and colleagues to include polyamine addition (i.e., the covalent attachment of 6-11 repeats of N-methylpropylamine), ε-amine dimethylation, or phosphorylation and ε-amine trimethylation.34,36 The serine residues of silaffins-1A1, -1A2, -1B are phosphorylated in ViVo, a modification that adds negative charges to these peptides.36 Silaffins carrying their full complement of lysine and serine modifications are known as native silaffins (abbreviated natSil) in order to differentiate them from those silaffins extracted by early methods in which some of these fragile modifications were not preserved.36 The extensive post-translational modifications of natsil1A1, -1A2, -1B are believed to have a significant affect on the ability of these peptides to induce silica formation in ViVo.34,36 Under in Vitro conditions thought to reflect the acidic internal environment of the SDV, sil-1A1, -1A2 (generically referred to as sil-1A), which carry methylated and polyamine-modified lysine residues, were found to possess significant silica precipitation activity.34 Under similar acidic conditions, a synthetic analog to sil-1A, peptide R5 (which did not carry post-translational modifications), was not observed to induce silica precipitation.34 This indicated that the numerous modifications to the lysine residues of sil-1A and -1B are required for silica precipitation activity within the proposed environment of the SDV.34 Similarly, Kro¨ger and colleagues found that the phosphorylation of native silaffin serine residues played a significant


role in the ability of these peptides to induce silica condensation.36 Silaffins lacking these native phosphate groups were incapable of precipitating silica in solutions lacking phosphate ions.36 Paralleling the activity of the LCPAs discussed above, the ability of silaffin-1A (the dephosphorylated version of natSil-1A) to induce silica condensation was restored when multivalent phosphate anions were added to the reaction solution.33,36

2.2.3. Diatom Silica-Regulation Proteins Kro¨ger and colleagues also discovered that the silica frustules of the C. fusiformis diatom contained the protein silaffin-2 (sil-2).34 Silaffin-2 was found to lack silica precipitation activity, but was observed to act as a modifier of the in Vitro silica-precipitation activity of LCPAs.37 Although the exact sequence of sil-2 is currently unknown, Poulsen et. al have characterized the amino acid composition and post-translational modifications of this protein.37 Much like the natSil-1 proteins, natSil-2 carries methylated and polyamine-modified lysine residues.37 Unlike natSil-1A and -1B, however, natSil-2 was found to possess an overabundance of negatively charged residue modifications, making the protein anionic in nature.37 In addition to phosphoserines, natSil-2 was found to possess phosphothreonines and phosphorylated hydroxyprolines, as well as sulfate- and saccharide-modified (e.g., glucuronic acid) residues.37 The anionic character introduced to natSil-2 through these amino acid modifications apparently inhibited the silica precipitation activity of this protein and is thought to influence the silica formation behavior of the cationic LCPAs.37 As discussed above, LCPAs require multivalent anions to self-assemble in solution and precipitate silica. NatSil-2, with its multitude of negatively charged modified residues, may fill this role. The effects of natSil-2 on silica precipitation, however, are more complicated than previously reported for simple inorganic multivalent ions.37 NatSil-2 was found to promote LCPA-induced silica formation when this protein is present in solution at low concentrations, but it inhibited LCPA-induced silica precipitation when present in high concentrations.37 Additionally, Kro¨ger and colleagues noted that natSil-2 inhibited the silica-precipitation activity of natSil-1A in a concentration-dependent manner.37 Beyond regulating the amount of material precipitated by LCPAs or natSil-1A, natSil-2 was also found to influence the morphology of the silica produced by these cationic biomolecules.37 Indeed, when combined in appropriate ratios, the silica formed in the presence of natSil-2 and natSil-1A possessed a pear-shaped or porous sheet morphology.37 Proteins that act as regulators or modifiers of silica formation are not unique to the C. fusiformis diatom. Poulsen and Kro¨ger have recently isolated and characterized natSil2-like silaffins from a second diatom species, Thalassiosira pseudonana.31 These T. pseudonana silaffins, tpSil-1H, -2H, and -3, like all silaffins identified to date, were found to possess substantial post-translational modifications.31 Analogous to natSil-2, the amino acid residues of the regulatory tpSils carry anionic modifications, such as appended phosphates and sulfates, and possess acidic isoelectric points.31,37 The modifications of tpSil-1/2H and tpSil-3 also include gylcosylation with a complex mixture of saccharides and the addition of short polyamine chains to the lysine residues of these proteins.31,38 Recent work by Sumper and colleagues


suggests that the primary structure of tpSil-3 dictates the types and positions of lysine modifications within the protein.38 Mirroring the regulatory role of natSil-2, the addition of tpSil-1/2H and tpSil-3 to reaction solutions containing LCPAs first enabled and then inhibited the in Vitro formation of silica with increasing concentration.31 The silica formed in the presence of tpSil-1/2H and tpSil-3 also possessed a morphology differing from the microspheres typically produced by LCPAs in phosphate-containing solutions.31 Kro¨ger and colleagues hypothesized that these regulatory silaffins may play a role in guiding the formation of diatom nanostructures in ViVo.31 This may occur through the formation of a silica-templating matrix assembled from the diatom’s cationic (e.g., natsil-1A, -1B, and LCPAs) and anionic (e.g., natSil-2 or tpSils) components within the SDV.31 The most recently discovered examples of proteins isolated from within the diatom cell wall that are able to modify the in Vitro silica precipitation activity of LCPAs are the silacidins.28 Named for their residence within T. pseudonana biosilica and their acidic nature, the silacidins are enriched in aspartic acid, glutamic acid, and phosphoserine residues.28 The silacidins (A, B, and C) contain no lysine residues and, thus, lack the extensive polyamine modifications native to the silaffins.28 Like natSil-2, tpSil-1/2H, and tpSil-3, the silacidins lack silica-precipitation activity but are able to induce LCPA-mediated silica formation in a concentrationdependent manner.28,31,37 Unlike natSil-2, tpSil-1/2H, and tpSil-3, the silacidins were not observed to inhibit the silicaprecipitation activity of the LCPAs or modify precipitate morphology.28,31,37

2.3. Silica-Associated Proteins in Sponges On an evolutionary time scale, the diatoms are relatively recent contributors to the biogenesis of silica, with frustules appearing in the fossil record only within the last 110-115 million years.3 Indeed, fossils containing biosilicified skeletal elements have been identified in fossil beds dating to the Early Cambrian period, approximately 549 million years ago.39 The animals preserved in these Early Cambrian fossil beds are members of a taxon that still exists today, the sponges (i.e., phylum porifera).39 Two classes of sponges, Hexactinellida and Demospongiae, produce skeletal structures composed of amorphous silica termed spicules.20,21 While outward spicule morphology may appear rather plain when compared to the intricate diatom frustule ultrastructure, interesting shapes are possible (Figure 3).40 What silica structures may lack in fine detail may be made up for in size, as spicules may grow up to 3 m in length (e.g., for the giant spicules of the sponge Monorhaphis chuni).40 Though these “living fossils” have been producing silica structures for hundreds of millions of years, the biomolecules and processes involved in spicule formation have only recently been evaluated through the application of modern molecular biology methods.20-22

2.3.1. Silica Precipiting Proteins: The Silicateins Sponge spicules are not simple monolithic bodies of silica, but instead contain several features, including an axial channel running through the center of the spicule and concentric rings that can be seen in spicule cross sections (Figure 3D).22 The first major biomolecular insights into


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originated through the development and observations of a cell culture system derived from the demosponge Suberites domuncula.46-49 An important discovery obtained through research conducted with the S. domuncula cell culture system was that spicule growth was genetically controlled.44,48,50 This was deduced from the upregulation of several genes coding for proteins associated with spicule growth, such as silicatein and galectin, upon exposure of the demosponge cells to silicate solutions.44,48,50

2.3.2. Silica-Precipiting LCPAs

Figure 3. SEM images of silica spicules from the sponge M. chuni. Spicules depicted in (A), (B), and (C) originated from the choanosomal body of the sponge while a cross section of the giant basal spicule is shown in (D). The axial channel (ac), axial center (cy) formed by a dense layer of silica, and concentric layers of silica (la) are visible in the polished cross section in (D). Reprinted with permission from Figures 2 and 3 of ref 12 (Mu¨ller, W. E. G.; Eckert, C.; Kropf, K.; Wang, X.; Schloβmacher, U.; Seckert, C.; Wolf, S. E.; Tremel, W.; Schro¨der, H. C. Formation of giant spicules in the deep sea hexactinellid Monorhaphis chuni (Schulze 1904): Electron-microscopic and biochemical studies. Cell Tissue Res. 2007, 329, 363). Copyright 2007 Springer Science + Business Media.

sponge silicification were made by Shimizu et al. and Cha et al., who isolated and characterized the proteins contained as a filament within the axial channel of the spicule.41,42 Following the dissolution of the silicaceous portion of the spicule in buffered hydrofluoric acid, these authors found that the axial filament of spicules from the demosponge Tethya aurantia was composed of three proteins, referred to as silicateins R, β, and γ.41 These isolated silicatein filaments were observed to initiate the condensation of silica from silicon alkoxide precursors in Vitro.42 Two of the isolated silicateins, R and β, were cloned from T. aurantia and found to be homologous to the papain family of proteolytic enzymes (i.e., silicatein R exhibited 52% sequence identity and 75% sequence similarity to human cathepsin L).41 Analogous to active sites of the papain proteases that contain the residues cysteine, histidine, and asparagine, the silica-condensation activity of the silicateins is thought to lie in a catalytic His-Ser-Asn triad.41,42 Specifically, it has been proposed that the histidine-serine pair found within the active site of the silicateins serves as a general acid/base catalyst for the hydrolysis of silicon alkoxides.42,43 The suggested role of this histidine-serine active site was supported by the observation that the silicacondensation activity of silicatein R was significantly reduced in recombinant variants in which either the histidine or serine was replaced with alanine.43 Expanding on the discoveries of Shimizu et al. and Cha et al., Mu¨ller and colleagues have isolated, from two additional demosponge species, silicateins that possess high homology to T. aurantia silicatein R.44,45 Mu¨ller et al. have reported that sponges are equipped with a complement of different silicateins (at least two per species) that may exist with differing degrees of phosphorylation.46 in Vitro studies conducted with recombinant and spiculeisolated silicateins have shed light upon the biochemical processes associated with sponge biosilicification.42-44 Additional insights into the growth of sponge spicules have

Given the vast phylogenetic divide between sponges and diatoms (i.e., these organisms are members of the kingdoms Animalia and Protista, respectively), one would expect that the sponges may have developed biosilicification mechanisms distinct from those believed to occur in diatoms. However, LCPAs, which are molecules associated with the formation of diatom frustules, have also recently been isolated from the spicules of the sponge Axinyssa aculeata by Matsunaga and colleagues.2,3,18,19,51 Like the diatom LCPAs, these A. aculeate LCPAs are composed of propylamines that range in length from 5-15 units and are capable of inducing silica precipitation in Vitro in the presence of multivalent anions.2,3,18,19,51 In this same study, Matsunaga and colleagues also reported the isolation of a silicatein-like protein from A. aculeate and LCPAs from the spicules of an additional species of sponge.51 This interesting initial report provides strong motivation for evaluating distribution of polyamines in additional sponge species and for determining the role these LCPAs play in in vivo spiculogenesis.

3. Sources of Biomolecules for the Biomimetic Synthesis of Materials 3.1. Biomolecules Isolated or Derived from Biomineralizing Organisms The biomolecules recruited to initiate and control the synthesis of materials have, to date, been extremely diverse in their origins, compositions, and structures. The application of proteins and peptides for the in Vitro production of materials has been inspired by the prominent role that these biomolecules are thought to play in biomineralization. Perhaps the most direct approach for obtaining biomolecules with materials synthesis activity for in Vitro reactions is to extract proteins and peptides from biomineralizing organisms. In fact, proteins and peptides isolated from biologically produced inorganic structures have exhibited in Vitro mineralization activity.2,3,18,20-22 Biomolecules extracted from biominerals may also be utilized to synthesize materials beyond those produced by the source organism. This is exemplified by the silicatein-induced formation of titanium dioxide and gallium oxide.52,53 The use of biomineral-isolated proteins and peptides has several drawbacks, however. Biomineralizing specimens may be difficult to obtain, require specialized facilities to grow, often yield limited quantities of the desired biomolecules, and may provide few if any opportunities to modify or engineer protein sequences. Many of these difficulties may be overcome through the recombinant expression and subsequent purification of mineralizing proteins from bacterial cells.43,54-56 This approach has been successfully applied to produce silicifying biomolecules, such as silicateins and


silaffins, in relatively high yield from genetically modified Escherichia coli.43,54-56 As an alternative to expressing fulllength gene products, truncated sections of the diatom sil1 gene have been chemically synthesized to obtain silicifying peptides, such as the R5 peptide.34,57,58 However, the production of proteins and peptides derived from biomineralizing proteins is restricted by the limited numbers and compositions of biomineralizing proteins that have been sequenced to date.2,3,18,20-22

3.2. Biomineralizing Protein Analogs The involvement of specialized biomolecules to guide the formation of inorganic structures in ViVo has spawned considerable interest in identifying readily available molecules that might mimic the activities of these proteins in Vitro. The sequence or characteristics of the biomolecule to be emulated typically serves as the criteria for selecting synthetic analogs. For example, the LCPAs isolated from the silica of diatom frustules and sponge spicules are highly cationic and possess a large number of 1′, 2′, 3′, and 4′ amines.2,18,51,59 Some of these characteristics are shared by several commercially available polymers (e.g., poly(allylamine)), which have been utilized to investigate the in Vitro synthesis of silica, as well as germania and titania.32,60,61 In addition to homopolymers, block copolymers that incorporate two or more functionalities of biomineralizing proteins have also been utilized to guide the formation of silica. The research of Cha and colleagues represents a specific example of this approach, where cysteine-lysine copolypeptides were designed to mimic the active site of silicatein and used to mediate silica formation in Vitro.62 The use of hydrophilic-hydrophilic block copolymers, like the cysteine-lysine polypeptide, to direct the formation of materials has recently been reviewed by Xu and colleagues.17 Some of the sequence characteristics native to biomineralizing proteins may also be found in readily available and inexpensive proteins, such as hen egg white lysozyme (HEWL) or bovine serum albumin (BSA), making them popular candidates for biomimetic studies.2,3,18,20-22,63-69 Hen egg white lysozyme possesses an excess of positively charged residues, making it an adequate analog to the LCPAs or the R5 peptide. Indeed, HEWL has found application in the synthesis of silica, as well as titania (a nonbiogenic material).64-66 In addition to lysozyme, a variety of other commercially available enzymes, including R-amylase and pepsin, have been utilized as templates for the formation of nanomaterials.67,70 BSA contains a diverse complement of amino acids, making it a versatile protein for the directed synthesis of a number of different materials. For example, the tyrosine content of BSA has been exploited to reduce gold and silver ions to metallic nanoparticles, whereas the cysteine residues of BSA have been utilized to cap the growth of quantum dots.63,69

3.3. Peptides Identified Through Biopanning Of all the sources of biomolecules utilized for the synthesis of materials, peptide libraries have offered the greatest diversity of sequences. A brief summary of the screening of peptide libraries and the application of this technology to materials synthesis is provided here, as a thorough treatment of this subject has recently been offered in comprehensive review articles authored by Sarikaya, Baneyx, Kirplani, Feldheim, and their respective colleagues.71-75 Detailed


information on the construction and use of phage-displayed peptide libraries is also offered in review articles by Kehoe and Kay, and Smith and Petrenko, as well as in a laboratory manual written by Barbaras and colleagues.76-78 To date, the peptide libraries utilized to identify peptides for the biomimetic synthesis of materials have been displayed on the surface of biological systems, such as bacteria, yeast, and bacteriophage (bacteria-infecting viruses).71-75 These biologically displayed peptide libraries are constructed through the modification of the host organism’s genome.78 Small genes inserted into the bacteria, yeast, or phage code for the amino acid sequence of the peptide that is displayed on the cell or capsid surface.78 The number of peptides of unique sequence that are displayed by these biological systems can be quite large (e.g., ∼3 × 109 for the phagedisplayed Ph.D.-12 library commercially available through New England Biolabs), allowing for extremely diverse peptide libraries.75,78 Because of the connection of the genotype and phenotype in these genetically engineered peptide libraries, the amino acid sequence of a given peptide may easily be determined through the sequencing of the small gene inserted into the host’s DNA.78 Additional advantages of these phage- and cell surface-displayed libraries are replicability and clonability.78 The construction of phage- or cell-surface displayed libraries requires considerable molecular biology expertise.78 Fortunately, several phage-displayed peptide libraries are commercially available for use in identifying peptides that direct the formation of materials.74,75 Because of the popularity of phage-displayed libraries, the screening of these libraries is briefly described here. Procedures for the screening of cell surface-displayed libraries are similar to those for their phage-displayed counterparts and are described elsewhere.71,72

3.3.1. Screening of Peptide Libraries The general procedure utilized for the screening of phagedisplayed peptide libraries is presented in Figure 4. As depicted in Figure 4, the phage library is diluted into a buffer solution and incubated with a target material (e.g., silica, titania, or silver) that is typically contained in a multiwelled plate or plastic centrifuge tube.78 During this incubation period, phage that display peptides having an affinity for the target material will bind to the target.78 Following incubation, unbound phages (i.e., phage that display peptides that lack affinity for the target material) are removed from the target by extensive washing with a detergent-containing solution.78 The target-bound, phage-displayed peptides are then removed from the target material through the disruption of the targetpeptide interaction.78 This is commonly accomplished by subjecting the target-peptide/phage pairing to a low pH buffer that disrupts ionic interactions and may partially denature peptides.78 These selected viruses are introduced into E. coli culture (i.e., liquid or plate) where they replicate in their infected host cells.78 The replicated phages carry the same peptide and corresponding gene insert as the target eluted phage.78 The replication of the selected phages represents the end of the first round of the screening or biopanning procedure.78 Additional rounds of screening (i.e., typically 2-4 more rounds) are conducted by reincubating the selected and replicated phages with the target.78 These additional biopanning rounds are typically conducted with increased washing stringency (e.g., increased concentrations of detergent) in order to exclude marginal target binders.78 Following


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Figure 4. Procedure for the isolation of peptides possessing a high affinity for an inorganic material from a phage-displayed library. Several (3-5) rounds of stringent washing and elution are used to remove weakly binding peptides from the final screened phage population. The amino acid sequence of the displayed peptide is determined through the DNA sequencing of the final phage clones.

the final round of selection, individual phage clones are isolated and sequenced to reveal the primary structure of the target binding peptide.78 These phage clones, or the chemically synthesized phage-free peptide, may then be assessed for their ability to initiate and direct the formation of the target material from an appropriate precursor solution.

3.3.2. Application of Biopanning to Functional Inorganic Materials The first application of the biopanning method to identify peptides that bind to inorganic materials was conducted by Brown in 1992.79 In this first study, Brown isolated peptides that selectively adhered to Fe2O3 surfaces.79 In this case, selectivity refers to the preference of the peptides to bind to Fe2O3 but not the related compounds Cr2O3 or Fe3O4.79 While Brown also identified gold-binding peptides in 1997, it was not until 2000 that other investigators began to apply the technique to additional inorganic materials.80,81 At that time, Whaley et al. utilized a phage-displayed library to identify peptides that specifically recognized GaAs, InP, and Si semiconductor surfaces.81 In addition to the use of phage-displayed libraries to screen for peptides that bind selectively to the inorganic target, the observation that the identified peptides may, in turn, initiate or guide the formation of the target material (from an appropriate precursor solution) has significantly advanced the bioenabled syntheses of materials. This important concept was first demonstrated in 2000 by Brown et al. and Gaskin et al. in studies focusing on the influence of library-identified peptides on the formation of gold and calcium carbonate, respectively.82,83 Several additional examples of the ability of library-identified peptides to direct the in Vitro formation of inorganic materials natively produced in biology soon followed the Brown and Gaskin studies.82-88 These later studies included the precipitation of noble metals (e.g., Au and Ag), silica, CaCO3, and CdS under the influence of library-identified peptides.84-88 Phage- and cell surface-displayed peptide libraries have also been important tools in the identification of peptide sequences that both bind to and direct the formation of nonbiogenic inorganic materials. The biopanning procedure has been successfully utilized to identify ZnS, TiO2, ZnO, CoPt, and FePt nanoparticle-forming peptides.85,88-92 Peptides identified through the biopanning process have also been found to induce and direct the syntheses of the binary ceramic compounds, CaMoO4 and BaTiO3.54,89 An important modification of the phage-displayed library screening process has been developed by Naik and col-

leagues.85 This alteration was made to allow for identification of peptides that possess such an affinity to the target that they are not removed by the typical low pH elution protocol.85 Naik et al. introduced the polymerase chain reaction (PCR)-driven biopanning method, in which the DNA of the target-bound phage was recovered through the disassembly of the capsid in a boiling detergent solution.85 The released phage DNA was then amplified by the PCR process and subsequently transformed into E. coli to generate individual clones.85 The phage DNA was then extracted from these E. coli clones utilizing standard molecular biology protocols and sequenced in order to obtain the identity of the phage’s high-affinity peptide.85 The influence that subtle changes in sequence exert on the binding behavior of biopanned peptides has been examined by Peele and colleagues.93 Utilizing yeast genetically engineered to display short peptides composed of one type of amino acid (e.g., His6, Ala6, or Lys6), these authors determined that only a select subset of residues (His, Trp, Cys, and Met) possessed an affinity to the surfaces of the materials in their study (i.e., CdS, CdSe, ZnS, ZnSe, and gold).93 A new library composed of peptides of the general form XHXHXHX (where H is histidine and X is one of the 20 common amino acids) was then created to assess the contribution of neighboring amino acids on the binding activity of histidine.93 Within the context of these designed peptides, the amino acids Gly, Lys, Arg, His, Trp, Cys, and Met were observed to enhance the inorganic binding activity of the designed peptides, relative to Ala, which was designated as neutral.93 Conversely, acidic, hydrogen bonding, and hydrophobic residues were found to reduce the affinity of the interdigitated histidine peptides for the inorganic surfaces tested.93 This systematic study demonstrated the dramatic impacts that minor protein sequence changes can have upon peptide-binding activity.93

4. Oxides Produced Under the Influence of Proteins and Peptides 4.1. Silicon Dioxide 4.1.1. Introduction The biomimetic synthesis of silica has been inspired by the biological production of silica by organisms such as sponges, diatoms, and higher plants (see discussion in section 2). An aim of biomimetic silicification research has been to augment knowledge gained from studies with biomineralizing organisms and to exploit this knowledge to build nano- and


microscaled silica materials from the bottom up. A large variety of synthetic molecules and biomolecules, including those derived from the genomes of silicifying organisms, have been evaluated for the ability to produce silica in Vitro under relatively benign conditions (i.e., near neutral pH, aqueous environment, and ambient temperature and pressure). Specific examples of such biomolecules include fulllength sponge and diatom gene products, such as recombinant silicateins and silaffins, as well as truncated gene products such as the synthetic R5 peptide, which corresponds to the repeat sequence of the silaffin precursor polypeptide sil1p.42,43,54,55,57,58,94 Synthetic analogues of the highly cationic sil1p-derived silaffins, including poly-L-lysine (PLL), poly(allylamine) (PAA), and polyethyleneimine (PEI), have also been extensively investigated for silica-precipitation activity.24,26,94-96 Inspired by the LCPAs recovered from diatom and sponge silica, biomimetic studies have also made use of readily available short-chain polyamines (SCPAs) such as spermine and putrescine.26,97-99 Peptides isolated from phage-display libraries, designed amphiphilic peptides, homoand block-copolymer poly(amino acids), and readily available proteins (e.g., BSA, HEWL, cytochrome C, and collagen) have also been evaluated for the biomimetic synthesis of silica.24,72,86,96 As aspects of the bioinspired production of silica have been previously summarized by several authors, this review will offer a brief initial overview of biomimetic silica formation.24,26,95-97 Subsequent sections of this review will then summarize recent research related to the dramatic programmability and flexibility of biomimetic silicification processes. The application of the biomimetic silicification to the encapsulation of enzymes and nanoparticles is described in section 8 of this review.

4.1.2. Silica Formation in the Presence of Biomolecules and Biomolecule Analogues A critical question in the field of biomimetic silicification has been: what characteristics endow a molecule with the ability to induce silica formation under biological conditions? A number of authors have found that a molecule’s biomimetic silica formation activity is strongly dependent upon the presence of amines.24,26,95-97 These amines may be primary amines (e.g., the side chain of the amino acid lysine), secondary amines (e.g., those found in the backbone of SCPAs), or quaternary amines (e.g., post-translationally modified lysines in natsil-1A).24,26,95-97 Amine groups are thought to play multiple roles in the biomimetic synthesis of silica, the first of which may be to catalyze the hydrolysis of organosilicate precursors.24,98,99 The influence of amines on such catalysis has been found to be dependent upon amine type, the pH of the reaction system, and the silica precursor utilized.24,95,97-99 Hydrolysis of organosilicate species such as tetramethoxysilicon (Si(OCH3)4) results in the release of alcohols and yields silanol-bearing molecules (i.e., R3Si-OH). In many studies, this initial amine-based hydrolysis step is circumvented through the selection of silanol-rich species such as silicic acid (Si(OH)4).24,26,95-97These silanol groups may polymerize through condensation reactions to produce Si-O-Si bonds, eventually producing silica through the formation of a network of such bonds.24,26,95-97 As discussed above (see section 2 of this review) for cationic silaffins and LCPAs, amine groups may also promote the aggregation of negatively charged silicic acid species, which polymerize to produce silica precipitates.18,24,26,95-97 While the presence of amines


Figure 5. SEM images of silica formed in the presence of (A) T. pseudonana LCPAs and (B) poly-L-lysine (MW) 22 kDa). Scale bars correspond to1 µm in (A) and (B). Image (A) reprinted from ref 31 (Poulsen, N.; Kro¨ger, N. J. Biol. Chem. 2004, 279, 42993). Copyright 2004 American Society for Biochemistry and Molecular Biology. Image (B) reprinted with kind permission from Figure 3 of Patwardhan, S. V.; Raab, C.; Hu¨sing, N.; Clarson, S. J. Macromolecule mediated bioinspired silica synthesis using a diolmodified silane precursor. Silicon Chem. 2003, 2, 279-285. Copyright 2003 Springer Science+Business Media.

appears to be critical for the biomimetic synthesis of silica, the relative precipitation activity of an amine-bearing biomolecule or synthetic molecule can be influenced by other factors. For example, silica-precipitation activity has been observed to be highly dependent on the ability of aminebearing polymers to self-assemble in solution.100 The presence of other chemical moieties, such as hydroxyl groups that may aid in the hydrogen bonding of silicic acid species to amine-rich biomolecules, has also been observed to influence silica-precipitation activity.86

4.1.3. Exploiting Environmental Influences in Biomimetic Silicification Silica precipitates prepared with biomolecules or synthetic amines under physiological conditions are often found to be formed as solid spheres with diameters that can range from 50 nm to over 1 µm (Figure 5).95,96 The size and morphology of biomimetically prepared silica precipitates may be modified, however, through the careful control of chemical and/ or physical influences in the reaction environment. Sumper and Brunner found that the size of polyamineprecipitated silica particles was strongly affected by both the type and concentration of anionic species present in the reaction solution (Figure 6).95 The influence that phosphate and sulfate ions exert on precipitate size appears to stem from their ability to induce polyamine aggregation and to control aggregate size in solution.33,95,101 Indeed, polyamine aggregation and silica precipitation did not occur in the absence of such anionic species.95 A key finding of Lutz and colleagues was that multivalent anions must be capable of both hydrogen bonding to, and undergoing electrostatic interactions with, cationic polyamines in order for phase separation (i.e., aggregation) to occur.100 Rodrı´guez and colleagues found that alcohols and saccharide-based chemical additives, which are capable of hydrogen bonding, could also be used to modify polyamine-induced silica precipitate size.94 Unlike the action of phosphate and sulfate ions, addition of these hydroxyl-bearing additives served to decrease the average silica precipitate diameter.94,95 The spherical silica precipitates resulting from the interaction of polyamines with anionic or alcoholic species represent but two examples of a variety of silica structures that may be produced through the introduction of chemical additives into the biomimetic silicification process. In their 2003 study,


Figure 6. Effect of multivalent anion concentration and type on the size of silica spheres formed in the presence of polyamines extracted from the diatom Stephanopyxis turris. Reactions were conducted in a sodium acetate buffered solution with increasing concentrations of orthophosphate and pyrophosphate anions. The responses to orthophosphate and pyrophosphate are shown by the dark gray and light gray lines, respectively. SEM images of resulting particles are shown in the insets, with scale bars corresponding to 1 µm. Image reprinted with permission from ref 33 (Sumper, M.; Lorenz, S.; Brunner, E. Biomimetic control of size in the polyaminedirected formation of silica nanospheres. Angew. Chem., Int. Ed. 2003, 42, 5192-5195). Copyright 2003 Wiley-VCH Verlag GMBH & Co. KGA.

Sumper and colleagues found that strings of silica nanoparticles could be produced by exchanging the simple multivalent anions used in their previous work with negatively charged double-stranded DNA (Figure 7A).33 Furthermore, collapsed vesicular or spongy silica could be created with polyamines in the presence of the anionic surfactant, sodium dodecyl sulfate (SDS), at concentrations of 0.6 or 1.2 mM, respectively (Figure 7B).33 A reverse micelle system based on a negatively charged surfactant and a synthetic polyamine (PEI) has also been successfully utilized to biomimetically synthesize hollow silica spheres of controlled size (Figure 7C).102 Externally applied physical forces may also be used to influence the morphology of biomimetically formed silica.58,65 In research that exemplifies the utility of combining biomimetic and traditional materials synthesis approaches, Shiomi and colleagues produced hollow silica spheres through the sonication of an aqueous solution containing HEWL and a silicon alkoxide (parts A and B of Figure 8).65 It should be noted that the silica precipitates formed from identical lysozyme and silicon alkoxide solutions under static or stirred conditions were solid spheres.65 Shiomi and colleagues reported that hollow silica spheres could also be produced under sonochemical conditions with the enzyme ribonuclease A.65 Dependent on protein concentrations, silica formed under sonication was also observed to adopt a spongelike architecture (Figure 8C).65 Hydrodynamic environments created through shear flow rather than sonication have also been utilized to modify biomimetic silica architectures.58 Among the most dramatically altered biomimetic silica morphologies reported to date are the woven fiberlike structures generated through the application of a shear force to a solution containing silicic acid and the R5 peptide (Figure 9A).58 Rodrı´guez and colleagues have also utilized electrostatic and/or hydrodynamic fields to modify the silica precipitate morphology generated by poly-L-lysine from spheres, to dendritic or rosettelike morphologies (parts B and C of Figure 9).94

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Figure 7. SEM images of silica morphologies formed through the interaction of polyamines with anionic (A) dsDNA, (B) SDS, and (C) detergent micelles. Scale bars correspond to 200 nm (A and B) and 3 µm (C). Images (A) and (B) reprinted with permission from ref 33 (Sumper, M.; Lorenz, S.; Brunner, E. Biomimetic control of size in the polyamine-directed formation of silica nanospheres. Angew. Chem., Int. Ed. 2003, 42, 5192-5195). Copyright 2003 Wiley-VCH Verlag GMBH & Co. KGA. Image (C) reprinted with permission from ref 103 (Bauer, C. A.; Robinson, D. B.; Simmons, B. A. Silica particle formation in confined environments via bioinspired polyamine catalysis at near-neutral pH. Small 2007, 3, 58-62). Copyright 2007 Wiley-VCH Verlag GMBH & Co. KGA.

4.1.4. Peptide Sequence Programmed Silica Architecture While the exploitation of chemical and physical factors to produce unique precipitate morphologies demonstrates the flexibility of biomimetic silica synthesis, silica architectures may also be controlled through the tailoring of the peptide/ protein sequence and resulting peptide/protein structure. For example, Jan and co-workers designed and employed lysinephenylalanine block copolypeptides to produce hollow spherical silica precipitates.103 The copolypeptides utilized by Jan et al. were designed to be amphiphilic, having an N-terminal block of hydrophilic lysine residues and a C-terminal domain composed entirely of hydrophobic phenylalanine residues.103 Because of their amphiphilic nature, these lysine-phenylalanine block copolypeptides self-assembled in solution to form vesicles.103 This vesicle structure then acted as a template for the deposition/polymerization of silicic acid on its surface, resulting in the formation of hollow silica particles.103 Amphiphilic peptides may be designed to assemble into a range of morphologies for the subsequent templated deposition of silica. For example, silica nanotubes have been successfully synthesized with amphiphilic peptides that possessed a silica precipitating (e.g., polyhistidine or polylysine) headgroup attached to a palmitic acid-based tail through a polyalanine spacer region (Figure 10).104 While block hydrophilic-hydrophobic designs have proven successful, other peptide designs are also capable of tem-



Figure 10. TEM images of (A) negatively stained nanotubes selfassembled from palmitic acid conjugated cationic peptides and (B) silica-coated peptide nanotubes following exposure to a silicon alkoxide solution. Reprinted with permission from ref 105 (Yuwono, V. M.; Hartgerink, J. D. Langmuir 2007, 23, 5033). Copyright 2007 American Chemical Society.

Figure 8. (A) TEM and (B) SEM images of hollow silica produced by lysozyme under sonication. (C) SEM image of spongy silica formed in the presence of lysozyme at higher concentration under sonication. Scale bars correspond to 3, 5, and 10 µm in (A), (B), and (C), respectively. Reprinted with permission from ref 65 (Shiomi, T.; Tsunoda, T.; Kawai, A.; Mizukami, F.; Sakaguchi, K. Chem. Mater. 2007, 19, 4486). Copyright 2007 American Chemical Society.

Figure 11. TEM images of (A) negatively stained fibrils selfassembled from β-sheet forming cationic peptides and (B) fibrils encrusted with silica following exposure to a silicon alkoxide solution. Reproduced with permission from ref 106 (Meegan, J. E.; Aggeli, A.; Boden, N.; Brydson, R.; Brown, A. P.; Carrick, L.; Brough, A. R.; Hussain, A.; Ansell, R. J. Designed Self-Assembled β-Sheet Peptide Fibrils as Templates for Silica Nanotubes. AdV. Funct. Mater. 2004, 14, 31-37). Copyright 2004 Wiley-VCH Verlag GMBH & Co. KGA.

Figure 9. SEM images of silica morphologies created in the presence of (A) the R5 peptide under shear flow conditions, (B) 2.5 kDa average MW PLL, and (C) 62 kDa average MW PLL under nonuniform electrostatic and hydrodynamic fields. Scale bars correspond to 1 µm in (A) and (B) and 2 µm in (C). Image (A) reprinted with permission from ref 58 (Naik, R. R.; Whitlock, P. W.; Rodrı´guez, F.; Brott, L. L.; Glawe, D. D.; Clarson, S. J.; Stone, M. O. Chem. Commun. 2003, 238). Copyright 2003 Royal Society of Chemistry. Images (B) and (C) reprinted with permission from ref 95 (Rodrı´guez, F.; Glawe, D. D.; Naik, R. R.; Hallinan, K. P.; Stone, M. O. Biomacromolecules 2004, 5, 261). Copyright 2004 American Chemical Society.

plating sophisticated silica structures. In an elegant study, Meegan and colleagues designed peptides that adopt an antiparallel β-sheet structure.105 With this particular secondary structure, the peptide’s hydrophobic and hydrophilic residues primarily reside on opposite sides of the sheet.105 This segregation of hydrophobic and hydrophilic residues facilitated the self-assembly of many copies of the peptide into fibrils (Figure 11A).105 Upon exposure to a silicon alkoxide solution, these cationic peptide-based fibrils were

coated with silica, producing nanotube structures (Figure 11B).105 The self-assembling capsid proteins of the M13 and tobacco mosaic viruses, which possess cationic character at low pH, as well as the surfaces of collagen fibers (collagen pI ) 8), have also been utilized for the templated formation of silica nanotubes.106-109

4.1.5. Synergistic Co-Self-Assembly of Silica/Template Structures Several research teams have discovered well-defined structures that are created through the dynamic co-selfassembly of a biomolecular template and a condensing silica precursor. Early observations of one such distinctive silica/ biomolecule architecture, hexagonal platelets (parts A and B of Figure 12), were reported in 2003 and 2004 by Patwardhan et al. and Rodrı´quez et al., respectively.94,110 In each of these studies, the biomimetic synthesis of silica was explored through the interaction of poly-L-lysine (PLL) with silica precursor molecules under phosphate-buffered, neutral pH conditions.94,110 While Rodrı´quez and colleagues observed that the appearance of silica platelets was dependent on PLL precursor molecular weight, it was not until 2005 that a more complete model was developed that described the mechanism by which these structures formed.94,111 Tomczak and colleagues reported that the formation of this


Figure 12. SEM images of peptide/silica composites synergistically formed through the interaction of PLL, phosphate ions, and silicic acid species in (A) and (B). A low magnification image of platelets is shown as an inset in (B). Silica overgrown on preformed PLL crystals, with steps presumably formed around a screw dislocation, is shown in the SEM image in (C). Image (A) reprinted with permission from ref 111 (Patwardhan, S. V.; Mukherjee, N.; Steinitz-Kannan, M.; Clarson, S. J. Chem. Commun. 2003, 1122). Copyright 2003 Royal Society of Chemistry. Images (B) and (C) reprinted with permission from ref 112 (Tomczak, M. M.; Glawe, D. D.; Drummy, L. F.; Lawrence, C. G.; Stone, M. O.; Perry, C. C.; Pochan, D. J.; Deming, T. J.; Naik, R. R. J. Am. Chem. Soc. 2005, 127, 12577). Copyright 2005 American Chemical Society.

Figure 13. Schematic representation of the proposed mechanism of silica hexagon formation through the synergistic self-assembly of PLL, phosphate ions, and silicic acid species. Reprinted with permission from ref 112 (Tomczak, M. M.; Glawe, D. D.; Drummy, L. F.; Lawrence, C. G.; Stone, M. O.; Perry, C. C.; Pochan, D. J.; Deming, T. J.; Naik, R. R. J. Am. Chem. Soc. 2005, 127, 12577). Copyright 2005 American Chemical Society.

distinctive hexagonal platelet morphology arose from a synergistic interaction between PLL, phosphate ions from the buffer, and silicic acid (Figure 13).111 When present together in solution, silicic acid and phosphate ions were observed to induce a random coil to R-helix transition in the secondary structure of PLL at neutral pH.111 Clarifying the earlier observations of Rodrı´quez et al., this conformational change was determined to be dependent on PLL chain length, where only peptides with >100 residues exhibited R-helical character.94,111 Tomczak and colleagues suggested that once formed, these PLL R-helices self-assemble into a hexagonal lattice.111 Neighboring PLL helices within this lattice were thought to be bound together through multiple interactions, including the condensation of their associated silicic acid species, resulting in hexagonal PLL/silica hybrid materials (Figure 13).111 Reinforcing their assertion that dynamically self-assembled R-helical PLL was the origin of

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Figure 14. (A) SEM images of individual siliceous hexagons formed through the self-assembly of the KP peptide and silicic acid. Stepped morphology is apparently due to the appearance of screwdislocations within the composite structures. SEM images of (B) randomly oriented and (C) magnetically aligned composite monoliths composed of silica/KP hexagons. Scale bars correspond to 10 µm in (A) and 20 µm in (B) and (C). Arrow in (C) indicates direction of applied magnetic field. Reprinted with permission from ref 114 (Bellomo, E. G.; Deming, T. J. J. Am. Chem. Soc. 2006, 128, 2276). Copyright 2006 American Chemical Society.

this unique silica morphology, Tomczak et al. also found that silica hexagons could also be produced by exposing preformed crystals of R-helical PLL to silicic acid (Figure 12C).111 Hybrid silica materials possessing a hexagonal platelet morphology have also been observed in a study examining the silicification of linear poly(ethelenimine) (PEI) crystals.112 Building on and supporting this first demonstration of cooperative self-assembly and biomimetic mineralization, Bellomo and Deming have utilized ethylene glycol-modified PLL (abbreviated as KP) to synthesize siliceous hexagons (Figure 14A).113 Much like the PLL-based system discussed above, the organization of KP into hexagonal lattices was determined to be dependent on chain length (>200 residues) and on the presence of silicic acid.111,113 While KP- and PLLbased systems share some characteristics, Bellomo and Deming report that KP may be utilized for biomimetic silica formation at much higher concentrations than its unmodified PLL counterpart.113 Utilizing highly concentrated KP solutions, Bellomo and Deming were able to biomimetically produce monoliths composed of a disordered mass of hexagons (Figure 14B).113 Composite bodies composed of well-oriented KP/silica hexagonal platelets were also produced by orienting the inherent dipole of the KP helical structure with a strong magnetic field during the silicification reaction (Figure 14C).113 Silica structures possessing a nanotube morphology have also been created through the synergistic interactions of peptides with condensing silica.114 The peptide utilized by Pouget and colleagues, Lanreotide (NH2-(D)Naph-Cys-Tyr(D)Trp-Lys-Val-Cys-Thr-CONH2), possessed only 8 residues, making it relatively small compared to the co-selfassembling PLL and KP systems discussed above.34 Similar to the peptide explored by Meegan et al. (see discussion in section 4.1.4), Lanreotide spontaneously assembled into β-sheets, segregating its hydrophobic and hydrophilic residues on opposite sides of its β-strands.105,114 Unlike the Meegan peptide, however, the end product of Lanreotide β-sheet assembly is not fibrils but nanotubes.105,114 These



Figure 15. TEM images of double-walled silica nanotubes produced through the dynamic assembly of Lanreotide and silicic acid species. Cross-sectional views are shown in (A) looking down a direction parallel to the nanotube length and (B) perpendicular to the nanotube length. Scale bars correspond to 30 and 50 nm. Reprinted with permission from ref 115 (Pouget, E.; Dujardin, E.; Cavalier, A.; Moreac, A.; Vale´ry, C.; Marchi-Artzner, V.; Weiss, T.; Renault, A.; Paternostre, M.; Artzner, F. Nat. Mater. 2007, 6, 434). Copyright 2007 Macmillan Publishers Ltd.

Lanreotide nanotubes were reported to be over 100 nm in length and possess diameters and wall thicknesses of 24.4 and 1.8 nm, respectively.34 Quite surprisingly, although these preformed nanotubes are cationic (i.e., Lanreotide charge is +2 at pH 7), they were not coated with silica upon exposure to an aqueous silicon alkoxide solution.114 The syntheses of double-walled nanotubes possessing silica/Lanreotide/silica wall architecture have, however, been reported by Pouget and colleagues (Figure 15).114 These seemingly conflicting observations were reconciled by Pouget and colleagues through the elucidation of a synergistic interaction between Lanreotide and the condensing silica phase.34 Pouget et al. proposed that the preformed Lanreotide nanotubes, present as a gel in their study, begin to disassociate into individual octapeptides upon the addition of a silica precursor solution.114 These authors suggested that anionic silicate species facilitate the reassembly of the free Lanreotides into nanotubes through the electrostatic interaction with the positively charged peptides.114 Silica polymerization was thought to occur simultaneously on the interior and exterior surfaces of the growing nanotube, producing the characteristic doublewalled morphology observed by Pouget and colleagues.114 The silica/Lanreotide nanotubes created through this proposed dynamic dissolution and co-self-assembly process were reported to be quite uniform in diameter (29 ( 2 nm) and typically 1-3 µm in length.114 In an impressive example of the “bottom up” biomimetic syntheses of hierarchically structured materials, Pouget et al. found that these individual double-walled siliceous nanotubes could self-organize into bundles exceeding 7 µm and into well-ordered fibers over 1 cm long.114

4.1.6. Summary of the Bioenabled Synthesis of Silica Over roughly a decade, studies centered on the biomimetic syntheses of silica have yielded important insights regarding the characteristics required for biomolecules and their close analogues to induce the formation of controlled silica structures under gentle reaction conditions in Vitro. The morphology of these biomimetically generated silica precipitates has proven to be malleable under the influences of both the reaction environment and the peptide/protein sequence. Research in biomimetic silicification has also yielded a synergistic self-assembly mechanism by which hierarchically organized siliceous structures are formed from the “bottom up” through the co-self-assembly of peptides with silica.111,113,114 As the archetype for the biomimetic syntheses of ceramic oxides, the studies reviewed above may

Figure 16. Silicatein-catalyzed synthesis of TiO2. (A) SEM micrograph of silicatein filaments as isolated from sponge spicules. (B) SEM micrograph of TiO2 encrusted silicatein filaments following exposure to a neutral pH aqueous solution containing TiBALDH. (C) TEM and electron diffraction (as inset) characterization of the titania coating composed of both amorphous and crystalline titania on a silicatein filament. Reprinted with permission from ref 52 (Sumerel, J. L.; Yang, W.; Kisailus, D.; Weaver, J. C.; Choi, J. H.; Morse, D. E. Chem. Mater. 2003, 15, 4804). Copyright 2003 American Chemical Society.

provide strategies for potential advances in the biomimetic production of other metal oxides.

4.2. Titanium Dioxide In a 2003 study, proteinaceous filaments isolated from the spicules of the sponge T. aurantia were utilized to synthesize titanium dioxide (titania) under gentle reaction conditions from a water-stable titanium complex.52 This work by Sumerel et al. provided the first demonstration that biomolecules implicated in biosilicification could be utilized to induce the formation a nonbiogenic inorganic oxide. In addition to being a nonbiological material, titanium dioxide is a technologically significant compound, finding use in a myriad of applications from photovoltaic cells to gas sensors.115-117 Titania may exist as a noncrystalline solid or as crystalline anatase, rutile, or brookite.115 The properties of TiO2 are dependent on the crystal structure of this inorganic compound; for example, anatase titania is often preferred for (photo)catalysis, while rutile titania possesses a high index of refraction and is often preferred for optical applications.115 In the work of Sumerel and colleagues, silicatein filaments were coated with titania (parts B and C of Figure 16) following incubation (24 h at 20 °C) in a neutral pH aqueous solution containing the Tiprecursor compound, titanium(IV) bis-(ammonium lactato)dihydroxide (TiBALDH).52 This TiO2 coating was found to be composed of nanocrystalline anatase near the silicatein filament and amorphous titania in regions away from the filament/inorganic interface. In keeping with prior in Vitro observations of the fold-dependent silica-precipitation activity of the silicateins, heat-denatured spicule filaments were unable to induce the formation of any titanium-containing materials.42,43,52 Though the mechanism by which silicatein filaments interacted with TiBALDH was not examined in this initial study, Sumerel and co-workers suggested that silicateins may be hydrolyzing the TiBALDH complex in a manner similar to that proposed for the interaction of the protein with silicon alkoxides.42,43,52


Following the initial study by Sumerel et al., titania has become one of the most extensively studied nonbiological inorganic materials to be synthesized by biomolecules.52,54,56,61,66,89,118,119 Following this initial in Vitro synthesis of titania with the aid of protein filaments isolated from sponge silica, the majority of the biomolecules investigated in this system have been derived from, or are analogues to, biomolecules believed to be associated with biosilicification.52,54,56,61,66,89,118,119 These molecules have included a recombinant Suberites domuncula sponge silicatein, the R5 peptide, recombinant silaffins derived fromdiatomgenes,PLL,PAA,andseveralSCPAs.54,56,61,66,89,118,119 The enzyme lysozyme and several peptides identified through the screening of a phage-displayed peptide library have also been explored for the biomimetic production of TiO2.66,89,90,120 While many of these biomolecules are capable of inducing the formation of titania, their relative effectiveness in doing so varies considerably.52,54,56,61,66,89,118,119 Many of the peptides identified to date through biopanning, along with a truncated R5 peptide, two recombinant silaffins, and two SCPAs, failed to yield any detectable Ti-bearing mineral precipitates under reaction conditions that proved productive for other biomolecules.54,89,118,119 As noted by Cole et al., the induction of titania precipitation in the presence of a titanium-containing precursor is not a general property of biopolymers.61,119 Hence, evaluation of the characteristics that endow biomolecules with TiO2-precipitation activity is of critical importance. While the mechanism(s) by which biomolecules induce the formation of TiO2 is not yet well-understood, the molecular characteristics required for such biomimetic titania synthesis have begun to be identified.52,54,56,61,66,89,118,119 Similar to several materials systems for which the sequence requirements for peptide-induced syntheses were initially unknown, significant insights into the characteristics required for titania synthesis have been gained through the screening of TiO2 with phage-displayed peptide libraries. Utilizing a constrained heptapeptide library, Chen et al. identified two anatase TiO2-binding peptides that were heavily enriched in basic amino acid residues.121 Consistent with these results, peptides identified through biopanning against rutile TiO2 substrates with a phage-displayed dodecapeptide library were noted to possess increased lysine, arginine, and histidine residue contents as compared to the unscreened peptide library.89 A number of peptides identified in this latter study were evaluated for titania-precipitation activity.89 Of the peptides tested, only those that possessed more than two net positive charges were observed to induce appreciable formation of TiO2 from a TiBALDH solution.89 Evaluating both biopanning-identified and subsequently designed peptides, Dickerson and colleagues also noted that the amount of TiO2 formed by a given peptide increased with the number of positive charges (i.e., Lys, Arg, or protonated His residues) carried by the peptide.89 This correlation between peptide positive charge and titaniaprecipitation activity was also observed in studies recently conducted by Sewell et al. and Kro¨ger et al.54,118 Sewell and Wright noted a decrease in precipitation activity of two truncated R5 peptides that possessed a +4 charge, relative to the +6 charged (full-length) R5 peptide.118 A third R5derived peptide that carried only 2 lysine residues (i.e., +2 charge) failed to induce appreciable TiO2 synthesis.118 The relative importance of protein charge is also apparent in the work of Kro¨ger et al., where only the most cationic recombinant silaffins, rSilC and rSil1L, were observed to initiate titania precipitation from TiBALDH.54 These authors

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Figure 17. Titania morphologies produced under the influence of biomolecules. (A) Spherical particles synthesized through the interaction of the R5 peptide with TiBALDH. Spherical particles were also reported to be formed in the presence of poly-L-lysine and poly(alyllamine). (B) An interconnected network of TiO2 particles produced with the aid of spermine. Fused particulate structures were also noted to be produced in the presence of rSil1L, spermidine, and peptides isolated by biopanning or designed from phage-displayed library isolated peptides. Scale bars correspond to 10 µm in (A) and 1 µm in (B). Reprinted with permission from refs 61 (Cole, K. E.; Ortiz, A. N.; Schoonen, M. A.; Valentine, A. M. Chem. Mater. 2006, 18, 4592) and 120 (Cole, K. E.; Valentine, A. M. Biomacromolecules 2007, 8, 1641). Copyright 2006 and 2007 American Chemical Society.

also observed that the lysine-enriched but only slightly positively charged (i.e., +3) protein rSil3 did not initiate titania precipitation.54 Other polypeptides successfully utilized to biomimetically produce TiO2, including HEWL, poly-L-lysine, and chimeric ferritin proteins that display 24 copies of the minTBP-1 peptide on the assembled apoferritin cage (see section 7.2), are also quite cationic.61,66,120 Such prior work strongly implies that the ability of a protein or peptide to hydrolyze the TiBALDH molecule is tied to the net positive charge carried by the biomolecule. While cationic character appears to be a dominant factor, research with R5 and R5-derived peptides also indicates that TiO2-precipitation activity may be modulated by peptide self-assembly.61,118 Cole and Valentine reported that SCPAs must contain at least 3 amines (i.e., 2 primary and 1 or more secondary amines) to initiate TiO2 precipitation, implying possible roles for Ti-nitrogen complexation or biomolecule length in titania synthesis.119 It should also be noted that, while a high positive charge density may be somewhat predictive of TiO2precipitation activity, proteins that possess an acidic pI, such as silicateins, may possess enzymatic activity capable of destabilizing the TiBALDH molecule.52,56 The titanium dioxide produced through the interaction of biomolecules with TiBALDH tends to be composed of spherical or interconnected networks of irregular particles that range in particle size from 50 nm to 10 µm (Figure 17).52,54,56,61,66,89,118,119,122 The crystalline or amorphous structure of biomimetically formed TiO2 has been biomolecule dependent.52,54,56,61,66,89,118,119,122 The titania produced by R5 and R5-derived peptides, poly-L-lysine, HEWL, and poly(alyllamine) has been reported to be amorphous by X-ray diffraction analyses.61,66,118 An amorphous phase was also present in the TiO2 precipitates synthesized in the presence of several designed and biopanning isolated peptides.89 However, much like silicatein titania, these materials also contained TiO2 nanocrystals (Figure 18).52,89 The X-ray diffraction pattern obtained from titania prepared at room temperature under the influence of the SCPA spermidine contained broad yet distinct peaks, consistent with the presence of nanocrystalline titania.119 A mixture of amorphous and nanocrystalline titania was also noted in the TiO2 produced from a TiBALDH by the recombinant silaffin rSil1L.54


Figure 18. High-resolution TEM image and electron diffraction pattern (inset) of a titania precipitate formed from a TiBALDH precursor in the presence of a designed peptide (dTi-1(H/R)) induced titania-precipitate cross section. This image reveals the mixed crystalline and amorphous TiO2 typically observed for titania prepared in the presence of peptides explored by Dickerson and colleagues. The lattice fringe spacings indicated by arrows were consistent with both the (101) plane spacing of anatase and the (110) plane spacing of monoclinic TiO2. The scale bar corresponds to 5 nm. Reprinted with permission from ref 89 (Dickerson, M. B.; Jones, S. E.; Cai, Y.; Ahmad, G.; Naik, R. R.; Kro¨ger, N.; Sandhage, K. H. Chem. Mater. 2008, 20, 1578). Copyright 2008 American Chemical Society.

Figure 19. SEM micrographs of hierarchically structured rutile TiO2 microspheres generated through the dehydration of rSilC titania. Arrow heads in (D) highlight elongated rectangular pores in the cross section of a rutile microsphere. Scale bars correspond to 20 µm in (A), 5 µm in (B), 1 µm in (C), and 500 nm in (D). Reprinted with permission from ref 54 (Kro¨ger, N.; Dickerson, M. B.; Ahmad, G.; Cai, Y.; Haluska, M. S.; Sandhage, K. H.; Poulsen, N.; Sheppard, V. C. Bioenabled Synthesis of Rutile (TiO2) at Ambient Temperature and Neutral pH. Angew. Chem., Int. Ed. 2006, 45, 7239-7243). Copyright 2006 Wiley-VCH Verlag GMBH & Co. KGA.

In one of the most dramatic demonstrations of a biomolecule’s ability to control the crystal structure of a nonbiogenic material, the TiO2 initially formed from TiBALDH in the presence of rSilC was observed to transform into highly crystalline rutile when exposed to dehydrating conditions (Figure 19).54 This newly formed titania consisted of microspheres composed of radially arranged columnar crystals of rutile (Figure 19C).54 The external surfaces of


these rutile microspheres were covered with rectangular pores.54 Attempts to replicate this result through the dehydration of rSil1L or poly-L-lysine TiO2 failed, indicating that such rutile-formation activity is specific to rSilC.54 While the mechanism by which rSilC guided the transformation of TiO2 into the rutile structure at room temperature is currently unclear, Kro¨ger et al. proposed that the surprising rutileforming activity of this protein may lie within its primary structure.54 The sequence of rSilC contains many lysine residues that may enable this silaffin to act as an acid-base catalyst in the condensation of TiO6 octahedra.54 Kro¨ger et al. theorize that the highly repetitive primary structure of rSilC may serve to guide the rearrangement of TiO6 octahedra into the rutile crystal lattice.54 In addition to the TiO2 produced through the interaction of biomolecules with TiBALDH, a few authors have investigated the use of Ti-alkoxide chemicals as precursors for the bioenabled synthesis of titania.123,124 In one example, Banerjee and colleagues exposed bolaamphiphile nanotubes functionalized with Ti metal-binding peptides to a titanium isoproproxide-containing solution.124 The interaction of these peptides with the titanium alkoxide resulted in the formation of anatase nanoparticles that coated the nanotubes.124

4.3. Germanium Oxide Given the successes attained in the biomimetic formation of silica, as well as the relative chemical similarity of silica and germania (GeO2), germanium oxide was an obvious early candidate for exploring the biomimetic formation of “synthetic” materials. While trace quantities of germania are known to be incorporated into the silica produced by diatoms and sponges, germania is not a major constituent of these biogenic sturctures.125 Indeed, elevated concentrations of germanic acid (i.e., the soluble form of germania) have been observed to poison diatoms and sponges (i.e., to induce cell death) and to produce gross distortions in the silica structures formed by these organisms.125,126 As proteins specifically evolved for the formation of germania appear to be absent in nature, the first foray into the bioinspired synthesis of this material was conducted with germanium-binding peptides identified from a phagedisplayed library.127 Of the identified peptides, a subset was selected and evaluated for germania-synthesis activity.127 Peptides that demonstrated enhanced germania-synthesis activity were found to possess sequence characteristics similar to biopanned peptides that exhibited high silicaprecipitation activity.86,127 Specifically, germania-precipitating peptides contained histidine and/or basic residues (i.e., lysine or arginine) and, to a lesser extent, one or more of the hydroxyl-bearing residues, serine, threonine, or tyrosine.127 Subsequent research with homo-poly(amino acids) supported the relative importance of histidine, basic, and hydroxyl-containing residues in germania formation.128 In contrast, homo-poly(amino acids) composed of acidic or hydrophobic residues were not observed to induce germania precipitation.128 The germania precipitates produced in the presence of peptides identified through biopanning and homo-poly(amino acids) were found to be amorphous by X-ray and electron diffraction analyses.60,127,128 Germania synthesized in the presence of these peptides was observed to be composed of an interconnected network of particles approximately 50 nm in diameter.60,127,128 Germania precipitates that possessed a spherical morphology have also been observed in a biomi-


Figure 20. Germania precipitate formed through the interaction of poly(allylamine) with a germanium alkoxide precursor in a buffered aqueous solution under stirred conditions. Arrows denote the elongated structures likely caused by the imposed hydrodynamic conditions. The highlighted area in (A) is presented at higher magnification in (B). Scale bars correspond to 5 µm in (A) and 3 µm in (B). Reprinted with permission from ref 60 (Patwardhan, S. V.; Clarson, S. J. Bioinspired Mineralisation: Macromolecule Mediated Synthesis of Amorphous Germania Structures. Polymer 2005, 46, 4474-4479). Copyright 2005 Elsevier.

metic utilizing PAA.60 In that work, Patwardhan et al. also observed that germania prepared in stirred solutions of PAA adopted an elongated structure (Figure 20), which indicates that biomimetically formed germania may exhibit morphological flexibility akin to that previously observed (see sections 4.1.3-4.1.5) for silica.60 Following these initial studies, germania produced with one of the library-identified peptides, Ge34 (TGHQSPGAYAAH), has found use as a host matrix for the growth of metal nanoparticles.129,130 The biomimetic room-temperature synthesis of infrared transparent germania-based amorphous materials may also find application in the future development of integrated optical lasers, sensors, display devices, and amplifiers.131-134

4.4. Gallium Oxide and Zinc Oxide The repertoire of oxide semiconductor materials synthesized at room temperature through the direct influence of biomolecules includes gallium oxide (Ga2O3), zinc oxide (ZnO), and cuprous oxide (Cu2O, discussed in section 7.3).91,135-137 As with titania, the biomimetic formation of gallium oxide was first explored with silicatein filaments isolated from the sponge T. aurantia.52,135 In this pioneering work, Kisailus and colleagues found that, under proper solution conditions, native silicatein filaments became coated with a dispersed coating of cubic γ-Ga2O3 nanocrystallites (75-200 nm in diameter) (Figure 21).135 The silicateininduced formation of these nanocrystals was proposed by the Kisailus et al. to occur through a multistep process.135 First, the silicateins contained within the isolated filaments were proposed to hydrolyze the precursor chemical, hydrated gallium nitrate, to produce an intermediate gallium oxohydroxide compound.135 This intermediate compound was thought to transform into crystalline γ-Ga2O3 via dissolution and reprecipitation on the silicatein surface or through a dehydration and condensation process commonly observed in Al-based minerals.135 While native silicatein filaments produced nanocrystalline gallium oxide, heat-denatured filaments proved ineffective at such materials synthesis.135 This result suggested that the gallium oxide-formation activity of the silicatein filaments was dependent upon protein structure or folding.42,52,135 In addition to their role in the synthesis

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Figure 21. SEM images of (A) native and (B) heat-denatured silicatein filaments following incubation in a gallium nitrate solution. The particles on the filament surface in (A) are composed of nanocrystalline Ga2O3. Such crystals did not appear on the heatdenatured filament shown in (B). Reproduced with permission from ref 53 (Kisailus, D.; Choi, J. H.; Weaver, J. C.; Yang, W.; Morse, D. E. Enzymatic Synthesis and Nanostructural Control of Gallium Oxide at Low Temperature. AdV. Mater. 2005, 17, 314-318). Copyright 2005 Wiley-VCH Verlag GMBH & Co. KGA.

of gallium oxide, silicatein filaments were also reported to impart crystallographic orientation to the developing nanocrystals.135 Given the interest in Ga2O3 for optoelectronic devices and the limited availability of silicatein, it would be regrettable if gallium oxide synthesis was an activity unique to these sponge-derived filaments. Fortunately, Lee and colleagues have found that monodisperse nanoparticles of gallium oxide could be produced, under relatively mild solution conditions, within the nanocavities of doughnut-shaped peptide assemblies.136 Echoing a mechanism proposed by Kisailus et al., Lee and colleagues suggested that Ga2O3 may form through the dehydration of a GaOOH intermediate product (Figure 22).136 Lee et al. further suggested that this loss of water from gallium oxo-hydroxide may be facilitated by the specific environment (i.e., confined space and chemical moieties) provided by the peptide nanorings.136 The gallium oxide generated through this process was observed to coalesce over time and fuse into crystalline particles with diameters of approximately 50 nm.136 Monoclinic β-Ga2O3 was synthesized in the presence of these peptide nanorings, whereas silicatein filaments generated the cubic (i.e., γ-Ga2O3) phase of gallium oxide, suggesting that bimolecular control over the precipitation process may be utilized for the selective formation of a particular gallium oxide polymorph.135,136 Zinc oxide is a widely studied material that has been used, or is under development for use, in a number of devices (e.g., solarcells,bluelight-emittingdiodes(LEDs),andnanolasers).138,139 The gentle reaction conditions inherent to biomimetic processing may allow for the direct synthesis and integration of ZnO with soft materials (e.g., organic semiconductors or dyes). To date, inroads into the biomimetic production of ZnO have been made exclusively through the screening of peptide libraries. Although a number of studies have identified peptides with a specific affinity for ZnO, there is currently only one report of such a peptide being utilized to synthesize zinc oxide.91,140,141 In this work by Umetsu and colleagues, flowerlike structures built from nanometer-sized crystallites of ZnO (Figure 23) were observed to form in the presence of a modified version of the library identified ZnO-1 peptide (EAHVMHKVAPRP).91 The sequence of the ZnO-1 peptide contained a glutamic acid and two histidine residues, amino acids that are well-known to coordinate with Zn2+ ions within the active sites of several enzymes.91,142 While the ZnO-1 peptide was found to bind ZnO, it was not until the peptide was modified with a C-terminal Gly-Gly-



Figure 22. Schematic of proposed Ga2O3 growth inside of peptide nanorings presented in (A). (B) TEM micrographs of monodisperse Ga2O3 synthesized within peptide nanorings after 4 weeks. Higher magnification image of Ga2O3/peptide composites as an inset. Scale bars correspond to 50 nm. Ga2O3 nanocrystals appear as dark black cores surrounded by a lighter ring of assembled peptide. Reprinted with permission from ref 136 (Lee, S. Y.; Gao, X.; Matsui, H. J. Am. Chem. Soc. 2007, 129, 2954). Copyright 2007 American Chemical Society.

Figure 23. (A) SEM image of ZnO assemblies synthesized under the influence of a GGGSC-tagged ZnO-specific peptide. (B) TEM image of peptide-synthesized ZnO nanoparticles that agglomerate to form the SEM-observable flowerlike morphologies seen in (A). Reprinted with permission from ref 91 (Umetsu, M.; Mizuta, M.; Tsumoto, K.; Ohara, S.; Takami, S.; Watanabe, H.; Kumagai, I.; Adschiri, T. Bioassisted Room-Temperature Immobilization and Mineralization of Zinc OxidesThe Structural Ordering of ZnO Nanoparticles into a Flower-Type Morphology. AdV. Mater. 2005, 17, 2571-2575). Copyright 2005 Wiley-VCH Verlag GMBH & Co. KGA.

Gly-Ser-Cys tag that ZnO synthesis activity was observed.91 Although cysteine is an extremely effective binder of zinc ions, the GGGSC sequence failed to produce ZnO when utilized alone or when appended to a non-ZnO specific oligopeptide.91 Umetsu and colleagues proposed that, in presence of the ZnO-1 peptide, cysteine may facilitate the dehydration of the Zn(OH)2 precursor to yield ZnO.91 However, the exact mechanism by which these additional five residues (GGGSC) initiate the synthesis of ZnO is currently unclear.91 Given the current and potential applications of oxide semiconductors, as well as these initial important demonstrations of the ability of biomolecules to synthesize these materials, the gallium oxide and zinc oxide systems provide fertile ground for additional biomimetic studies.

4.5. Mixed-Valence Oxides of Iron and Cobalt (Fe3O4 and Co3O4) Iron is essential to biology, and its presence in redox, transport, and storage proteins is pervasive.142 One family of iron storage proteins, the ferritins, have been used outside of their biological context to produce magnetic mixedvalence oxides of iron (Fe3O4) and cobalt (Co3O4), as well as other materials.98,143-145 While the ferritin-based production of Fe3O4 and Co3O4 is briefly discussed here, the application of apoferritin cages for the biomimetic synthesis

Figure 24. Schematic representation of the production of magnetic oxide nanoparticles within the confines of a ferritin cage. Adapted with permission from ref 143 (Klem, M. T.; Young, M.; Douglas, T. Biomimetic Magnetic Nanoparticles. Mater. Today 2005, 8, 28-37). Copyright 2005 Elsevier.

of these and other materials have been described more extensively in several book chapters and reviews.98,143-145 Ferritins are a ubiquitous family of proteins that are assembled from R-helical protein subunits into a hollow, cagelike structure.98,143-145 The interior cavity of this structure serves as the site of iron storage in ViVo and is of primary importance to the utility of the protein for biomimetic materials fabrication.98,143-145 In the absence of the iron-containing material natively stored in the core of ferritin, the empty protein (apoferritin) may be utilized as a sizeconstrained reaction environment for nanoparticle synthesis. In a typical reaction, apoferritin is incubated and filled with Fe or Co ions, which are then partially oxidized through the addition of H2O2 at a slightly elevated temperature (i.e., e65 °C) to yield Fe3O4 or Co3O4 (Figure 24).98,143,144 Reaction solution conditions including buffer chemistry and pH have been found to exert strong influences over the success and yield of Co3O4 synthesized within apoferritin cages.146 The sizes of the Fe3O4 and Co3O4 nanoparticles formed within the assembled apoferritin complex have been observed to possess diameters similar to that of the apoferritin interior cavity (e.g., ∼7 nm for horse spleen apoferritin) and to be quite monodisperse due to the confined space within this cavity.98,143-146 While the internal cavity of apoferritin is of great utility in forming nanoparticles of well-controlled size, the protein’s chemistry is also equally critical to Fe3O4 and Co3O4 nanoparticle synthesis.98,143-146 The internal wall of the assembled apoferritin cage possesses clusters of acidic residues (i.e., Glu and Asp) that are known to bind ferrous


ions and are believed to serve as the initial nucleation sites for the growth of iron and cobalt oxides.98,143-146 The magnetic properties of the resulting Fe3O4/ferritin and Co3O4/ ferritin nanocomposites have been extensively characterized and explored for a number of potential applications by Klem and colleagues.143 A number of research groups have also taken advantage of the constrained reaction environments of apoferritin and apoferritin-like proteins to produce iron or cobalt oxide nanoparticles with narrow size ranges that, in turn, can be used to catalyze the growth of carbon nanotubes with very narrow diameter distributions.147-152 The success found in manipulating apoferritin for the production of magnetite is not entirely surprising, as a number of organisms have utilized confined reaction environments and specialized proteins to synthesize magnetic nanocrystals for millions of years.2,3,5,6,153 Indeed, magnetic particles are found widely throughout biology and have been used by organisms as diverse as algae, fish, pigeons, and magnetotactic bacteria for navigation.2,3,6 It is this latter group of organisms, magnetotactic bacteria, that have increasingly gained attention from biologists, materials scientists, and chemists as model systems for biomineralization and biomimetic studies.2,3,5,6,154-156 Magnetotactic bacteria are a diverse group of microorganisms that can align andmoveinresponsetomagneticfields(i.e.,magnetotaxis).2,3,5,6Currently, magnetotaxis is thought to assist these bacteria in their search for and migration to areas of optimal oxygen concentration within their aquatic habitat (i.e., magneto-aerotaxis).2,3,5,6 The orientation of magnetotactic bacteria in an external field is facilitated by a specialized organelle known as the magnetosome.2,3,5,6 The magnetosome consists of magnetic nanocrystals (i.e., typically magnetite or, less frequently, iron sulfides) enveloped by a membrane that contains specialized proteins.5,6,146,153 The nanocrystals are typically organized in single or multiple chains within the cell.5,6,146,153 Magnetosome organization and magnetic nanoparticle size and morphology have been observed to be species- or strainspecific, which suggests that magnetosome formation is under genetic control.2,3,5,6 While aspects of the molecular biology of the magnetosome and magnetotactic bacteria are currently under study and not yet fully understood, a protein identified from one of these bacteria has been utilized in the biomimetic synthesis of magnetite.154-156 In a pioneering study, Arakaki and colleagues identified a number of proteins that were tightly associated with the magnetite nanoparticles created by the magnetotactic bacteria Magnetospirillum magneticum strain AMB-1.154 These proteins, which could only be removed from AMB-1 magnetite by boiling in a sodium dodecyl sulfate (SDS) solution, possessed a common N-terminal region thought to be a transmembrane (i.e., magnetosome membrane) domain.154 The full sequences of these proteins were deduced from the genome of AMB-1, and several were found to be encoded by genes within the magnetosome island, which is a large genomic region associated with magnetosome formation.154 The C-terminal region of the proteins encoded by the magnetosome island were found to be enriched in acidic residues.154 Arakaki et al. selected one of these acidic proteins, Mms6, to explore for in Vitro magnetite formation.154 Recombinant Mms6 was found to bind iron ions from solution and to guide the formation of magnetite in Vitro.154,155 Indeed, the magnetite nanoparticles produced in the presence of this recombinant protein were similar in size and

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Figure 25. TEM images of (A) magnetic nanocrystals extracted from M. magneticum AMB-1 and magnetite grown in the presence (B) or absence (C) of the AMB-1 derived protein Mms6. Reprinted with permission from ref 156 (Amemiya, Y.; Arakaki, A.; Staniland, S. S.; Tanaka, T.; Matsunaga, T. Controlled Formation of Magnetite Crystal by Partial Oxidation of Ferrous Hydroxide in the Presence of Recombinant Magnetotactic Bacterial Protein Mms6. Biomaterials 2007, 28, 5381-5389). Copyright 2007 Elsevier.

morphology to those natively produced by AMB-1 (Figure 25).154,155 Conversely, magnetite particles formed under similar reaction conditions, but in the absence of Mms6, exhibited less uniform morphologies or size (Figure 25C).154,155 Although the exact mechanism by which this protein controls Fe3O4 nanoparticle synthesis is currently unclear, Arakaki and colleagues proposed that Mms6 may serve as a template to guide the shape and size of the magnetite crystals formed.154 Prozorov et al. reported that the in Vitro production of AMB-1-like magnetite is specific to Mms6, as several proteins with known iron-binding activity (i.e., apoferritin, lipocalin, and BSA) failed to generate Fe3O4 of this morphology.156 In an approach that may find utility in the biomimetic synthesis of other materials, Prozorov and colleagues also performed their reactions in viscous media (i.e., in a polymeric aqueous gel), which may better mimic the internal environment of the magnetotactic bacteria.156 The ferrous ion binding residues (i.e., Glu and Asp) native to ferritin and Mms6 may also be utilized outside of the confines of these proteins to generate magnetic oxide materials.157 Nam and colleagues modified the major coat protein (pVIII) of the M13 phage to display a tetraglutamate peptide (Glu-Glu-Glu-Glu).157 The modified M13 phage displayed ∼2700 copies of this acidic peptide, providing ample ligands for the binding of Co2+ ions.157 Uniform Co3O4 nanocrystals (Figure 26) were reported to be formed along the length of the phage through a reaction scheme involving reduction by NaBH4 and spontaneous aqueous oxidation.157

4.6. Multicomponent Oxides: BaTiO3, BaTiOF4, CaMoO4, and (Fe,Co)3O4 Multicomponent oxides are of widespread technological interest due to their magnetic, catalytic, electrical, chemical, and optical properties.158 Only very recently has the use of biomolecules been extended to direct the room-temperature



Figure 26. TEM image of Co3O4 nanocrystals grown on the surface of an M13 phage displaying a short tetraglutamate peptide on its pVIII capsid protein. Reprinted with permission from ref 157 (Nam, K. T.; Kim, D.W.; Yoo, P. J.; Chiang, C.Y.; Meethong, N.; Hammond, P. T.; Chiang, Y.-M.; Belcher, A. M. Science 2006, 312, 885-888). Copyright 2006 AAAS (www.sciencemag.org).

synthesisofthisimportantclassoffunctionalceramics.54,93,159,160 To date, the binary oxide compounds barium titanate (BaTiO3), calcium molybdate (CaMoO4), and cobalt-doped magnetite (Co,Fe)3O4, which are commonly processed at high temperature or under extreme conditions (i.e., elevated pressures, temperatures, and/or caustic pH), have been synthesized with the aid of biomolecules.54,93,158-160 The first of these compounds, BaTiO3, was initially produced by Nuraje and colleagues within the confines of peptide nanorings.159 The formation of BaTiO3 in this study was described as a synergistic process, in which the precursor chemical (i.e., a BaTi-alkoxide) facilitated the assembly of the peptides into nanorings that acted to hydrolyze the precursor.159 This simultaneous aggregation and hydrolysis yielded highly monodisperse peptide/inorganic composite particles containing a core of BaTiO3 between 6 and 12 nm in diameter (i.e., core size was observed to be dependent on reaction solution pH) surrounded by peptide (Figure 27A).159 Nuraje et al. reported that these barium titanate nanoparticles possessed tetragonal character and were accordingly observed to exhibit ferroelectric behavior.159 Expanding on this initial work by Nuraje et al., Ahmad and colleagues have also recently identified a peptide that was capable of initiating and directing the synthesis of tetragonal, ferroelectric barium titanate.93 This peptide, BT2, was isolated through the screening of BaTiO3 with a phage-displayed library.93 A second peptide identified through the biopanning process, BT-1, also yielded BaTiO3, although the degree of tetragonality of these precipitates was less than that formed in the presence of BT-2.93 It is interesting to note that both the BT-2 peptide and the bolaamphiphile peptide utilized by Nuraje and colleagues possessed two carboxylic acid moieties; however, the possible significance of this similarity and its relevance to BaTiO3 formation remains unknown at this time.93,159 The BaTiO3 produced by BT-2 was morphologically distinct from that originating from within bolaamphiphile peptide nanorings (parts A and B of Figure 27). The BT-2 BaTiO3 precipitates were larger in size (typically 50-100 nm) and exhibited a faceted, rather than spherical, morphology (Figure 27B).93 Brutchey and colleagues have found that silicatein filaments are capable of templating BaTiOF4, a compound chemically related to BaTiO3.161 This barium titanium oxyfluoride compound was observed to form on the surface of T. aurantia-isolated silicatein filaments as dispersed florets of nanocrystals (Figure 27C).161 Unlike previous silicateincatalyzed reactions involving the formation of silica, titania, or gallium oxide, the synthesis of BaTiOF4 required a cofactor, H3BO3, which was proposed to scavenge excess F- ions generated by the hydrolysis of the precursor salt

Figure 27. TEM micrographs of tetragonal BaTiO3 produced (A) within peptide nanorings and (B) by a peptide identified through biopanning. In (A) BaTiO3 is seen as a dark center surrounded by a peptide doughnut, which is less electron dense (dark gray). (C) TEM image and associated electron diffraction pattern (inset) of BaTiOF4 produced at room temperature by silicatein filaments and a F- scavenging cofactor. Image (A) reprinted with permission from ref 159 (Nuraje, N.; Su, K.; Haboosheh, A.; Samson, J.; Manning, E. P.; Yang, N.; Matsui, H. Room Temperature Synthesis of Ferroelectric Barium Titanate Nanoparticles Using Peptide Nanorings as Templates. AdV. Mater. 2006, 18, 807-811). Copyright 2006 Wiley-VCH Verlag GMBH & Co. KGA. Images (B) and (C) reprinted with permission from refs 93 (Ahmad, G.; Dickerson, M. B.; Cai, Y.; Jones, S. E.; Ernst, E. M.; Vernon, J. P.; Haluska, M. S.; Fang, Y.; Wang, J.; Subrarnanyarn, G.; Naik, R. R.; Sandhage, K. H. J. Am. Chem. Soc. 2008, 130, 4) and 161 (Brutchey, R. L.; Yoo, E. S.; Morse, D. E. J. Am. Chem. Soc. 2006, 128, 10288). Copyright 2008 and 2006 American Chemical Society.

BaTiF6.161 Barium titanium oxyfluoride production was not observed by these researchers under conditions in which the silicatein filament was absent or denatured, indicating that the presence of properly folded silicatein was necessary for the room-temperature synthesis of BaTiOF4.161 Peptides identified through the screening of phagedisplayed libraries have also been utilized to synthesize phase-pure calcium molybdate, CaMoO4.54 Calcium molybdate-based materials have been used and may potentially find new application in a number of roles, such as phosphors in light-emitting diodes (LEDs).162-164 Ahmad and colleagues reported that the CaMoO4 precipitation activity of the biopanning identified peptides, CM-3 and CM-4, was affected by serine content, amino acid residue position, and peptide acidity/alkalinity.54 Calcium molybdate formed in the presence of the identified peptides was found to be crystalline (powellite structure), composed of large spherical aggregates containing submicron platelets (Figure 28), and to exhibit green photoluminescence.54 It should be noted that, although the study by Ahmad and colleagues marks the first use of a peptide identified by the biopanning process to produce a multicomponent oxide, compounds similar to CaMoO4 have also been biomimetically synthesized with the aid of block copolymers.17 The most recent addition to the list of biomolecule-induced multicomponent oxides is cobalt-doped magnetite.160 As


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Figure 28. SEM micrographs of CaMoO4 synthesized by the CM3 peptide. (A) Low-magnification overview of precipitate morphology and (B) higher-magnification detail of microsphere surfaces. Previously unpublished data by Ahmad, G.; Dickerson, M. B.; and Sandhage, K. H.

reviewed in section 4.5, apoferritin cages have been utilized to produce both Co3O4 and Fe3O4; a natural extension of this work is to utilize apoferritin to synthesize iron/cobalt mixed-oxides. Highlighting the merit in pursuing the biomimetic synthesis of multicomponent ceramics, Klem and colleagues reported that CoxFe3-xO4/ferritin nanoparticles possessed enhanced blocking temperatures and magnetic anisotropy relative to Co3O4 or Fe3O4, which can make CoxFe3-xO4 moreattractiveformagneticstorageapplications.160,165 Klem et al. also reported that nanocomposites composed of distinct phases of antiferromagnetic Co3O4 and ferromagnetic Fe3O4 could be formed within the confines of the apoferritin cage through the manipulation of reaction conditions.160 Given that the first demonstration of the biomolecule-directed synthesis of multicomponent oxides appeared only within the last 2 years, and considering the widespread use of these materials in current and emerging technologies, the most remarkable advances in this field are likely to be seen.

5. Protein- and Peptide-Directed Syntheses of Non-Oxide Semiconductors 5.1. Introduction Quantum dots (QDs) are nanocrystallites of particular semiconducting materials that possess unique electrical and optical properties that differ from those of their bulk counterparts.166 The unique properties of QDs arise due to their small sizes and associated quantum confinement effects.166 Quantum dots are extremely bright photoluminescent materials that do not photobleach and possess broad absorption yet narrow emission bands.167,168 Quantum dot energy levels, and thus luminescence energies, are tunable and can be adjusted by varying QD size, chemistry, or applying coatings to the QDs.167,169 These characteristics make QDs ideal for a number of applications including light-emitting diodes (LEDs), fluorescent labels for biological research and detection,solarcellapplications,anduseaslasercomponents.167,168,170 Solution-based synthesis approaches are currently the preferred means of producing QDs.167,171 Typical reactions involve the heating of precursors at temperatures exceeding 200 °C in an organic solvent in the presence of a surfactant, such as trioctylphosphine oxide (TOPO).167,171 Synthesis techniques have been evolved to produce semiconductor nanocrystallites with narrow size distributions ((9%), controlled chemistries, and unique shapes that extend beyond the spherical “dot” morphology to include rods, disks, and even dendrititic structures.167,171,172 While QD synthesis and applications appear to be a product of modern times, recent research suggests that our production and use of QDs reaches back to antiquity.173 Exploring a hair-dyeing recipe described in Greco-Roman

Figure 29. TEM micrograph of PbS nanocrystals formed with the aid of keratin via reaction with an ancient Pb-based hair dye. PbS nanocrystals appear as dark spots that are linearly organized by the supramolecular structure native to hair. Inset is a highermagnification image of one of these PbS nanocrystals. Reprinted with permission from ref 173 (Walter, P.; Welcomme, E.; Halle´got, P.; Zaluzec, N. J.; Deeb, C.; Castaing, J.; Veyssie`re, P.; Breńiaux, R.; Le´veˆque, J. L.; Tsoucaris, G. Nano Lett. 2006, 6, 2215). Copyright 2006 American Chemical Society.

period texts over 2000 years ago, Walter and colleagues found that the application of a paste of lead oxide and Ca(OH)2 to human hair resulted in the formation of lead sulfide nanocrystals.173 The Ca(OH)2 included in this recipe creates a highly caustic solution (i.e., pH ) 12.5) that degrades the cysteine residues of the keratin proteins found in hair.173 The sulfur released form the degraded cysteine residues then reacted with lead ions available from the lead oxide component of the dye, yielding PbS.173 In addition to providing half of the required nanoparticle chemistry, the keratins possess a supramolecular organization that directed the arrangement of the growing PbS crystallites, which were observed to decorate specific areas of the treated hairs (Figure 29).173 The PbS nanoparticles formed through this proteinenabled process serve to darken hair by substituting for the melanin clusters that typically fill this role.173 This combined action of PbS synthesis and templated deposition likely makes this technique one of the earliest examples of the use of proteins in the synthesis of nanoparticles. It is important to note that, while this lead-based hair-darkening treatment accomplishes its desired aesthetic effect, lead-based cosmetics such as kohl, an eye makeup used in developing nations, have been identified as health hazards.174

5.2. Biogenic and Biologically-Derived Production of Semiconductor Nanoparticles Plants and microorganisms such as yeast may also be exposed to elevated levels of toxic heavy metal ions such as Pb2+ and Cd2+ in their environments.175 The method these organisms have evolved to contend with these detrimental ions has resulted in the biogenic production of QDs.175 As recently reviewed by Carney and colleagues, the binding of Cd2+ by cysteine-containing peptides, called phytochelatins, represents the first step in the detoxification of these ions in plants and yeast.175 The phytochelatins (PCs) possess the general structure (γ-Glu-Cys)n-Gly (n ) 2-11) that contains at least two cysteine residues that are capable of coordinating Cd2+ through their thiol moieties.175 These PC-bound cadmium ions are transported to vesicles where they combine with physiologically produced sulfide ions to produce CdS nanocrystals.175 Phytochelatins adsorb to the surfaces of the growing CdS QDs, inhibiting the influx of ions and arresting


the growth of the particles so that they do not become so large as to rupture the vesicle.175 PC capping results in the production of fairly monodisperse QDs; for example, CdS nanoparticles recovered from the yeast Candida glabrata were noted to possess diameters of 2.0 ( 0.3 nm.176 The detoxification process ends with the exportation of the PCcapped CdS particles out of the cell.175 While microorganisms such as yeast can be exploited to produce QDs in ViVo, their phytochelatin peptides may be utilized to synthesize semiconductor nanocrystallites in Vitro.176,177 In research conducted by Bae and Mehra, phytochelatins extracted from yeast, as well as glutathione, γ-Glu-Cys-Gly (a peptide known to bind Cd2+ in mice, abbreviated GSH), were found to be effective in limiting the size of solution-grown CdS particles.177,178 The CdS particles produced in the presence of PCs were reported to be limited to 2.4 nm in diameter with high monodispersity, whereas glutathione-capped CdS crystallites were found to be more polydisperse, ranging in diameter from 2.2 to 3.5 nm.177 Working with a small library of peptides, Spoerke and Voigt observed that the size of CdS nanoparticles was dependent upon peptide composition and structure.179 The presence of cysteine residues was found to be of paramount importance.179 Peptides that lacked cysteine failed to control the growth of CdS, producing large (5.9 nm average diameter), nonfluorescent particles similar to those produced in the absence of peptides.179 Cysteine-containing peptides were observed to produce CdS nanocrystallites with sizes smaller than CdS grown in free solution.179 Additionally, peptides containing two or more cysteines were found to be more effective in capping CdS growth than those with a single cysteine.179 When peptides containing a single cysteine disengage from the nanoparticle surface, they may diffuse away, compromising the peptide cap and creating a route for ion influx and crystal growth.179 As discussed by Spoerke and Voigt, the bonding of cysteine residues to CdS is dynamic and peptides displaying multiple cysteine residues may remain anchored to a CdS nanoparticle even when one of these residues disassociates from the surface.179 This mechanism may explain the effectiveness of phytochelatins, which possess two or more cysteines, in controlling CdS particle size. It is interesting to note that, in addition to cysteine, Spoerke and Voigt found that another component of native PCs, aspartic acid residues, played a role in controlling the size and dispersion of CdS nanoparticles synthesized with their engineered peptides.179 QDs fabricated with neutral peptides were observed to quickly (within 30 min) precipitate from solution, while CdS particles capped with negatively charged peptides remained dispersed even after centrifugation and were smaller and possessed tighter size distributions than their uncharged counterparts.179 One of the small peptide dendrimers designed by Spoerke and Voigt, which contained 2 aspartic acid and 2 cysteine residues, was highly effective at controlling CdS growth, producing QDs with size homogeneity (2.6 ( 0.21 nm) surpassingthatachievedinmodernsynthetictechniques.171,172,179 Spreitzer and colleagues have also reported that the sequence of phytochelatins plays an important role in determining the size of CdS nanocrystals produced in biomimetic synthesis processes.180 Glutathione plays many roles in ViVo, including Cd2+ detoxification.175,178 The applications of this tripeptide have been extended in Vitro to include the capping of semiconduc-


tor nanocrystals with noncadmium-based chemistries.181,182 Torres-Martıńez et al. and Brelle et al. have used glutathione to influence the formation of zinc and silver sulfide nanoparticles (ZnS and Ag2S, respectively).181,182 While glutathione is attractive as a relatively inexpensive peptide, this biomolecule failed to stringently control the sizes of ZnS and Ag2S nanoparticles synthesized in its presence; that is, these particles possessed sizes of 3.45 ( 0.5 nm and ca. 9 ( 2.25 nm, respectively.181,182 The monodispersity of these glutathione-produced QDs was improved, however, through theuseofpostsynthesissize-selectiveprecipitationprocedures.181,182

5.3. Syntheses of Sulfide Semiconductors with Zinc Fingerlike Peptides and Nanotubes As detailed above, Cd2+ binding and the capping of growing nanocrystals are activities linked specifically to the cysteine residues of glutathione and phytochelatin peptides.175 Indeed, the ability of cysteines to bind divalent ions has been widely observed in the structural motifs of proteins, one of the most common of which is the zinc finger.183 Banerjee and colleagues have explored the influence of the M1 peptide, which contains the zinc fingerlike Cys2His2 structure, on the growth of ZnS nanocrystals.184,185 Under moderate pH conditions (pH 5.5-7.4), M1 peptides adsorbed onto the surface of bolaamphiphile nanotubes were found to direct the formation of monodisperse, wurtzite-type ZnS nanocrystals with average diameter of 4 nm.185 Increasing the pH of the reaction solution into the basic regime (pH > 8), however, resulted in the formation of amorphous ZnS on the M1 coated nanotubes.185 Banerjee and colleagues attribute this transition in inorganic structure to a pH-induced structural transition in the M1 peptide.184,185 Banjeree et al. proposed that the unfolding of M1 with increasing pH favored singly coordinated Zn2+, whereas bidentate chelation was preferred with the more restrictive R-helical structure adopted by the peptide at lower pH.185 These authors reported that, while M1 functionalized nanotubes were effective in influencing the growth of ZnS, unbound M1 peptides or neat bolaamphiphile nanotubes were incapable of producing monodisperse QDs.185 Banerjee and colleagues have also utilized peptides capable of histidine-mediated chelation to explore the biomimetic synthesis of Cu2S nanoparticles.184,186 In this study, Banerjee and colleagues used the HG12 peptide, which contains four histidines and eight highly flexible glycine residues.184,186 The HG12 peptide was adsorbed onto preformed nanotubes prior to Cu2S mineralization trials.186 The average size of the synthesized nanoparticles was reported to be dependent on the pH of the reaction solution (Figure 30), although the synthesized Cu2S nanoparticles possessed crystal structure independent of pH.186 Banjeree et al. propose that HG12 structure changes as a function of pH, which, in turn, influences inorganic nanocrystal size.186 Cu2S nanoparticles synthesized in the presence of free HG12 peptide were noted to be more polydisperse than those produced by nanotubeadsorbed HG12.186 Slocik and colleagues have also utilized a histidine-rich peptide (i.e., HRE) derived from the histidine-rich protein II (HRP II) of the malarial parasite Plasmodium falciparum to direct the synthesis of semiconducting sulfide nanocrystals.123 The HRE peptide was utilized to cap ZnS and Ag2S nanoparticles to limit growth so as to generate spherical particles with average diameters of 3.1 and 11.3 nm, respectively.123


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Figure 30. (A) Schematic representation of the proposed modification of nanotube-conjugated peptide structure and Cu2S particle size with changes in pH. TEM images of peptide-templated Cu2S nanoparticles formed at (B) pH 5 and (C) pH 7 decorating the surface of bionanotubes. Electron diffraction patterns corresponding to hexagonal Cu2S are shown as insets in (B) and (C). Scale bars correspond to 70 nm. Reprinted with permission from ref 124 (Banerjee, I. A.; Muniz, G.; Lee, S. Y.; Matsui, H. J. Nanosci. Nanotechnol. 2007, 7, 2287). Copyright 2007 American Scientific Publishers.

5.4. Syntheses of Sulfide Semiconductors with Amphiphilic Peptides The work of Banjeree and colleagues demonstrated that properly functionalized bolaamphiphile nanotubes (e.g., nanotubes conjugated with His and/or Cys-containing peptides) may serve as effective scaffolds for the nucleation and growth of semiconductor nanoparticles.184,185 Nanotubes may also be functionalized, through the incorporation of ionbinding chemistries directly into their molecular building blocks. This second strategy has proven successful in producing nanotubes and nanowires decorated with CdS nanocrystals.187-189 Relatively simple peptide amphiphile building blocks that have recently been examined for CdS synthesis are described as peptide lipids, possessing a 14carbon myristic acid-based tail and a Gly-Gly-COOheadgroup.187 The surfaces of the lipid bilayer walls that comprise the assembled nanotubes display a high density of carboxylic acid moieties that effectively bind Cd2+ ions from solution.187 These bound cadmium ions are proposed to serve as reactants and nucleation sites for the growth of CdS upon the introduction of sulfide ions into the system.187 The CdS nanoparticles synthesized in this work by Zhou and colleagues were observed to possess average diameters of 4-5 nm and were evenly distributed throughout the peptide lipid nanotubes.187 Sone et al. and Rabatic et al. investigated the synthesis of CdS, utilizing peptide amphiphiles displaying S(P)RGD-COO- and KKK-COO- sequences, where S(P) was phosphoserine.188,189 The sequences of these two peptide amphiphiles provided charged moieties that may interact and bind cadmium ions from solution.188,189 The addition of H2S gas to the Cd2+ charged peptide amphiphile nanowires resulted in the generation of zinc blende structured CdS nanocrystals that coated the nanowires (Figure 31).188,189

5.5. Apoferritin-Templated Semiconductor Nanoparticles The properties and applications of quantum dots are dependent upon the size, size distribution, and in-solution stability of these semiconductor nanocrystals.167,171 Given these requirements, the aqueous dispersion stability and constrained internal reaction cavity of apoferritins make these

Figure 31. TEM image of self-assembled nanowires of peptide amphiphiles decorated with CdS nanocrystals. An enlargement of a section of the CdS coated nanowire and an associated electron diffraction pattern matching the zinc blende polymorph of CdS are presented as insets. The scale bar corresponds to 100 nm. Reprinted with permission from ref 188 (Sone, E. D.; Stupp, S. I. J. Am. Chem. Soc. 2004, 126, 12756). Copyright 2004 American Chemical Society.

proteins excellent candidates for biotemplated quantum dot syntheses. The use of an apoferritin to produce CdS nanoparticles was first reported in 1996 by Wong and Mann.190 This pioneering study demonstrated that apoferritin is capable of binding large amounts of Cd2+, that CdS synthesis occurs exclusively within the inner cavity of the protein and not on its outer surface, and that ferritin-CdS nanocomposites were stable in aqueous solution for extended periods of time.190 However, the apoferritin-generated CdS nanoparticles formed in this early study were irregularly shaped and possessed broad size distributions.190 Expanding on the initial work of Wong and Mann, Iwahori et al. and Yamashita et al. have successfully exploited the unique architecture of mammalian and bacterial apoferritins to synthesize well-controlled CdS, CdSe, and ZnSe quantum dots.191-194 Utilizing recombinant apoferritins with specific mutations, these authors deduced that the negatively charged residues lining the pore channels and interior cavity of the protein acted to locally concentrate Cd2+ or Zn2+ ions that could then react with S2- or Se2- ions to generate nuclei for semiconductor growth.194 The relative importance of the residues lining the interior of the apoferritin cavity was also well-supported by the observations of Wong and Mann, who found that CdS growth was inhibited when the protein



Figure 33. (A) UV-vis absorption, (B) photoluminescence emission curve, and (C) TEM image of Ag2S nanorods produced under the influence of BSA. Reprinted with permission from ref 63 (Yang, L.; Xing, R.; Shen, Q.; Jiang, K.; Ye, F.; Wang, J.; Ren, Q. J. Phys. Chem. B 2006, 110, 10534). Copyright 2006 American Chemical Society.

Figure 32. Series of TEM images of ZnSe synthesized within apoferritin cages utilizing a slow chemical reaction process. Apoferritin cages are stained in (A) and appear as bright regions in the image. Arrowheads call attention to the few apoferritin shells not filled by ZnSe. (B) Unstained apoferritin-ZnSe showing ZnSe cores; the protein shell is not visible. A higher-magnification image of one of these ZnSe nanoparticles appears in (C). Reprinted with permission from ref 194 (Iwahori, K.; Yashizawa, M.; Yamashita, I. Inorg. Chem. 2005, 44, 6393). Copyright 2005 American Chemical Society.

interior was blocked with its native iron-containing core.190 Enhanced use of the unique composition and structure of the apoferritin proteins for QD synthesis was realized through the development of slow chemical reaction systems by Iwahori et al. and Yamashita et al.191-194 The slow chemical reaction system concept, outlined nicely in the 2005 study of Iwahori et al., employs ammonium-coordinated Cd2+ or Zn2+ and a reactant that slowly degrades to release sulfide or selenide ions, such as selenourea.194 Iwahori and colleagues proposed that the critical function of ammonium was to block the interaction of anionic reactants with cadmium or zinc outside of the apoferritin cavity, whereas Cd2+ or Zn2+ conjugated to the interior wall of the apoferritin cavity are available for reaction.194 By utilizing the slow chemical reaction approach, horse spleen apoferritin templated CdSe and ZnSe polycrystalline nanoparticles with uniform 7 nm diameters and recombinant Dps (a ferritin-like protein) templated luminescent CdS QDs with a 4.2 nm average diameter have been synthesized (Figure 32).191-194

5.6. Semiconductor Nanoparticles from Transport Proteins and Enzymes Paralleling the approach taken for apoferritin, other proteins with no known ability to influence semiconductor material growth in ViVo, including bovine serum albumin (BSA) and pepsin, have recently been explored in this role.63,68,195 Yang et al. observed that monoclinic R-Ag2S nanorods 40 ( 2.5 nm in diameter and 220 ( 35 nm in length could be formed in the presence of BSA (Figure 33).63 While the Ag2S/BSA nanorods were observed to possess a wide UV-vis absorption spectrum, these nanorods exhibited a narrow photoluminescent emission band centered at 474

nm (Figure 33).63 In contrast, nanorods of Ag2S were not synthesized in control experiments conducted in the absence of BSA, but instead resulted in the formation of aggregated elliptical particles.63 Yang and colleagues proposed that BSA-chelated Ag+ ions served as nuclei for the growth of Ag2S nanorods and that the protein coated the mature inorganic structures.63 Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy indicated that structural transitions in BSA occurred with the binding of Ag+ and the formation of Ag2S, which further supported the notion that BSA was involved in nanorod synthesis.63 Brelle and colleagues report that BSA may also be used to limit CuS and Cu2S growth, so as to yield materials suitable for electron spin resonance measurements.195 The commercially available enzyme pepsin has been studied by Yang and colleagues for its ability to nucleate and control the growth of CdS.68 These authors selected pepsin for the abundance of aspartic acid residues located in the active site of this protein.68 CdS formed in the presence of pepsin possessed the wurtzite structure, exhibited a sharp photoluminescence emission band centered at ∼450 nm, possessed an average diameter of ∼6.3 nm with a narrow size distribution, and exhibited an irregular morphology.68 CdS synthesized in the absence of pepsin possessed a morphology distinctly different from that produced in the presence of the enzyme, which indicated that pepsin was actively involved in the growth process for these nanoparticles.68 Yang and colleagues also suggested that pepsin likely chelates Cd2+, as the secondary structure of this enzyme was found to be altered in solutions containing cadmium ions.68

5.7. Oriented QDs from Genetically-Modified Phage As discussed previously (see section 5.2), organisms such as yeast have developed specialized peptides, capable of controlling the growth of semiconductor nanocrystals in ViVo, and these phytochelatins have been utilized to produce quantum dots in Vitro.175,177 Flynn et al. have screened phage-displayed libraries for other peptides (i.e., nonphytochelatins) displaying an affinity for the surfaces of ZnS, CdS, and PbS.196 The ZnS- and CdS-specific peptides isolated through this screening process were found to possess a level of control over in Vitro QD synthesis, surpassing that demonstrated for the phytochelatins. Of the peptides identified during the screening process, Flynn et al. selected four peptides, two ZnS-specific (i.e., peptides Z8 and A7) and two CdS-specific sequences (i.e., peptides J182 and J140),


for further study.196 Although the sequences of these four peptides were unique, they may be generalized as disulfidebond constrained 7-mers (A7 and J182) and linear 12-mer peptides (Z8 and J140).196 Displayed as fusions to either the pIII or pVIII M13 capsid proteins, Flynn and colleagues observed that the selected peptides induced the formation of specific crystal structures of ZnS or CdS.196 Specifically, Z8 templated the zinc blende form of ZnS, A7 produced wurtzite ZnS, J182 yielded zinc blende CdS, and J140 formed wurtzite CdS.196 This result provided an excellent example of the control of the inorganic crystal structure that may be achieved through biomimetic syntheses. It is interesting to note, however, that the peptides were isolated from libraries screened against single polymorphs of CdS (wurtzite form) and ZnS (zinc blende form).196 In addition to influencing crystal structure, pIII fusions of the ZnS- or CdS-specific peptides were also reported to limit the growth of QDs to sizes of approximately 4 nm.196 Conversely, ZnS grown in the absence of phage or with wild-type M13 were noted to be 100-500 nm in size and noncrystalline.196 Displaying A7 on the pIII protein of the M13 capsid allowed for the phage-driven assembly of A7-synthesized and bound QDs into well-ordered, self-supporting hybrid materials.196,197 Peptides inserted into the sequence of the pVIII capsid protein were observed to produce nanocrystals preferentially oriented with respect to the phage surface.196,198 This orientation relationship between the phage capsid and the A7-produced ZnS was observed not only on the local scale for a handful of phage by high-resolution TEM and selected area electron diffraction (SAED) characterization, but also on a global scale by X-ray diffraction (XRD) analysis.196,198 Z8 and J140 peptides displayed on the M13 capsid as pVIII-fusions were also found to be equally adept as A7 in producing QDs (i.e., ZnS and CdS for Z8 and J140, respectively) with preferred orientation relative to the virus surface.196,198 These peptides were also found to exhibit chemical specificity.198 Mao and colleagues observed that phage displaying ZnS-isolated peptides did not form CdS when exposed to CdS mineralizing conditions, and similarly, CdS peptides did not exhibit ZnS formation activity.198 Taking advantage of this chemical specificity, Mao et al. engineered phage displaying both A7-pVIII and J140-pVIII fusions, which were utilized to produce phage-templated ZnS/CdS composite nanowires.198 In a later study, Mao and colleagues demonstrated that the well-aligned QDs produced by A7-pVIII and J140-pVIII could be annealed at 400-500 °C to produce single crystal whiskers of ZnS and CdS that were 20 nm in diameter and up to 650 nm in length (Figure 34).199 Although such high-temperature treatment of M13/ QD nanobiocomposites negates one of the major advantages of biomimetic processing (i.e., room-temperature synthesis), this methodology does present a facile route to the production of highly crystalline, one-dimensional, functional inorganic materials of controlled chemistry.199

5.8. Biomimetic Synthesis of an Elemental Semiconductor To date, there has been but a single demonstration of the biomolecule-enabled synthesis of an elemental semiconductor.124 In this pioneering work, Ge nanoparticles were formed on nanotubes functionalized with a previously identified Gespecific peptide.124,127 Germanium synthesis was conducted by incubating peptide-functionalized nanotubes with a germanium salt in a mixed aqueous-organic solvent, allowing

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Figure 34. (A) Dark-field TEM image using the (100) reflection, depicting well-aligned ZnS nanocrystals templated onto peptidedisplaying phage. A SAED pattern consistent with wurtzite ZnS is shown as an inset. g ) (100)* denotes the reciprocal vector of (100) crystal planes. (B) TEM image of the lattice fringes that run throughout the ZnS whiskers produced by heat treating the ZnS phage depicted in (A). Lower-magnification TEM image of the annealed whiskers provided as an inset in (B). Reprinted with permission from ref 199 (Mao, C.; Solis, D. J.; Reiss, B. D.; Kottmann, S. T.; Sweeney, R. Y.; Hayhurst, A.; Georgiou, G.; Iverson, B.; Belcher, A. M. Science 2004, 303, 213-217). Copyright 2004 AAAS (www.sciencemag.org).

the peptide to complex Ge ions that were then reduced with hydrazine.124 Although these reaction conditions are not as mild as those utilized in many biomimetic studies, the peptide-functionalized nanotubes were successfully utilized to form crystalline Ge nanoparticles.124 Banerjee and colleagues found that the germanium nanoparticles that coated the nanotube surface were monodisperse in size and possessed pH-tunable diameters.124

5.9. Summary of the Bio-Enabled Synthesis of Semiconductor Nanocrystals Yeast-derived peptides, zinc fingerlike peptides, protein cages, engineered dendritic and amphiphilic peptides, phagedisplayed library-identified peptides, and commercially available proteins have all been utilized to produce semiconductor nanoparticles with varying degrees of success. A select number of these biomolecules have synthesized quantumconfined semiconductors with size distributions equal to or finer than those achieved with the most modern synthetic techniques, while avoiding the use of exotic organic solvents and high temperatures. The chemistry and crystal structure (polymorph) control achieved through biomimetic mineralization is also notable. While such biomolecule-controlled syntheses of semiconductor nanomaterials have been achieved, further advances rivaling or exceeding synthetic approaches seem possible and likely. For example, one particular area in which biomimetic engineering may make inroads relative to synthetic chemistry is in the production of core-shell or hybrid quantum structures, wherein protein or peptide templates can be used to direct/template the growth of several semiconductor materials.169

6. Biomimetic Syntheses of Metallic Nanoparticles, Nanorods, and Nanowires 6.1. Application and Chemical Syntheses of Metal Nanostructures The past decade has witnessed an explosive growth in research aimed at the synthesis of metallic nanoparticles, nanorods, and nanowires.200-202 This increased research tempo has been spurred, in part, by interest in utilizing these metallic nanomaterials as components of highly sensitive and


selective diagnostic assays for the detection of toxic chemical species (e.g., Hg+, Pb2+) or biomolecules (e.g., proteins, DNA) indicative of disease.200,203 Additionally, metallic nanomaterials are being investigated for use in ViVo in the targeted delivery of drugs and genes, as well as the directed imaging and treatment of cancers.204,205 Beyond these emerging applications, metal nanoparticles are widely used as catalysts and are being increasingly used as device components (e.g., magnetic data storage).200,206,207 Metallic nanomaterials are attractive for these sensing, imaging, transport, and cancer therapy applications because of their unique surface plasmon resonance (SPR) enhanced light scattering and absorption.200,201,203,205 The SPR and SPR-related properties of nanomaterials can be tailored through the adjustment of metal nanoparticle size, shape, and chemistry.200,208 Furthermore, the surface chemistry of metal nanoparticles (e.g., gold) enables the facile functionalization of these materials with a variety of ligands.200,203-205 Such nanoparticle-conjugated species may include therapeutic DNA or drug molecules, selective targeting agents, such as antibodies or peptides, as well as a variety of other proteins, molecules, and synthetic polymers.200,203-205 Currently, the fabrication of metal nanoparticles, nanorods, andnanowiresisdominatedbysolution-basedtechniques.200-202 Modern metal nanomaterial synthesis methods seek to control theshape,size,size-rangedispersity,andsolutionstability.200-202 In order to synthesize stable metal colloids from metal saltbearing solutions, reducing and stabilizing agents are needed.200-202 The reducing and stabilizing agents in a reaction need not be two different reagents, because certain chemical species, such as the citrate ion, may fulfill both of these roles.200,202 The reaction conditions utilized in the syntheses of metal nanoparticles cover a wide spectrum in terms of their relative severity. Some of these synthesis methods employ elevated temperatures (e.g., 200 °C for Au formed in the Brust method), organic solvents (e.g., toluene, ethylene glycol, or hexadecylamine), and extremely strong reducing agents (e.g., solvated Mg).200-202 Other techniques for the synthesis of certain metallic nanoparticle chemistries utilize neutral pH, aqueous solutions containing small biomolecules (e.g., ascorbic acid), or common biological buffering agents (e.g., HEPES) that act as reducing and/or stabilizing agents.200-202,209 If relatively gentle reaction conditions can be achieved in the synthetic production of metal nanoparticles, nanorods, and nanowires, then what are the advantages of the biomimetic or biomolecule-enabled approach? In some cases, biomimetic syntheses can be used to control the shape, size, size distribution, and solution stability of metal nanoparticles. Biomolecules have been utilized in the high-yield production metal nanoparticle prisms or intermetallic particles under relatively mild conditions that are difficult to synthesize chemically. The “green” aspects of bioenabled syntheses are particularly relevant for certain potential in Vitro or in ViVo applications. The surfactants and stabilizers that are used in the chemical syntheses of metal nanoparticles may persist as contaminants in the final product, are often toxic, and may need to be replaced to incorporate new functional groups, such as biomolecules, onto the material surface.210 A more straightforward and potentially less deleterious route to producing biomolecule-conjugated metal nanoparticles is to directly synthesize metal nanostructures on the proteins or peptides of interest. Biomolecules can also act as self-assembling and nanoparticle organizing agents.211


Figure 35. TEM image of a P. stutzeri AG259 cell containing silver-based nanoparticles. Reprinted with permission from ref 213 (Klaus, T.; Joerger, R.; Olsson, E.; Granqvist, C. G. Silver-Based Crystalline Nanoparticles, Microbially Fabricated. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 13611-13614). Copyright 1999 National Academy of Sciences, U.S.A.

6.2. Biogenic and Biologically-Derived Production of Metal Nanoparticles 6.2.1. Production of Metal Nanoparticles by Bacteria, Fungi, and Plants No review of the biomimetic syntheses of metal nanostructures would be complete without considering the inspiration for this body of work: the biological production of gold and silver nanoparticles. As recently summarized by Carney and colleagues, organisms including plants, bacteria, and fungi reduce noble metal ions to produce metal nanoparticles as a detoxification strategy.175 Although noble metal ions are not typically perceived as toxic elements, these metals have no known biological role but may deactivate enzymes, interrupt protein and DNA structure, and disrupt electron transport within an organism.175,212 Specific examples of the biological production of metal nanoparticles include the intracellular synthesis of polygonal silver and silver-based platelets up to 200 nm in size by the bacteria Pseudomonas stutzeri AG259 (Figure 35), the extracellular formation of gold nanoparticles (2-40 nm in size) in alfalfa plants, and the intracellular creation of gold nanoparticles of varying size and morphology in Verticillium fungi.175,213-215 While these results are possibly significant for precious metal mining operations, the organisms surveyed to date exert relatively little control (as compared with advanced synthetic methods) over the size, shape, and chemistry of the gold or silver nanoparticles they form.175,213-215 Additionally, the possible biomolecules and biochemical processes involved in the cellular reduction and detoxification of metal ions remains unclear.175,213-215

6.2.2. Cell-Free Extracts for in vitro Gold Nanoprism Synthesis Several research groups have pursued the in Vitro synthesis of gold nanoparticles utilizing extracts derived from the cells of plants and fungi.175,214,216-218 In one such study, Gericke and Pinches explored the use of material extracted from the ruptured cells of the gold-reducing fungus Verticillium luteoalbum to produce a mixture of gold nanoparticles and nanoprisms.214 Shankar and colleagues have made extensive use of this biological extract-based approach to synthesize gold and silver nanoparticles.216,219,220 For example, these authors demonstrated the synthesis of gold nanoprisms in moderate yield (∼45% of the nanoparticle population)


Figure 36. Gold nanoprisms synthesized under the influence of cellular extracts from (A) brown seaweed and (B) green algae. Image (A) reprinted with permission from ref 217 (Liu, B.; Xie, J.; Lee, J. Y.; Ting, Y. P.; Chen, J. P. J. Phys. Chem. B 2005, 109, 15256). Copyright 2005 American Chemical Society. Image (B) reprinted with permission from ref 218 (Xie, J.; Lee, J. Y.; Wang, D. I. C.; Ting, Y. P. Identification of Active Biomolecules in the High-Yield Synthesis of Single-Crystalline Gold Nanoplates in Algal Solutions. Small 2007, 3, 672-678). Copyright 2007 WileyVCH Verlag GMBH & Co. KGA.

utilizing extracts from the lemongrass plant (Cymbopogon flexuosus).216 Liu et al. utilized extracts from brown seaweed to yield gold nanoprisms and polygons (Figure 36A) of controlled sizes at yields exceeding 80%.217 It should be noted here that the production of gold nanoprisms at such high yields is significant, as the synthetic production of these materials provides yields of ∼70%.221,222 In each of the studies described in this section, the specific biomolecules acting as reducing and capping agents in the synthesis of gold nanoprisms were not specifically identified but were suggested to be sugars, terpenoids, or proteins.214,216,217,219,220 A more definitive identification of a plant-derived biomolecule that produces gold nanoprisms has recently been reported by Xie and colleagues.218 These researchers observed that cellular extracts from the green algae, Chlorella Vulgaris, were also capable of producing gold nanoprisms.218 Subsequent fractionation and purification of this algal extract resulted in the isolation of a protein that acted as a reducing, shape-directing, and dispersing agent.218 This protein was determined to be the only component of the C. Vulgaris extract that could produce prism- and polygonal-shaped gold nanoparticles (Figure 36B) and was observed to do so in high yield (∼90%).218 The amino acid sequence of this gold nanoplate-directing protein was unfortunately not reported by Xie and co-workers.218

6.3. Amino Acids Influencing the Biomimetic Syntheses of Metal Nanoparticles A number of research groups have investigated the ability of amino acids to act as reducing and stabilizing agents for the syntheses of gold nanoparticles.223-225 Aspartic acid, lysine, arginine, tyrosine, and tryptophan have been found to initiate and control the syntheses of gold nanostructures at room temperature.223-225 While aspartic acid was found to produce some polygonal gold nanoplatelets,223 these results are not directly applicable to proteins, as the R-carbon carboxylic acid and amine functionalities of individual amino acids are unavailable following the polymerization of the residues into the peptide backbone. Working with short peptides, Mandal and colleagues have found that tyrosine and tryptophan residues reduced gold and silver ions in solution, resulting in the formation of gold nanoparticles.226,227 Slocik and colleagues found that the gold-reducing activity of the A3 peptide was greatly reduced when its lone tyrosine residue was replaced by a serine residue in a modified version of A3.228 Similarly, gold-reducing activity was endowed to

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peptides lacking such intrinsic activity when tyrosine residues were added to the peptide sequence.229 Tyrosine residues have also been implicated in the reduction of silver ions.230 The thiol side group of cysteine has been suggested to be a source of electrons for the reduction of gold and is wellknown to bind with gold and other noble metals.67,200-202 The lysine, arginine, aspartate, glutamate, and histidine residues found in many proteins and peptides may sequester metal ions or metal ion complexes.142 These amino acid residue-chelated ions may subsequently be reduced to metal nanoparticles through the addition of external reducing agents, such as sodium borohydride (NaBH4).

6.4. Peptide-Induced Syntheses of Gold Nanoparticles and Nanotubes Brown and colleagues were the first to explore the possible influence of peptides in the formation of gold nanoparticles.82 In this initial study, repeating polypeptides identified through the screening of a cell-surface displayed peptide library were added to solutions containing gold ions and sodium ascorbate (i.e., a reducing agent).80,82 Of the 50 peptides screened by Brown and colleagues, 3 polypeptides were found to accelerate nanoparticle growth and to modify the morphology of the gold crystals.82 These 3 polypeptides were proposed to influence gold nanoparticle growth by binding to gold embryos or nanoparticles in solution and acidifying the local solution surrounding these inorganics.82 Polypeptides containing a higher number of tandem peptide repeats were found to be more effective at controlling nanoparticle growth, and these peptides were not incorporated into the growing gold crystals.82 Utilizing one of the peptides studied by Brown and colleagues, Wang et al. have recently determined that the morphology of gold nanocrystals formed in the presence of this peptide also depends on the solution conditions such as pH and gold ion concentration.231 Slocik and colleagues have also explored the use of a number of peptides, including a library-identified peptide (i.e.,theA3peptide)inthesynthesisofgoldnanoparticles.123,228,232 Peptides containing tyrosine residues (i.e., A3 and FLG) were utilized as reducing and shape-directing agents, while peptides lacking tyrosine residues (i.e., GSH and HRE) were used to modify the shape and size of gold nanoparticles produced with the aid of sodium borohydride.123,228,232 The use of the FLG, HRE, and GSH peptides by Slocik and colleagues is particularly significant, as these peptides are epitopes and were readily recognized by antibodies following in situ nanoparticle growth.123,228,232 These antigen/antibody interactions were exploited to assemble hybrid nanostructures of gold nanoparticles with quantum dots and magnetic particles that were functionalized with antibodies reactive to the FLG and GSH peptides (Figure 37).232,233 Furthermore, the histidine-rich sequence (AHHAHHAAD) of the HRE peptide could be used to conjugate HRE-produced gold nanoparticles to Ni2+ nitrilotriacetic acid (Ni-NTA) functionalized surfaces.232 The in situ growth of gold nanoparticles on peptide epitopes and Ni-NTA binding sequences is a pioneering development that may potentially impact biomedical diagnoses and treatments, as well as the directed assembly of multifunctional nanostructures. The ability of the HRE peptide to direct the formation of gold nanostructures has not gone unnoticed, and its use has been extended to the synthesis of gold-coated nanotubes.234,235 In research conducted by Djalali et al., HRE peptides were



6.5. Protein-Templated Production of Gold Nanoparticles and Nanowires An example of the protein-mediated synthesis of gold nanoparticles was discussed in section 6.2.2, where Xie and colleagues utilized an algal protein to produce nanoprisms in high yield.218 Gold nanoparticle synthesis is not confined to proteins extracted from green algae, as several examples of the protein-based reduction, shape modification, and organization of gold have recently been reported.

6.5.1. BSA-Au Nanoparticles

Figure 37. (A) Schematic depicting the Flg peptide-facilitated synthesis and assembly of gold nanoparticles. (B) TEM image of 4 Flg-Au nanoparticles surrounding an anti-Flg functionalized quantum dot. Reprinted with permission from ref 233 (Slocik, J. M.; Govorov, A. O.; Naik, R. R. Optical Characterization of Bioassembled Hybrid Nanostructures. Supramol. Chem. 2006, 18, 415-421). Copyright 2006 Taylor & Francis Ltd. (http://www.tandf.co.uk/journals).

adsorbed onto preformed bolaamphiphile nanotubes, where they were utilized to bind an organic gold complex (ClAuP(CH)3).234,235 The HRE-conjugated gold complex was reduced with a chemical agent, producing a high-density coating of gold nanocrystals (∼6 nm average diameter) on the peptide nanotubes.234,235 Djalali and colleagues observed that gold nanoparticles first appeared on the inside of the peptide nanotubes and subsequently grew on the outside of these peptide structures.235 While this growth mechanism is not currently understood, it presents opportunities for the production of nanotubes coated with multiple materials.235 Djalali et al. also reported that pH changes in the reaction solution influenced the packing density of gold nanoparticles grown on the peptide nanotubes.235 This may occur through protonation/deprotonation of the amino acid residues that comprise the HRE peptide.235 Peptide nanotube diameters may also be tailored to produce large (i.e., up to 300 nm in diameter, >1 µm in length) gold nanoparticle/peptide hybrid tubes or single chains of gold nanospheres organized on a peptide nanostring (i.e., peptide nanotubes 10 nm in diameter).236 The addition of the HRE peptide to bolaamphiphile nanotubes improved nanocrystal size distribution and packing density; however, the nanotubes themselves also possess the capacity (albeit greatly reduced) to template the growth of gold nanocrystals.234,235 The ability of the bolaamphiphile utilized by Djalali and colleagues to produce monodisperse gold nanoparticles was significantly enhanced when this peptide-analogue was not preassembled into nanotubes.237 When added into solution with ClAuP(CH)3, the bolaamphiphile peptide was observed to self-assemble into doughnutshaped reactors that served as size-restrictive environments for the growth of gold nanoparticles upon reduction with UV irradiation or hydrazine hydrate (NH2NH2 · H2O).237 The gold nanocrystals produced within the peptide nanoreactors were reported to be monodisperse and possessed average diameters of 12 or 23 nm, for particles reduced by UV or hydrazine, respectively.237

To date, one of the most common proteins investigated for gold nanoparticle synthesis has been bovine serum albumin.69,70,238,239 BSA is a strong candidate for the synthesis of gold nanoparticles because it contains a large number of cysteine, tyrosine, and charged residues and has a known propensity to bind to gold ion complexes.240 BSA is also attractive for metal nanoparticle synthesis research, as the protein is readily available and relatively inexpensive. Because of its popularity among biomimetic metallization researchers, BSA-based research provides interesting examples of the variety of methods and results that may be obtained with the use of a single protein in the production of metallic nanoparticles. Burt and colleagues were the first to explore the use of BSA in gold nanoparticle synthesis.238 In this initial study, NaBH4 was utilized to reduce gold ions in the presence of BSA and yielded well-dispersed nanoparticles less than 2 nm in size.238 Utilizing a variety of spectroscopic characterization techniques, Burt et al. determined that BSA was conjugated, most likely through its cysteine residues, to the surface of the gold nanoparticles.238 In a separate study, larger gold nanospheres (7.7 ( 0.9 nm in diameter under pH 7 reaction conditions) were obtained when gold ions were reduced in the presence of BSA with UV irradiation.70 Singh and colleagues utilized the surfactantlike properties of BSA to produce gold ion charged foams of BSA.239 These Au ion charged foams were reduced with hydrazine hydrate vapors, producing irregularly shaped gold nanoparticles 90% of the loaded BuChE and were easily confined within a commercial chromatography column.275 Bead/silica/BuChE containing columns were demonstrated to be capable of over 16 h of continuous operation without significant loss of enzyme activity or substrate conversion efficiency.275 The use of butyrylcholinesterase in these initial studies is significant because this enzyme has been reported to irreversibly bind to dangerous organophosphorus (OP) compounds used as pesticides and chemical warfare agents.286 The affinity of BuChE for OP agents may be utilized to great advantage in protecting humans and animals from OP toxicity.286-288 This may be accomplished in two ways: BuChE may scavenge OP compounds in ViVo or the OPbased inhibition of enzymatic activity may be monitored in Vitro and used as a sensor for the detection of these toxic compounds in the environment.286-288 Seeking to take advantage of the enhanced stability of enzymes within biomimetic silica structures, Luckarift and colleagues investigated the latter of these potential BuChE applications.288 In this work, an air-sampling device was mated with a chromatography system equipped with agarose bead/silica/ BuChE composites, and the reduction in the hydrolysis of a

substrate by BuChE was monitored.288 Additions of the OP pesticide, paraoxon, generated detectable inhibition of silicaimmobilized BuChE at concentrations as low as ∼0.2 µmol/ m3air.288 In addition to BuChE, biomimetically synthesized agarose bead/silica composites containing organophosphorus hydrolase (OPH), an OP-degrading enzyme, also proved to be suitable for the detection and destruction of a variety of OP agents.288 While the encapsulation of enzymes individually within a biomimetically synthesized inorganic matrix has proven advantageous, materials with additional functionality may be acquired through the simultaneous entrapment of multiple enzymes.66,289,290 The first examples of inorganic/multienzyme composites contained BuChE and the antibacterial enzyme, hen egg white lysozyme (HEWL).66 HEWL was used in these studies as the inorganic matrix precipitating species (i.e., fulfilling the role of the R5 peptide) as well as a bactericidal agent.66 The ability of lysozyme to induce the precipitation of silica and titania materials likely lies in the common characteristics that this enzyme shares with biomacromolecules believed to be involved in diatom biosilicification (see sections 2.2 and 4.1); specifically, HEWL is a highly basic protein, with a pI of 11.1, and carries a large positive charge (+9).18,64,291 High levels of enzymatic activity were achieved for both BuChE and HEWL when immobilized within precipitated inorganic matrices with lysozyme.66 In later developments, HEWL has been successfully used to synthesize silica/HEWL/OPH materials on gold surfaces.289 The coimmobilization of enzymes may be especially advantageous in systems where the functions of these proteins are dependent rather than disparate. Exploiting this strategy, Betancor and co-workers encapsulated nitrobenzene nitroreductase, a NADPH-dependent enzyme, with the NADPH-regenerating enzyme glucose-6-phosphate dehydrogenase, in biomimetically formed silica particles.290 Working in concert, these two enzymes facilitated the continuous conversion of nitrobenzene to hydroxylaminobenzene.290 In the event that the coimmobilization of enzymes within a single monolith is not possible or practical, multistep chemical reactions may be conducted through the use of enzymatically active materials that are physically connected in series. In one such system presented by Luckarift and colleagues, a reactant solution was drawn through a series


of reaction chambers containing zinc metal powder, immobilized hydroxylaminobenzene mutase, and immobilized soybean peroxidase (i.e., enzymes immobilized in biomimetically synthesized silica) that were connected together by microfluidic channels, to synthesize the compound 2-aminophenoxazin-3-one from the relatively simple reactant nitrobenzene.292 In research on the coencapsulation of nitrobenzene nitroreductase and glucose-6-phosphate dehydrogenase, Betancor and colleagues utilized the polymer PEI to direct silica formation in place of R5 or a protein.290 Additional synthetic biomolecule analogues, including amine dendrimers, cysteamine, or PAA, have also been successfully utilized to encapsulate a variety of biologicals (e.g., enzymes, fluorescent proteins, and live E. coli cells).61,293,294 In the few years since its inception, the biomimetic synthesis approach has been used to successfully encapsulate and stabilize a number of different enzymes. The sophistication of these bionanocomposites has also markedly increased with the emergence of materials that include functional nanoparticles and multiple coencapsulated enzymes.66,283,289,290 While only biomimetically produced silica or titania have been explored for the immobilization of enzymes to date, the increasing number of materials chemistries accessible to biomolecular-based synthesis provides opportunities to develop revolutionary multifunctional composites. Given recent emphases on “green” technologies and the established use of enzymes in the chemical and pharmaceutical industries, it seems likely that the entrapment of enzymes may be one of the first commercial applications of biomimetic materials processing.295,296

8.2. Virus Capsid-Templated Energy-Storage Materials An intriguing application recently explored for biotemplated synthesis is the fabrication of battery electrode materials. The utility of viruses for the production of battery materials was first demonstrated by Nam and colleagues in research with the M13 phage and has been expanded upon inTMV-basedworkconductedbyRoystonandco-workers.157,297 In each of these studies, the assembly and synthesis of the battery ion intercalation matrix was facilitated by specific modifications to the major coat protein of the virus capsid and the anisotropic morphology of the virus particles.157,297 Nam and colleagues engineered the major coat protein of the M13 phage (i.e., pVIII), which numbers ∼2700 per virus, so as to display a short peptide (E4) composed of four aspartic acid residues.157 Nam and colleagues observed that the negative charge and filamentous shape of these engineered phage (E4-M13) could be exploited to assemble the viruses, in 2D liquid crystalline patterns, on positively charged polymer surfaces.157 Following this electrostatic immobilization procedure, the E4 peptide was further exploited for its strong affinity for cobalt ions. These E4bound cobalt ions were chemically reduced and subsequently oxidized to yield Co3O4, a material that possesses a significant reversible storage capacity for Li ions.157 Depending on reaction conditions, the Co3O4 could be deposited on the E4-M13 capsid as discrete nanoparticles that were 2-3 nm in size (Figure 26) or as a continuous layer to yield a virustemplated Co3O4 nanowire.157 Test cells fabricated with an E4-M13 phage assembled and templated Co3O4 nanowire electrode were observed to deliver capacities of approximately 1000 mAh/g over six


charge and discharge cycles.157 Nam and colleagues proposed that the capacity of these electrodes may be improved through design changes, such as moving from a monolayer of Co3O4 nanowires to a stacked design through the alternating syntheses of nanowires and deposition of a cationic polymer.157 These authors also found that hybrid Co3O4 nanowires with embedded gold nanoparticles could be used to achieve ∼30% higher Li ion storage capacity than their monolithic counterparts.157 Viruses capable of producing hybrid Au-Co3O4 nanowires (AuE4-M13) were generated through the introduction of a relatively small number of pVIII proteins that displayed a gold-binding peptide into the M13 capsid.157 The synthesis of Au-Co3O4 nanowires then involved the additional step of binding gold nanoparticles from solution prior to (Co3O4) mineralization.157 Nam and colleagues proposed that the role these gold nanoparticles played in increasing the Li ion capacity of the M13-templated nanowires may be due to a catalytic effect or the improvement of the electronic conductivity of the composites.157 Royston and colleagues have manipulated the genome of the tobacco mosaic virus (TMV) to display an extra cysteine residue on the TMV capsid protein (∼2130 copies per capsid).297,298 The additional cysteine residues expressed on the TMV capsid (termed TMV1cys) were exploited to both facilitate the organization and syntheses of TMV-based electrode materials.157,297 Royston et al. proposed that the differential presentation of the engineered cysteine residues on the ends of the rod-shaped TMV1cys capsid resulted in the preferential attachment of the viruses to a gold substrate (i.e., through Au-thiol bonding) in a perpendicular orientation.297 These immobilized viruses were mineralized utilizing a Pd nanocluster “activation” and electroless nickel deposition process similar to that employed by Knez and colleagues (see section 6.8.2).260,261,297 The additional cysteine residues engineered into the TMV1cys capsid were expected to promote a relatively greater adhesion between the virus and the Pd nanoclusters, and subsequently deposited Ni-based materials than would have been achieved with the wild-type TMV.297 The metallization of the activated TMV1cys capsids resulted in the formation of a continuous and conformal coating approximately 20-40 nm in thickness that was composed of Ni-based materials.297 While metallic nickel was initially deposited by the electroless process, this material was subsequently oxidized under ambient conditions to yield a coating composed of a mixture of Ni, Ni(OH)2, and NiO on the surface of the gold substrate-immobilized capsids.297 Royston and colleagues explored the use of these TMV1cystemplated nickel oxide structures for electrodes in NiO-Zn batteries.297 For comparative purposes, control samples were fabricated through the electroless deposition and subsequent oxidation of nickel on bare gold-coated Si wafers and tested alongside the TMV1cys-based samples.297 Royston et al. reported that the TMV1cys-templated battery electrodes performed favorably compared to the control samples, exhibiting a final capacity twice that of the control electrodes.297 Such enhanced electrode performance is most likely due to the increased surface area afforded by the addition of the TMV1cys virus particles to the gold-coated Si substrates prior to electroless Ni deposition.297 Given the relative importance of energy-storage materials to modern technology, as well as the renewable nature of biological materials, there is likely to be continued interest in the biomimetic fabrication of ion intercalation and electrode materials for battery applications in the future.


9. Summary and Outlook Proteins and peptides have been successfully utilized to produce a variety of material chemistries under relatively benign solution conditions. The first steps in the field were made by mimicking the ability of biology to create SiO2, Fe3O4, CdS, Au, and Ag nanomaterials. The range of chemistries accessible by biomimetic processing has since been extended to include semiconducting materials, metal alloys, multicomponent oxides, and composites. In addition to controlling material chemistry, biomolecules have also been found to be able to direct the shapes, sizes, and crystal structures of the synthesized inorganic materials. Exemplars of such morphological and crystallographic control include the protein-directed synthesis of gold and silver nanoprisms, rutile TiO2, L10FePt, and γ-Ga2O3. The proteins and peptides utilized to direct the syntheses of these materials have been drawn or derived from a variety of sources including biomineralizing organisms (e.g., the R5 peptide, as well as recombinant silaffins, silicateins, and Mms6), plant tissues, phage and cell surface display peptide libraries, and commercial vendors (e.g., BSA, lysozyme, and pepsin). The architecture of the apoferritin cage has been exploited to produce inorganic nanoparticles of well-defined size, and the structures of virus capsids have facilitated the syntheses of inorganic nanotubes and nanowires. Virustemplated materials have been recently explored as ion intercalation matrices for energy-storage applications. The mineralizing activities of peptides and proteins have been combined with other functional amino acid sequences to produce unique multifunctional chimeric biomolecules. Chimeric proteins have enabled the syntheses of selfassembling hybrid materials that can bind to specific surfaces or assemble on strands of DNA. Modern molecular biology has also enabled the construction of chimeric biomolecules that combine high-strength biopolymers and mineralizing sequences, which may be utilized to create proteinsynthesized and -reinforced composite materials. The fusion of peptides to epitope tags or ferritin proteins has yielded biomolecules that are able to serially synthesize two different material chemistries, producing bionanocomposites. The gentle reaction conditions afforded by the proteinand peptide-based syntheses of materials have facilitated the production of enzymatically active composites. The enzymes entrapped within these composite materials display enhanced thermal and dry storage stability as compared to free enzymes and have been successfully utilized in a variety of flowthrough bioprocessing, biocatalysis, and biosensing applications. While enzymes may be encapsulated in silica through the action of a second biomolecule, such as the R5 peptide, a few enzymes (e.g., HEWL and R-amylase) have been demonstrated to direct the in situ synthesis of materials while maintaining their catalytic activity. With the continued attention and ingenuity of researchers from diverse disciplines, as well as sustained research and development funding, the future of biomimetic materials syntheses promises to be exciting, dynamic, and rich in applications. The past decade has witnessed great advances in identifying biomolecules that possess material-syntheses activity, deciphering the contributions of individual residues within these sequences, and expanding the number of inorganic chemistries accessible from proteins and peptides. While our fundamental understanding of these existing topics must be furthered in order to more fully harness the potential of biomolecules for material syntheses, there are also a

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number of interesting and powerful new concepts that have received only passing attention or remain unexplored. For example, the incorporation of rare amino acid residues (e.g., selenocysteine or pyrrolysine) or even synthetic amino acids into proteins or peptides may be used to affect the formation of inorganic materials.299,300 Relatively few studies (with the notable exceptions of those utilizing certain virus capsids, apoferritin cages, and enzymes) have sought to specifically take advantage of the secondary, tertiary, and quaternary structures of many proteins for material syntheses. Increasingly important contributions from bioinformatics and computational modeling approaches are expected to allow for the rational design of proteins or peptides for the programmed biomimetic syntheses and assembly of inorganic structures. Perhaps the next great frontier in this field will be a shift from biomimetic material syntheses with isolated peptides and proteins in Vitro to the in ViVo production of materials using whole-organism systems. Multifunctional materials of prescribed shape, size, chemistry, and crystal structure could possibly be produced inexpensively in bioreactors by bacteria that have been genetically modified to express inorganic synthesizing proteins or peptides. It would also be highly desirable for these materials to be formed or exported out of the cell, where they could possibly self-assemble into hierarchically organized structures. Although research focused on the syntheses of materials with fungal, bacterial, or yeast cultures has been conducted (see sections 5.2 and 6.2 for examples), these systems have not yet gained the sophistication required to achieve such ambitious objectives as those listed above. Predicting the course of biomimetic research beyond the near term may appear to fall into the realm of science fiction. However, the roots for possible future technologies are clearly evident today. For example, the term “self-replicating materials” may at some point become as commonly used as the word nanotechnology is today. A number of articles focusing on the modification of diatom frustules (e.g., through environmental manipulation, random genetic mutation, and genetic engineering) are especially interesting in relationtothepossiblegenesisofself-replicatingmaterials.27,301,302 Indeed, an entire issue of the Journal of Nanoscience and Nanotechnology has been devoted to diatom nanotechnology.303 Given the widespread use of viruses in biomimetic material syntheses, it may only be a matter of time before a virus has been engineered that possesses a hybrid inorganic/ protein or even a solely inorganic capsid that is generated within a host cell as a necessary step in its replication cycle. As noted by Mao and colleagues, the biopanning process effectively selects for viruses with information regarding the composition, phase, and crystallographic detail of materials stored at the DNA level.199 Roth and colleagues have utilized biomimetic silicification reactions to entrap bacteria within an inorganic matrix, with these cells remaining viable for a number of days.294 Perhaps these results could be extended to produce composites of complex functional inorganic materials containing living components that are capable of adapting to environmental stimuli and of self-repair.

10. Acknowledgments We thank the members of the AFRL/RX biotechnology group for their technical insights and AFRL for funding for this work. We are grateful for the assistance provided by W. J. Crookes-Goodson and J. M. Slocik in the critical


reading and editing of this article. M.B.D. is supported by a National Research Council Research Associateship award.

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