Examples of polypeptide sequences exhibiting affinity for various inorganics. (Reproduced from
Many organisms are capable of synthesizing organic/inorganic composites for protective or support purposes, such as bones, shells, and teeth. They exert a remarkable level of molecular control on particle size, structure, morphology, aggregation, and crystallographic orientation of these organic/inorganic structured materials. These materials often hierarchically arrange from nanoscale to macroscale (Feldheim and Eaton 2007; Dujardin and Mann 2002; Mann et al. 1993; Palmer et al. 2008; Estroff and Hamilton 2001; Aizenberg et al. 2005). For example, mollusks produce shells or nacres that contain a single distinct calcium carbonate crystalline phase, such as aragonite or calcite (Addadi et al. 2006). Magnetotactic bacteria produce Fe3O4 or Fe3S4 nanoparticles with well-defined sizes and shapes to recognize magnetic fields for alignment and migration (Dennis A. Bazylinski and Frankel 2004; Komeili 2007). Marine sponges produce silica spicules that have been demonstrated to possess light-guiding characteristics and may reach lengths of up to 3 m (Aizenberg et al. 2004; Sundar et al. 2003; Cattaneo-Vietti et al. 1996). In each of the examples listed above, and in many more examples in nature (Fig.1), specialized biomolecules, such as proteins, peptides, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and polysaccharides, have been found or are thought to play a critical role in directing the formation of these hierarchically assembled inorganic structures (Söllner et al. 2003; Müller et al. 2007). The participation of biomolecules in the nucleation and growth of crystals has attracted much research attention. Most notably, the proteins involved in directing the shape of these biomaterials have often evolved to recognize and bind selectively to one or more faces of the growing crystal. For instance, important matrix proteins involved in bone growth contain different function domains that orient the protein on hydroxyapatite nanocrystals and interact with target cell receptors (Gilbert et al. 2003). Nature has always been a source of inspiration for technical developments. Materials scientists consider the hierarchical structure of natural materials as a model for the development of new types of high-performance engineered materials (George and Ravindran 2010). The biomimetic approach could lead to the development of the controlled synthesis of inorganic nanophases, the crystal engineering of bulk solids, and the assembly of organized composite and ceramic materials (Mann et al. 1993).
During the past decades, many inorganic crystals or hybrid inorganic/organic materials with special sizes, shapes, organization, complex forms, and hierarchies have been synthesized via bioinspired methods with the assistance of various templates, such as synthetic polymers, self-assembling peptides, proteins, and some low mass surfactant molecules (Cai and Yao 2010; Xu et al. 2007). Routine and reliable synthesis of self-assembled hybrid materials with tunable functionalities are urgently required for real-life applications and economic commercialization (Patil and Mann 2008; Gower 2008).
There are mainly two mechanisms by which organisms control the self-assembled hierarchical organic/inorganic structures. First, the organic matrix serves as template on which to form a specific mineral. Second, inorganic materials usually appear in cells at the protoplasmic surface boundary layer. Therefore, the arrangement of the biominerals is controlled by the surface tension between the cells, the vesicles, and the growing mineral (Estroff and Hamilton 2001). Recent work in the field of bioinspired synthesis has achieved varying degrees of success in both of these strategies, especially the first mechanism, in which the self-assembling organic structures are used to template the growth of inorganic materials. The structural information from the organic assembly is directly transcribed to the inorganic materials, or used to modify the morphology of the inorganic phases.
This review will focus on the recent successes in using self-assembling biomolecules as the organic matrix templates to direct and facilitate the formation of different kinds of structured organic/inorganic composite materials. The biomolecules are either natural or synthetic, including proteins, peptides, DNA, RNA, and polysaccharides.
2. The advantages of using biomolecules for bioinspired synthesis, and their sources
The use of biomolecules to direct the
The primary sources to obtain the biomolecules used for the bioinspired synthesis of materials include: biomolecules isolated or derived from biomineralizing organisms, biomineralizing biomolecule analogs, and peptides identified for biomineralization (Dickerson et al. 2008). Biomolecules isolated or derived from biomineralizing organisms have been widely used for biomimetic synthesis of inorganic materials, however, the use of biomineral-isolated biomolecules has several drawbacks. For example, the biomolecules may be difficult to obtain or limited by the yield, may require specialized facilities to grow, 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 (Tahir et al. 2005). 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 (Yang et al. 2006; Shiomi et al. 2007). According to these recognized sequences, biomineralizing biomolecule analogs can be developed. The analogs are not restricted to biomolecules, for instance, synthetic polymers are also developed as templates for bioinspired inorganic synthesis (Enlow et al. 2007; Kanapathipillai et al. 2008). The development of peptide, DNA, and RNA identification, separation, and synthesis techniques provides more opportunity to design templates for the bioinspired inorganic material synthesis, for example, phage display is used for identifying peptides and systematic enrichment of ligands by exponential enrichment (SELEX) is used for recognize RNA (Feldheim and Eaton 2007).
Different types of biomolecules used in bioinspired synthesis can be broadly categorized into four categories: proteins, peptides, nucleic acids, and polysaccharides. The role of these different types of biomolecules in the bioinspired synthesis and fabrication process is discussed in greater detail using specific cases as examples in the following sections.
3. Protein-mediated bioinspired synthesis
Proteins provide functional building blocks for the development of multi-functional materials (Gajjeraman et al. 2008). The self-assembly property of proteins would allow controlled organization of the organic/inorganic interface based on molecular recognition, resulting in hierarchical organization with desirable properties at multiple length scales. Proteins have superior specificity for target binding with complex molecular recognition mechanism (de la Rica and Matsui 2010). Through their unique and specific interactions with other macromolecules and inorganics, they process the ability to control structures and functions of biological hard and soft tissues in organisms (Sarikaya et al. 2003). In the following sections, several examples of protein-mediated bioinspired synthesis of structured organic/inorganic materials
3.1. Protein mediated hydroxyapatite (HAp) formation
Bone is a highly ordered, dynamic, and highly vascularized tissue that exhibits excellent strength, hardness, and fracture toughness. It is a biocomposite of 70% mineral (mostly nanoscale calcium phosphate crystals) and 30% organics (including collagen, glycoproteins, proteoglycans, and sialoproteins) by dry weight (Salgado et al. 2004; Hu et al. 2010; Palmer et al. 2008). Calcium phosphates, notably HAp [Ca10(PO4)6(OH)2], exhibit many levels of hierarchical structures in bone from nano to macro scales (Rey et al. 2009). Mineralized collagen fibrils are the basic building block for bone formation. More than 20 human collagens have been reported. In collagens, the amino acids glycine, proline, and hydroxyproline account for more than 50% of the amino acid composition, often presented as the Gly-X-Y repeat unit (where X and Y are either proline or hydroxyproline) (Cui, et al. 2007). Most collagens display a 67 nm periodicity due to the axial packing of the individual collagen molecules (Prockop 1995). Collagens also serve as extracellular matrix molecules for many other soft and hard tissues, such as cartilage, tendons, and ligaments.
We highlight some recent studies focused on the collagen-HAp interactions in the bioinspired synthesis of HAp composite materials. A nanocomposite of collagen and HAp was prepared in a continuous flow system, mimicking the situation
Another collagen templated HAp nanocomposite showed equal or better biocompatibility than HAp ceramics, which was known to have excellent biocompatibility. The c-axes of HAp nanocrystals were regularly aligned along collagen fibrils, which was similar to natural bone orientation. The composite promoted the osteoclastic resorption, followed by new bone formation by osteoblasts, which was very similar to the reaction of a transplanted autogenous-bone. Therefore, the HAp/collagen composite can be potentially used as an artificial bone material in medical and dental fields (Kikuchi et al. 2004).
In another study, two different bioinspired methods were used to fabricate HAp on collagen templates: dispersion of synthetic HAp in a solution of telopeptide-free collagen molecules and direct nucleation of HAp into reconstituted collagen fibers during their assembly. Composite materials obtained by direct nucleation showed similar composition, morphology, and structure to natural bone, and also indicated an intimated interaction between the inorganic phase and protein components (Tampieri et al. 2003). This proved the template function of the collagen during the bone formation.
Proteins other than collagen are also used in bioinspired HAp synthesis. A novel human hair proteins and HAp composite was synthesized for using as a biomineral-scaffolding material. The human hair protein was soaked to a CaCl2 solution for fabrication into flat films. The flat films mainly consisted of α-keratin, which could bind 3 Ca2+ ions per 1 keratin molecule. The composite of the human hair protein and calcium phosphate was prepared via alternate soaking processes using CaCl2 and Na2HPO4 solutions. The diameters of deposited calcium phosphate particles were about 2–4 μm. The human hair proteins were not soluble and degraded during the soaking processes.(Fujii et al. 2009) Synthetic proteins have also been developed as templates for bioinspired synthesis. Self-assembled chimeric protein hydrogels comprising leucine zipper motifs flanking a dentin matrix protein 1 domain were developed to act as a HAp nucleator for the formation of highly oriented apatite similar to bone mineral (Gajjeraman et al. 2008).
3.2. Protein mediated magnetic materials formation
Magnetotactic bacteria form magnetite nanoparticles
Some inorganic magnetic materials which do not appear in living organisms, for example, cobalt ferrite (CoFe2O4) nanoparticles, were also synthesized
3.3. Protein mediated silica formation
Aizenberg et al. reported the structural hierarchy of biosilica observed in the hexactinellid sponge
Silicateins, or silica proteins, were found to be enzymes (structural and catalytic proteins) that promote biosilica formation in nature ( Wang et al. 2010 ). The silicateins exhibit catalytic activity at neutral pH and low temperature. They have also been used as templates to direct the growth of silica particles along the axial protein filament. It has been used to simultaneously catalyze and structurally direct the hydrolysis and condensation of tetraethyl orthosilicate
Amorphous silica (or silica glass) is widely used in different applications, such as membranes, columns, heat-proof materials, optical communication fibers, and catalysts in organic synthesis (Jensen et al. 2009). Silicatein from the freshwater sponge
Silicateins could be immobilized onto a template surface, while still preserving their catalytic activity. In a bioinspired approach, biosilica was synthesized on “inert” surfaces (matrices) from monomeric precursors (Tahir et al. 2004). The matrices were first functionalized with a reactive polymer that was subsequently able to chemisorb nitrilotriacetic acid (NTA), a required binder for His-tagged recombinant silicatein. Silicatein that had been immobilized onto this matrix using NTA-His tag linkage had the capability to synthesize nanoparticulate biosilica, biotitania, and biozirconia from monomeric precursors. The process is shown by Fig.6.
Fungi have been used in bioinspired synthesis of inorganic materials. Silica, zirconia, and titania nanoparticles were produced by mixing the fungus
4. Peptide-mediated bioinspired synthesis
Peptides consist of short amino acid sequences that have less intricate functionality than proteins. Although peptides may not perform highly specialized functions compared to proteins, they can be synthesized more easily with desired amino acid sequences by well-established chemical and genetic engineering techniques. Therefore, they are widely used in the applications ranging from controlled gene and drug release, nanofabrication, biomineralization, and membrane protein stabilization to three-dimensional (3D) cell culture and tissue engineering. Peptides are designed to be folded in desired conformations, such as α-helix, β-sheet, etc. These 3D building blocks may yield supramolecular structures through self-assembly process. Moreover, the supramolecular structures can be controlled by changing the physicochemical properties of the environment such as pH, temperature, and salt content, which makes peptides versatile smart materials for the design of structured materials (de la Rica and Matsui 2010; Zhao et al. 2010).
There are several possible ways of obtaining polypeptide sequences with specific affinity to inorganics. For example, well-established
A 12-residue peptide (NPYHPTIPQSVH-GGGK-biotin: CLP12 peptide) has been identified for HAp biomineralization using phage display. The sequence responsible for the mineralizing activity resembled the tripeptide repeat (Gly-Pro-Hyp) of type I collagen. This peptide was capable of binding to single crystal HAp and templating the nucleation and growth of crystalline HAp mineral in a sequence- and composition-dependent manner. (Chung et al.). In another study, polylysine and polyleucine based block copolypeptides (K170L30) were found to form gels at very low concentrations in aqueous media. The block copolypeptides have been used as templates for forming self-assembled calcium phosphate nanocomposites. The synthesis method allowed for simultaneous formation of the self-assembled block copolypeptide gel and of the inorganic phase. The inorganic contents accounted for over 50 wt% in the nanocomposite, approaching the inorganic content in bone (Hu et al. 2009). Thermoreversibly gelling block copolymers (Pluronic F127) conjugated to hydroxyapatite-nucleating peptides (DSKSDSSKSESDSS) were used to template the growth of inorganic calcium phosphate in aqueous solutions. The inorganic phase in the organic/inorganic nanocomposite was confirmed to be HAp. This work offered a route for the development of novel, self-assembling, injectable nanocomposite biomaterials for potential orthopedic applications (Yusufoglu et al. 2008).
Self-assembling peptide amphiphiles have great potential as templates for nanofabrication. In 2001, a lipopeptide was designed and synthesized for biomineralization by the Stupp group (Hartgerink et al. 2001). In Fig.7 a and b, the C16 tail was connected to the N terminal of a peptide sequence which contained four cysteines, three glycines, a single phosphorylated serine and a cell adhesion ligand RGD (C16–C4G3S(p)RGD–OH). The C16 tail was hydrophobic and the peptide sequences were hydrophilic. These peptide amphiphlies self-assembled into cylindrical micellar structures in aqueous phase. C16 acyl tails packed themselves in the center of the micelle, while the peptide sequences formed β-sheets at the outside. There were disulfide bonds that formed by cross-linking of the four cysteine residues in the middle of the molecules, making the self-assembled nanofibers robust and impervious to pH variation (Fig.7 c). The nanofibers were then used to direct the
mineralization of HAp. The HAp nucleated on the surfaces of the lipopeptide nanofibers and its crystals grew with their c-axes oriented along the long axes of the nanofibers. This alignment was the same as that observed between collagen fibers and HAp crystals in bone (Hartgerink et al. 2001; Zhao et al. 2010).
Shorter peptide I3K may form nanotubes with diameters about 10 nm and lengths over 5 mm. The nanostructure from this ultra-short peptide indicated that the amphiphilicity of a peptide amphiphile can be balanced between the length of a peptide sequence and the size of hydrophobic amino acids. I3K molecules were thought to initially interdigitate with each other through the hydrophobic interaction among the I3 tails, forming bilayer fragments. The self-assembly was driven by the hydrophobic affinity between isoleucine residues with the I3 tails packed in the middle and the K residues projected at the outside, facing the water. The peptide bilayer fragments then further assembled into twisted ribbons.
The fusion of the helical ribbons resulted in the formation of stable nanotubes, indicating the strong driving force along the main axial direction of the nanotubular structure. Fig.8 shows the self-assembling process. Because of their extreme stability against heating or exposure to organic solvents, I3K nanotubes were used as templates for silicification from the hydrolysis of TEOS (tetraethoxysilane) precusor. The lysine groups on the inner and outer nanotube surfaces catalyzed the silicification, leading to the formation of silica nanotubes (Xu et al. 2010).
5. DNA and RNA-mediated bioinspired synthesis
DNA and RNA can self-assemble into well-defined secondary and tertiary structures at the nanoscale, which provide an ideal template for the formation of nanocrystals (Loweth et al. 1999). DNA templated gold nanoparticles have attracted much attention, as the self-assembled DNA nanostructures offer programmable scaffolds to organize the gold nanoparticles (Ding et al. 2010; Zhang et al. 2006; Wang et al. 2010 ). A self-assembled two-dimensional (2D) DNA nanogrid was used as a template to grow 5-nm gold nanoparticles (Au NPs) into periodic square lattices, as shown by Fig. 9. The center-to-center distance between neighboring particles was about 38 nm. These accurate controlled Au NPs distribution may find applications in nanoelectronic and nanophotonic devices (Zhang et al. 2006).
A chemically well-defined bio-core in an inorganic shell nanohybrid material was recently reported. It consisted of a DNA molecule as the bio-core with a size of 100 nm and a spherical inorganic nanoshell reassembled with exfoliated layered metal hydroxide (MH) with an overall thickness of 10 nm. The negatively charged DNA molecules can be encapsulated into a positively charged inorganic nanocavity of self-assembled MH nanosheets, as illustrated in Fig.10. Due to the pH-dependent solubility of the MH nanoshell, the DNA can be encapsulated and released, which play a crucial role in maximizing the stability of base sequence-manipulated and probe-functionalized DNA molecules with designed information. Therefore, these hybrid materials could be used as advanced gene delivery systems and a biomedical diagnostics system for DNA-based information (Park et al. 2010).
A multi-lamellar structure was formed by complexes of anionic DNA and cationic liposomes self-assembly. The two-dimensional lipid sheets confined a periodic one-dimensional lattice of parallel DNA chains, as shown by Fig.11. These simple DNA-membrane templates could be used for controlled CdS crystalline synthesis. Cd2+ ions condensed on the DNA chains, and subsequently reacted with H2S to form CdS nanorods. Depending on the charge of the membrane, different concentrations of Cd2+ ions condense onto the template, and different morphologies of CdS were formed. The degree of template overcharging was determined by the relative stoichiometry of DNA and cationic membrane lipids (Liang et al. 2004).
One kind of DNA molecules were reported to mediate the nucleation and growth of the calcium carbonate particles. CaCO3 microspheres with different surface morphologies were prepared in the presence of the DNA, indicating that DNA could induce biomineralization in the biological system. It was found that the DNA concentration influenced on the surface structures of CaCO3 particles significantly. This research provided new insight into the morphological control of CaCO3 and other inorganic materials (Cheng et al. 2010).
The Kelley group investigated the role of RNA secondary structure on the growth of CdS nanocrystals. They showed that a folded wild-type tRNA (wtRNA) and an unfolded mutant tRNA (mtRNA) of identical length were both able to mediate the formation of CdS during its spontaneous precipitation from solution, but they saw differences in the average nanocrystal sizes and size distributions. A narrow distribution around 6 nm diameter particles was found for particles grown with wtRNA, while mtRNA generated a bimodal distribution of 7 and 11.5 nm diameter particles. This is a good illustration that a biomolecule can affect the nanocrystal size (Ma, Dooley, and Kelley 2006).
6. Polysaccharide-mediated bioinspired synthesis
A slow but increasing interest has been developing to explore the role of polysaccharides in biomineralization, despite the fact that they have been prevalent since the early stages of evolution. Single types of polysaccharides are typically not associated with biominerals. Only hydroxylated, carboxylated, or sulfated polysaccharides, or those containing a mixture of these functional moieties, are found in biominerals (Arias and Fernández 2008). Chitin is the second most abundant natural polymer after cellulose on earth. It is a linear polysaccharide of β-(1-4)-2-acetamido-2-deoxy-d-glucose. The chemical structure of chitin is very similar to that of cellulose, with a hydroxyl group replaced by an acetamido group. Pure chitin with 100% acetylation does not exist in nature. Chitin tends to form a co-polymer with its N-deacetylated derivative, chitosan. Chitosan is a polymer of β-(1-4)-2-amino-2-deoxy-d-glucose. The chemical structures of cellulose, chitin, and chitosan are shown in Fig.12 (Meyers et al. 2008).
Chitosan composite materials have attracted much research interest in bone tissue engineering due to their minimal foreign body reactions, intrinsic antibacterial nature, biocompatibility, biodegradability, and ability to be molded into various geometries and forms. Recently, grafted chitosan natural polymer with carbon nanotubes has been incorporated to increase the mechanical strength of artificial bone (Venkatesan and Kim 2010). Laminated HAp/chitosan nanocomposites and nano-HAp/chitosan-pectin composites were prepared and showed improved strength, especially in moist environments. This combination can be expanded to other HAp-biopolymer systems, thus offer a new insight for fabricating biomimetic nanocomposites (Li et al. 2010; Zuo et al. 2010).
Chitosan was also used as organic template to form HAp nanocrystals. Spindle shaped HAp with 30- 40 nm length and 7- 8 nm width was synthesized through the biomimetic method with chitosan as template. The spindle shaped nano HAp grew in a 0.5wt% chitosan solution for 7 days. The crystallinity of samples increased with the aging time. The HAp powders synthesized with chitosan as templates had good thermal stability up to 800 °C (He et al. 2007).
Design and synthesis of bacterial cellulose/HAp nanocomposites was reported for bone healing applications using a bioinspired approach. Bacterial cellulose with various surface morphologies (pellicles and tubes) was negatively charged by the adsorption of carboxymethyl cellulose to initiate nucleation of calcium-deficient hydroxyapatite (cdHAp). The cdHAp was grown
7. Conclusions and outlook
In summary, in the recent past, there has been remarkable progress in the development of bioinspired procedures for controlling inorganic crystal nucleation and growth, especially at the nanoscale. Biomolecules have been successfully utilized to produce a variety of self-assembled structured inorganic materials under relatively mild conditions. Biomolecules have been found to be able to direct or modify the shapes, sizes, crystal structures, and other properties of the synthesized inorganic materials. Examples of such bioinspired inorganic nanostructures include HAp, SiO2, Fe3O4, CdS, TiO2, ZrO2, gold and silver
In the recent past, many biomolecules promoting materials synthesis have been identified. The number of inorganic materials that could be used for bioinspired synthesis has also been expanded. However, our fundamental understanding of these existing topics must be furthered in order to more fully harness the potential of biomolecules for material synthesis. There are also a number of interesting and powerful new concepts that have received only a little attention or remain unexplored. Design of more hierarchically self-assembled biomolecules that could template and direct the inorganic formation is also required. With the continued attention and ingenuity of researchers from diverse disciplines, the future of biomimetic materials synthesis promises to be exciting, dynamic, and rich in applications.
This work was supported by the U.S. Department of Energy, Office of Basic Energy Science, Division of Materials Sciences and Engineering. The research was performed at the Ames Laboratory. Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract No. DE-AC02-07CH11358.