The ability of proteins to direct mineral formation is clearly recognisable by the simple observation that the shape, size, and mineral composition of seashells are faithfully reproduced by a species from generation to generation. This implies that the control of mineral formation is under some form of genetic control - most importantly at the level of protein expression. Many biomineral systems require the orchestration of multiple protein partners which have proved difficult to isolate but some clear examples exist of proteins which control biomineral formation [6, 7]. There are also some compelling examples, in the literature, of naturally occurring protein systems whose functions have been subverted to the formation of novel new materials [2, 8]. Other protein systems such as collagen gels (gelatine) have been responsible for much of our photographic technology through the encapsulation of nanocrystals of silver salts. Additionally, the synthesis of polypeptides of non-biological origin for inorganic materials applications is a new and novel direction in biomimetic synthesis.

Ferritin, an iron storage protein, is found in almost all biological systems [9]. It has 24 subunits surrounding an inner cavity of 60-80A diameter where the iron is sequestered as the mineral ferrihydrite (Fe2O3.nH2O - sometimes also containing phosphate). The subunits of mammalian ferritin are of two types, H (heavy) and L (light) chain which are present in varying proportions in the assembled protein. These join together forming channels into the interior cavity through which molecules can diffuse [ 10, 11]. The native ferrihydrite core is easily removed by reduction of the Fe(III) at low pH ~4.5, and subsequent chelation and removal of the Fe(II). It is also easily remineralised by the air oxidation of Fe(II) at pH> 6. The oxidation is facilitated by specific ferroxidase sites which have been identified by site directed mutagenesis studies, as E27, E62, His65, E107 and 414], which proceed through a diferric-^-peroxo intermediate [12, 13] on the pathway to the formation ofFe(III). These sites are conserved on H chain subunits but absent from L chain subunits. Similar studies have also identified mineral nucleation sites which are comprised of a cluster of glutamates (E57, E60, E61, E64, and 67) inside the cavity. These sites are conserved on both H and L chain subunits. The charged cluster of glutamates that is the nucleation site, probably serves to lower the activation energy of nucleation by strong electrostatic interaction with the incipient crystal nucleus.

Figure 1. The reaction pathways for nanoparticle synthesis using ferritin. (a) Mineralization,/demineralisation, (b) metathesis mineralization, (c) hydrolysis polymerisation. Source: Reprinted by permission from Nature [17] copyright 1991 MacmillanMgazinesLtd.

The intact, demineralised protein (apoferritin) provides a spatially constrained reaction environment for the formation of inorganic particles which are rendered stable to aggregation. Ferritin is able to withstand quite extreme conditions of pH (4.0-9.0) and temperature (up to 85°C) for limited periods of time and this has been used to advantage in the novel synthesis and entrapment of non-native minerals. Oxides of Fe(II/III) [14-16], Mn(III) [17, 18], Co(III) [19], a uranium oxy-hydroxide, an iron



Figure 1. The reaction pathways for nanoparticle synthesis using ferritin. (a) Mineralization,/demineralisation, (b) metathesis mineralization, (c) hydrolysis polymerisation. Source: Reprinted by permission from Nature [17] copyright 1991 MacmillanMgazinesLtd.


Apoferritin sulphide phase [20] prepared by treatment of the ferrihydrite core with H2S (or Na2S) as well as small semiconductor particles of CdS [21] have all been synthesised inside the constrained environment of the protein. The recent advances in site directed mutagenesis technology holds promise for the specific modification of the protein for the tailored formation of further novel materials.

3.1.2. Bacterial S-layers

The S-layer is a regularly ordered layer on the surface of prokaryotes comprising protein and glycoproteins. These layers can recrystalise as monolayers showing square, hexagonal or oblique symmetry on solid supports [22], with highly homogeneous and regular pore sizes in the range 2 to 8 nm. These proteins have also been implicated in biomineralisation of cell walls and their synthetic use is a great example of the biomimetic approach wherein an existing functionality is utilised for a nonbiological materials synthesis. The two-dimensional crystalline array of bacterial S-layers have been used as templates for ordered materials synthesis on the nanometer scale, both to initiate organised mineralization from solution [23, 24] as well as ordered templates for nanolithography [25]. Both techniques have produced ordered inorganic replicas of the organic (protein) structure.

Treatment of an ordered array of bacterial S-layers (having square, hexagonal or oblique geometry) to Cd2+ followed by exposure to H2S results in the formation of nanocrystalline CdS particles aligned in register with the periodicity of the s-layer. Ordered domains ofup to 1 ^m were observed (Figure 2).

Figure 2. Transmission electron micrographs of self-assembled Slayers: (a) S-layer prior to mineralization (stained), (b) after CdS mineralization (unstained). Scale bars = 60 nm. Reprinted by permission from Nature [23] copyright 1997 Macmillan Mgazines Ltd.

The interaction of S-layers with inorganic materials for the nanofabrication of a solid state heterostructure relies on the ability to crystallise these proteins into two-dimensional sheets. The crystallised protein was initially coated by a thin metal film of Ti which was allowed to oxidise to TiO2. By ion milling, the TiO2 was selectively removed from the sites adjacent to the protein leaving a hole with the underlying substrate exposed. Thus, the underlying hexagonal packing arrangement of the 2-d protein crystal layer has been used as a structural template for the synthesis of porous inorganic materials.

3.1.3. Anisotropic structures - Tobacco mosaic virus

It was recently reported that the protein shell of tobacco mosaic virus (TMV) could be used as a template for materials synthesis [26, 27]. The TMV assembly comprises approximately 2 130 protein subunits arranged as a helical rod around a single strand of RNA to produce a hollow tube 300 nm x 18 nm with a central cavity 4 nm in diameter. The exterior protein assembly of TMV provides a highly polar surface, which has successfully been used to initiate mineralization of iron oxyhydroxides, CdS, PbS and silica (Figure 3). These materials form as thin coatings at the protein solution interface through processes such as oxidative hydrolysis, sol-gel condensation and so-crystallisation and result in formation of mineral fibres, having diameters in the 20-30 nm range. In addition, there is evidence for ordered end-to-end assembly of individual TMV particles to form mineralised fibres with very high aspect ratios, of iron oxide or silica, over 1 ^m long and 20-30 nm in diameter.

Figure 3. Strategies for nanoparticle synthesis using tobacco mosaic virus. Reprinted by permissionfrom Adv. Mater. [26] copyright 1999 Wiley - VCH Verlag ,

Figure 3. Strategies for nanoparticle synthesis using tobacco mosaic virus. Reprinted by permissionfrom Adv. Mater. [26] copyright 1999 Wiley - VCH Verlag ,

3.1.4. Spherical virus protein cages

Spherical viruses such as cowpea chlorotic mottle virus (CCMV) have cage structures reminiscent of ferritin and they have been used as constrained reaction vessels for biomimetic materials synthesis [8, 27]. CCMV capsids are 26 nm in diameter and the protein shell defines an inner cavity approximately 20 nm in diameter. CCMV is composed of 180 identical coat protein subunits that can be easily assembled in vitro into empty cage structures. Each coat protein subunit presents at least nine basic residues (arginine and lysine) to the interior of the cavity, which creates a positively charged interior interface that is the binding site of nucleic acid in the native virus. The outer surface of the capsid is not highly charged, thus the inner and outer surfaces of this molecular cage provide electrostatically dissimilar environments.

Mineralized virion

Figure 4, Strategy for biomimetic synthesis using cowpea chlorotic mottle virus. Adapted from [8].

The protein cage of CCMV was used to mineralise polyoxometallate species such as NH4H2W12O42 at the interior protein-solution interface. It was suggested that mineralization was electrostatically induced at the basic interior surface of the protein where the negatively charged polyoxometalate ions aggregate, thus facilitating crystal nucleation. The protein shell therefore acts as a nucleation catalyst, similar to the biomineralisation reaction observed in ferritin, in addition to its role as a size constrained reaction vessel.


Some interesting synthetic polypeptides are emerging in the field of materials chemistry, in particular dendritic polymers based on poly(amidoamine) or PAMAM dendrimers. These polymers are protein mimics in that they too are polyamides, have fairly well defined structural characteristics (topology), and can accommodate a variety of surface functional groups. They are roughly spherical in shape and they can be terminated with amine, alcohol, carboxylate or ester functionalities. Two groups have demonstrated that pre treatment of either alcohol or amine terminated dendrimers with Pt(II), Pd(II), Cu(II) or HAuCl4 followed by chemical reduction using hydrazine or borohydride resulted in the stabilisation of nanoparticles of the metals [28-3 1]. These were originally suggested to be stabilised within the matrix of the dendrimer sphere. In addition it has also been shown that dendrimers having different surface functionalities are able to stabilise nanoparticles of CdS (amine terminated [32]) and ferrimagnetic iron oxides (carboxyl terminated [33]). In this regard the functionalised dendrimer acts as a nucleation site by selective binding of the precursor ions and additionally passivates the nanoparticle by steric bulk to prevent extended solid formation.

A gel is a loosely cross-linked extended three dimensional polymer permeated by water through interconnecting pores. Gels are used as reaction media for crystal growth when especially big, defect free crystals are desired. Solutes are allowed to diffuse toward each other from opposite ends of a gel-filled tube. This creates a concentration gradient as the two fronts diffuse through each other, giving rise to conditions of local supersaturation. The gel additionally serves to suppress nucleation that allows fewer crystals to form, thus reducing the competition between crystallites for solute molecules, and the result is larger and more perfect crystals. It also acts to suppress particle growth that might otherwise occur by aggregation. Gels are easily deformed and so exert little force on the growing crystal [34].

Gelatine is used extensively in the photographic process for the immobilisation of silver and silver halide micro crystals. The most commonly used photographic emulsion comprises a gelatine matrix with microcrystals of silver halides distributed throughout. While gelatine is the most common matrix, albumen, casein, agar-agar, cellulose derivatives, and synthetic polymers have all been used as gel matrices. The silver halide crystals vary in size from 0.05^m to 1.7^m depending on the film type. Exposure of the film to light forms a "latent image" (a small critical nucleus of silver metal) that will catalyse the reduction (and growth of a silver crystal) of that particular grain when the film is developed. The development process is the chemical reduction of the silver halide grains and the growth, in its place, of a microcrystal of silver metal. The matrix serves to keep these microcrystals separate and prevent their aggregation that would result in loss of image resolution.

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