Previous studies discovered the formation of unusual long Escherichia coli cell filaments induced by S-layer protein expression [2, 9]. Such biological structures provide a promising matrix for technical applications such as the development of microcontainers or hollow metallic microwires. Especially gram-negative cells like E. coli are attractive for such applications. They possess a comparatively fragile cell wall that can be easily destroyed. E. coli can be easily cultivated giving a high yield of biomass and can be used for multifaceted applications. In the present study we used the cells for the synthesis of polyelectrolyte hollow capsules and investigated the possibility to use them as substrate for the functionalisation with proteins and metal nanoparticles.
The development of polyelectrolyte capsules was investigated by several groups using different kinds of templates postulating that those capsules are ideal candidates for applications in the areas of drug delivery, sensing and catalysis . Sukhorukov and co-workers coated polystyrene and melamine formaldehyde latex particles with polyelectrolyte multilayers and dissolved the core , while Yu and others described the production of polymeric capsules with pre-loaded proteins based on mesoporous silica capsules which were finally removed . The encapsulation of spores was described by Balkundi and co-workers aiming the development of environmental compatible materials for agriculture . Franz and others investigated the encapsulation of microbes with different polyelectrolyte combinations and the following substrate uptake properties of enclosed bacteria . These studies used the benefit of layer-by-layer technique which enables the variation of thickness, composition, and function of these assemblies by tuning the layer number, the species deposited, and the assembly conditions .
The present study describes the development of polyelectrolyte hollow tubes based on S-layer expressing E. coli cells which were fixed in glutaraldehyde and combined with the polyelectrolytes PSS (sodium poly(styrene sulfonate)) and PAH (poly(allylamine hydrochloride)) and a final NaOCl treatment. Other papers that used cells as template described the combination of negatively charged surfaces which were afterwards coated with the polycation followed by washing steps and a polyanion . In contrast, the assembly of polyelectrolyte layers on E. coli filaments necessitated the starting with a polyanion to a probably negatively charged cell surface . The combination of the glutaraldehyde fixed cells with polycationic solution induced an irreversible agglomeration of the cells. In comparison they stayed in suspension well separated when they were initially incubated with a polyanionic solution. Responsible for cell agglomerations which were observed after polycation incubation are potentially single positive groups at the mainly negative charged bacterial cell surface. Potentially, in the presence of polycations very high attractive forces operate between these cells which lead to agglomerations. However, negative polymers will saturate the few positive groups at the bacterial cell surface resulting in a very consistent charge distribution. So, the negative polymer works potentially as solubiliser.
Moya et al. described that treatment of polyelectrolyte encapsulated cells with NaOCl solution changed the chemical composition of the capsules dramatically. They observed the oxidation of the amino groups of polyallylamine to nitriles, nitroso-, nitro-, azo- and carbonyl groups and the disappearance of positive charges. Coevally the polymer chains were cross-linked with covalent bonds. Finally, the amount of PSS is strongly reduced to 10% of the original value. Moya et al. justified the stability of these capsules with the combination of cross-linking and hydrophobic interaction . In our work, the use of the polyelectrolytes PSS and PAH in combination with sodium hypochlorite resulted evidently in the formation of uniformly coated stabile filamentous hollow capsules. However, round about 1% of the coated cells remain intact during NaOCl treatment. This observation leads to the assumption that these cells were not treated efficiently with NaOCl, perhaps because of their localisation in the lid of the reaction tube during incubation.
The surface coating of these tubes with surface layer polymer proteins aimed the synthesis of two dimensional crystal lattice which hold regular ordered nanopores with uniform bonding characteristics. Toca-Herrera and co-workers described the recrystallisation of S-layer proteins on polyelectrolyte surfaces and demonstrated by AFM that the combination of a final PAH layer with surface layer proteins hinder the recrystallisation of the proteins . However, our light microscopic studies indicate that the binding of S-layer polymer proteins to polyelectrolyte capsules is enhanced with PAH as final polyelectrolyte capsule coating. It can be assumed that the constitution of PAH was influenced by sodium hypochloride treatment. Probably the uniform negative charges of the polyelectrolyte surface support the binding of S-layer polymer proteins via electrostatic attractive forces. The complete S-layer coating of the polyelectrolyte capsule surface is not assumed. S-layers were used to bio-functionalise the new-designed polyelectrolyte tubes.
In previous works self-assembling of bio-molecules to capsules or filaments has been reported several times and methods to functionalise these structures have been established. Mbindyo and co-workers reported the DNA-directed assembly of gold nanowires 0.2 μm in diameter and up to 6 μm in length , while the recognition capabilities of DNA, which induced the targeted attachment of functional wires were described by Braun and others . Vauthey and co-workers described the molecular self-assembly of surfactant-like peptides to form nanotubes and nanovesicles . The ability of protein coated peptide tubules to recognise and bind the protein complementary molecules in solution was investigated by Douberly and co-workers , while Yang and others analysed microtubules as templates for fabricating metallic nanowires . Sugunan and others describe the formation of microwires of gold nanoparticle coated hyphea of Aspergillus strains while growing of initial spores in colloidal gold solution . The assembly of nanoparticles on filamentous fungi generates microwires with extraordinary length. However, the diameter of the distinct shorter E. coli filament based polyelectrolyte capsules is smaller. The removal of the inner organic material of the E. coli filaments is much easier than the one of gold nanoparticle encapsulated filamentous fungi. The final synthesis of palladium nanoparticles in the pores of S-layer polymer proteins seems to produce distinct smaller nanoparticles than the glutamate stabilized gold nanoparticles. Kahraman and others studied the polyelectrolyte encapsulation of E. coli and Staphylococcus cohnii with additional gold and silver nanoparticles  while Zhang and co-workers analysed the functionalisation of bacterial cell walls with magnetic nanoparticles . Fakhrullin and co-workers gave in their review a detailed overview over the studies which focus the functionalisation of living cells with polymers and nanoparticles .
The application of surface layer proteins as template for the synthesis of nanoparticles is a well established method [35, 37, 51]. S-layers are an interesting starting material for the synthesis of bio-inorganic composite materials that are promising for various applications, e.g. catalysts . The proteins that are decorated with catalytic active nanoparticles can be fixed on carrier materials. The S-layer properties (amino acid composition, array symmetry and pore size) determine the nanoparticle properties like size and distribution. In previous work EXAFS and ATR-FT-IR analyses proved that carboxyl groups of the proteins are involved in the binding of the Pd(II) complexes [35, 51]. In the present study we used S-layer coated polyelectrolyte filaments as carrier material for synthesis of Pd(0) particles. The immobilised S-layer proteins are able to bind Pd(II) complexes, thus enabling the synthesis of palladium particles by the addition of a reducing agent.
The newly designed bio-functionalised polyelectrolyte tubes that are described in this paper are unique due to its starting material. Specific regulations of template organism, temperature and amount of activator induce the formation of Escherichia coli filaments with defined diameter and cell wall stability. The template bacteria provide up to several 100 μm long structures with defined 0.8-1 μm in diameter which were encapsulated by layer-by-layer method with polyelectrolytes. After removing the bacterial core these polyelectrolyte hollow capsules can be bio-functionalised with S-layer polymer proteins which support the synthesis of metal nanoparticles in the protein pores. In conclusion, these filamentous polyelectrolyte tubes may provide an interesting matrix for the development of microcontainers and metal microwires with possibly novel physical and chemical properties. In combination with S-layer coupled palladium nanoparticles these materials could find application as novel catalysts or in the preparation of conductive metal microwires in electrical devices. Such developments are part of future work.