De Cuyper M., Bulte J.W.M. - Physics and chemistry basis of biotechnology (Vol. 7) (2002)(en)

Aleksey Nedoluzhko and Trevor Douglas

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 of up to 1 µm 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.

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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 twodimensional 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 socrystallisation 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 µm 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 ,

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Aleksey Nedoluzhko and Trevor Douglas

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.

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 NH4H2W12 O42 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.

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3.2. SYNTHETIC POLYAMIDES - DENDRIMERS

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.

3.3. GELS

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 0.05µm to 1.7µm 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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Aleksey Nedoluzhko and Trevor Douglas

3.4. COMPOSITE MATERIALS

Proteins that have been isolated from biominerals exhibit a number of the properties mentioned in the preceding sections such as oriented nucleation, and confined reaction environments. The production of biocomposite ceramics is a low temperature route to strong, lightweight materials that has not yet been fully exploited. In bone, hydroxyapatite crystals are found in spaces within the collagen fibril. Purified collagen serves as a matrix for calcium phosphate growth in attempts to study that process and to create synthetic bone-like material. Matrix proteins isolated from bivalves have been shown to mediate nucleation and growth of calcium carbonate [35-38]. These materials are composites of microscopic crystals held together by a protein "glue" and have the advantages of both the hardness of the inorganic material, and the flexibility of the organic matrix. Composite materials such as these often have high fracture toughness thought to arise from interruption, by the protein, of the cleavage planes in the inorganic crystals. For example, the calcite crystal cleaves easily along the (104) planes, In the sea urchin skeleton the crystal fractures conchoidaly (like glass) and not cleanly along the (104) planes of calcite. It is suggested that this is due to the protein that is occluded within the crystal, preventing the cleavage along the (104) plane and thereby increasing the strength of the inorganic phase. These proteins have been isolated and shown to produce the same conchoidal fracture in synthetic calcite crystals grown in its presence. These materials are the inspiration for a new generation of materials incorporating both natural and synthetic polymers.

Figure 5. Schematic representation of organised surfactant assemblies. Reprinted by permission from Chem.Rev. [15O] copyright 1987ACS Publications.

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3.5. ORGANIZED SURFACTANT ASSEMBLIES

Although surfactant assemblies are not ‘biological’ in the general sense of the word, they often give a good opportunity to mimic biomineralisation processes. These assemblies are schematically represented in Figure 5. With respect to the methods of their application in the synthesis of inorganic materials they may be separated into two groups on the basis of their geometry. Micelles, microemulsions and vesicles form one class of the assemblies, whose specific feature is maintenance of a confined environment for the crystal growth. Layered structures are the other class of assemblies, for which the periodicity of layered arrays is essential.

The phase behaviour of surfactant – water mixture depends on the water-to- surfactant ratio w. Another important parameter – cmc (critical micelle concentration) represents the constraint surfactant concentration for the formation of micelles.

3.5.1. Confined surfactant assemblies

3.5.1.1. Reverse micelles (water-in-oil microemulsions)

General principles for the synthesis of inorganic materials in these environments involve dissolution of reactants in the aqueous phase and the subsequent reactions, which occur due to micelle collisions, accompanied by the exchange of their aqueous phases.

Figure 6. The dependence of particle size on water-to-surfactant ratio. CdS (triangles), PbS [squares), CdyZn1-yS [circles), CdyMn1,yS (+), ZnS (X), Ag [octagons). Reprinted by permission from Langmuir [151] copyright 1997 ACS Publications.

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Aleksey Nedoluzhko and Trevor Douglas

First reports on the application of reverse micelles for the synthesis of inorganic nanoparticles were devoted to the synthesis of precious metals. In the study of Boutonnet et al [39] metal cations dissolved in water pools of water/CTAB/octanol or water/ pentaethylene glycol dodecyl ether/hexane micellar solution, were reduced by hydrazine. The formed particles (Pt, Pd, Rh and Ir) were reported to have a narrow size distribution (standard deviation 10%) being in the size range 30 – 50 Å The formation of gold particles in water-in-oil microemulsions was first reported by Kurihara et a1 [40] who studied the reaction of HAuCl4 reduction by laser photolysis and pulse radiolysis. Again, higher uniformity of Au particles obtained in micellar solution comparing to those obtained in homogeneous solution was reported.

Studies of particle formation in reverse micelles were initially oriented for the synthesis of highly dispersed catalysts, and in a similar vein was the work of Meyer et a1 [41] describing synthesis of cadmium sulphide nanoparticles. AOT micelles containing Cd2+ were prepared in isooctane and then exposed to H2S. The possibility to use formed CdS as a photosensitiser was demonstrated. In the study by Lianos and Thomas [42] CdS was obtained by mixing micellar (heptane/AOT/water) solutions of cadmium perchlorate and sodium sulphide. The increase of particle size with water-to- surfactant ratio w was reported. This dependence was shown to occur only at w < 15, with the particle size being almost constant at higher w values [43] (Figure 6). Maximum particle size was reported to be twice that of CdS particles prepared with a two-fold excess of S2-, than for those prepared in a two-fold excess of Cd2+ .

Figure 7, Changes in absorbance spectrum during CdSe crystal growth in reverse micelles. Reprinted by permission from J.Am.Chem.Soc. [44] Copyright 1988 ACS PubIications.

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In the paper of Steigenvald et a1 [44] the formation of CdSe in heptane/AOT/water micelles was reported. The synthesis was performed by the fast addition of bis(trimethylsily1)selenium to the micellar solution of Cd2+ . Particle growth was monitored by changes in light absorption spectrum, which exhibited characteristic onset shifting to longer wavelengths on subsequent additions of the selenium derivative (Figure 7). Also, the possibility to change the properties of the particle surface to strongly hydrophobic was demonstrated using phenyl-bis(trimethylsi1yl)selenium reacting with excess Cd2+ atoms on the surface of CdSe. Although precipitate was formed, it could be subsequently redissolved in non-polar solvents yielding CdSe colloid. Later, the authors demonstrated the use of water-in-oil microemulsions for the synthesis of CdSe/ZnS core-shell structures [45]. Difference in bond length for these compounds is 13 %, and the crystal lattices were found not to match each other. The important result of this study was the synthesis of 35 – 40 Å CdSe particles, covered with 4 Å thick ZnS layer. Deposited ZnS ‘fills’ deep surface trap states of CdSe, providing strong and stable luminescence of the composite particles.

In the work of Towey et a1 [46] changes in absorption spectrum during CdS particle growth under a variety of experimental conditions were monitored using stopped-flow technique, and the attempt to analyse growth kinetics quantitatively was presented. The authors concluded that inter-droplet exchange of solubilised reactants was the ratedetermining step.

Petit et a1 [47] proposed to use metal-substituted surfactants, such as cadmiumlauryl sulphate or cadmium-AOT for the synthesis of CdS, as the source of the metal cation. Later, the same approach was employed by the authors for the synthesis of Cu [48] and Ag [49] metal particles. Copper metal particles were obtained by reduction of Cu(AOT)2 in water/isooctane with either hydrazine (added by injection) or borohydride (introduced in the form of water/AOT/isooctane micellar solution). The properties of the particles formed in the reaction were found to depend greatly on the nature of the reducing agent. While in the reaction with hydrazine small (20 - 100 Å) metal particles were formed, whose size, as usual, increased with w value, the reaction with borohydride yielded large (up to 28 nm) particles exhibiting anomalous dependence on w. It was found that the increase of w in the case of borohydride led to a progressive formation of copper oxide instead of Cu metal. Above w = 8 pure CuO was reported to appear even in the absence of oxygen.

The synthesis of silver metal particles was demonstrated using the same general approach. Although the average particle size was clearly dependent on w (from 30 Å (w = 5) to 70 Å, (w = 15)) the size distribution was rather broad (s= 30 - 40 %).

Chang et a1 [50] demonstrated synthesis of silica particles by the hydrolysis of tetraethoxysilane in water-in-oil microemulsions in the presence of ammonium hydroxide and hexanol, acting as co-surfactant. SiO2 had a spherical structure. The size of the formed spheres could be controlled by the reaction conditions in the range 40 - 300 nm with standard deviation of 5%. Also, the possibility to synthesise mixed SiO2- CdS spheres was demonstrated, with CdS being incorporated in different ways as core, shell, or intermediate sphere, or small (24 Å size) inclusions (volume or surface) (Figure 8), or surface patches.

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Figure 8. Silica spheres containing homogeneously distributed CdS inclusions,formed in water-in-oil microemulsions. Reprinted by permission fromJ.Am.Chem.Soc. [50] Copyright 1994 ACS Publications.

General strategies described above have been successfully employed for the synthesis of other semiconductors (PbS and CuS [51], TiO2 [52], Se [53]) as well as magnetic materials (Fe3O4 [54], barium ferrite [55], iron ferrite [56], cobalt metal [57]) and other solids (zincophosphate [58], BaSO4 [59]).

Figure 9. Mechanism for the formation of elongated BaSO4 crystal. Reprinted by permission from Chem. Mater. [59] Copyright 1997 ACS Publications.

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The microemulsion method for the inorganic synthesis was shown to allow, in some cases, control of the shape of the inorganic particles. It was demonstrated that the shape of copper metal particles formed in isooctane/AOT/water micelles depends on w value [60]. Both at w < 5.5 and at w > 34 nearly all particles are spherical, however, elongated particles are formed at the intermediate values of w. Within this region there is a further dependence of the particle shape on w. For example, at w = 12 highly elongated cylinders are formed, and at w = 18 particles of different shapes and sizes appear. The authors explain these data by changes in micellar solution phase with the variation of w.

In the study of Hopwood and Mann [59] on the synthesis of BaSO4 using isooctane/AOT/water reverse micelles the variation of w value led to even more drastic changes in the shape of the formed crystals. While at w = 5 particles of amorphous BaSO4 were formed, micellar systems with w > 10 led to the formation of highly elongated filaments, with lengths up to 100 µm and aspect ratios of 1000. Formation of the filaments was not observed when AOT had been changed to another surfactant. The authors proposed a mechanism for the filaments formation, which involved the anisotropic binding of the surfactant to BaSO4. Since the crystal growth occurs only on the unbound crystal surface, the crystal progressively elongates. (Figure 9).

Recently, the possibility for a self-assembly of nanoparticles formed in reverse micelles was reported [61]. Nanocrystals of barium chromate synthesised in AOT reverse micelles were shown to form periodic arrays due to the interdigitation of surfactant monolayers (Figure 10). By variation of reactant molar ratio it was possible to change the shape of formed BaCrO4 nanoparticles, which, simultaneously, lead to different structures of assembled aggregates.

Figure 10. Rectangular supperlattice of BaCrO4 nanoparticles prepared in AOT microemulsions (w = 10)from equimolar amounts ofBa2+ and CrO42-. Scale bar = 50 nm. Reprintedby permissionfrom Nature [61]. Copyright 1999Macmillan MgazinesLtd.

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