Final Report Summary - MEM-S (Bottom-up design and fabrication of industrial bio-inorganic nano-porous membranes with novel functionalities based on principles of protein self-assembly and biomineralization)
Executive Summary:
There is strong interest in the development of novel functionalized membranes which can be used as microsieves, as a component of integrated analytical systems, in food processing, drug discovery and diagnostic applications. This project was based on a combination of three break-through technologies, developed by the applicants in the past, with high impact for nano(bio)technological application: (i) the S-layer technology allowing the construction of nanoporous protein lattices, (ii) the biocatalytic formation of inorganic materials by silicatein, a group of unique enzymes capable to catalyze the formation of porous silica from soluble precursors, and (iii) the sol-gel technique for encapsulation (immobilization) of biomolecules serving as biocatalyst or as a component of sensors. The goal of this project was to design and fabricate - based on molecular biology inspired approaches - nano-porous bio-inorganic membranes with novel functionalities for industrial application. These membranes were formed by S-layer proteins, which are able to assemble to highly ordered structures of defined pore-size, and recombinant silicateins or silicatein fusion proteins. The innovative functionalized membranes developed in this project exploit two principles: (i) protein self-assembly and (ii) enzymatic (silicatein-mediated) deposition of inorganic material used for reinforcement of the membranes as well as for encasing biomolecules, providing the membranes with new functionalities. The S-layer-coated surfaces could be functionalized by binding of streptavidin and used for binding of biotinylated silicatein. The developed functionalized membranes could be immobilized on inert silicon-nitride waveguide core material. The methods developed were used for encapsulation of biocatalysts (enzymes) and antibodies directed against small molecules in optically transparent silica matrices. Using this approach, we could design an enzymatic biosensor within silica thin films through the sol-gel technology. The silica matrix was formed either enzymatically (via silicatein) or via sol-gel techniques. The new technologies were applied for the preparation of absorbers for drinking water systems. The aim was to build absorbers based on S-layer technology functionalized with selected antibodies against Legionella surface antigens, which reduce the number of Legionella possibly present in the water systems of e.g. homes, nursing homes, hospitals and hotels. Moreover, a bacterial antigen (L. pneumophila) biosensor was constructed by the binding of a specific antibody to the S-layer functionalized nitride surface of a micro ring resonator sensor connected to a read-out system. The S-layer technology was also used for the generation of microsieves with additional functionality, such as protein repellence and smaller pore sizes. Our results revealed that the biofunctionalized microsieves can be used for the removal of potential pathogens from house-hold drinking water. A proof of concept has been performed for an integrated membrane biosensor platform based on functionalised silicon nitride microsieves. The dissemination and communication activities in this project also included activities to improve the public acceptance of nanotechnology. The new techniques were exploited by three research-based SMEs and the enduser involved in the project, in microfluidics based sample processing and micro-array development, in industrial nanosieves, as well as in sensors in drinking water systems.
Project Context and Objectives:
The production of new micro/nanoporous membranes, which can be functionalised, is in rapid development and has been shown to have a high potential in a wide range of industrial applications. The immobilization of biomolecules serving as biocatalysts or as a component of sensors into organised structures presents many advantages in terms of cost reduction, high-throughput sample processing, integration, and automation. Bioactive molecules can be oriented onto a nanostructured surface or encased in an inorganic matrix. This project aimed to develop novel functionalised inorganic-organic membranes, with selective and defined physico-chemical properties, which can be used as nanosieves, as a component of integrated analytical systems, in food processing, drug discovery and diagnostic applications. Bioactive molecules such as enzymes and antibodies, immobilized within the inorganic-organic hybrid architecture of the membrane, maintain their biological activity and are more stable, and/or are only accessible for a selected (size) range of substrates compared to their freely soluble counterparts. The manufacture of nanoporous membranes reinforced by an inorganic skeleton and combined with bioactive biomolecules is a highly innovative field. This project aimed to combine two parallel approaches for the formation of functional inorganic-organic membranes. The first one regarded the formation of a single-layered structure derived from bacteria and archaea, named S-layer, in which the self-assembling property of the subunits into a porous “paracrystalline” structure is exploited as a template on which other molecules, such as silicateins, can bind. The second approach regarded the encapsulation of biomolecules into the porous silica glass formed either biocatalytically (via silicateins, a special group of enzymes catalyzing the formation of inorganic metal oxides) or by sol-gel process. While the first approach was completely biological, being the S-layer derived from living organisms, the second one resulted in an inorganic-organic hybrid composition of the membrane, in which organic molecules can be encapsulated. Both nanobiotechnology approaches are inspired by molecular biology and are based on processes of self-organisation at mild environmental conditions.
The specific objectives of this project were:
1. Design and synthesis of the basic building blocks (modules) of the new membranes by molecular biology methods: S-layer proteins, silicateins, fusion proteins
2. Self-assembly of these modules to nanoporous membranes
3. Reinforcement of the generated membranes by silica (metal oxide) nanoparticle formation / deposition
4. Physical-chemical characterization of the generated functional membranes
5. Applying encapsulation technology for the design of nanoporous membranes used as biocatalysts and / or sensors
6. Exploiting the newly developed technique for the manufacture of novel micro/nanosieves and capsules
Project Results:
The experimental part of this project was performed in eight work packages (WP1 – WP8) concerning S & T activities. The results can be summarized as follows.
WP1: Design and synthesis of the basic building blocks (modules) of the new membranes by molecular biology methods: S-layer proteins, silicateins, fusion proteins
The objective of this work package was the expression, purification and characterization of the building blocks (modules) used for the construction of the functional membranes. The specific objectives were: (1) Expression and purification of recombinant silicateins; (2) Expression and purification of S-layer proteins; (3) Expression and purification of fusion proteins; and (3) Characterization of the recombinant proteins.
The silicatein-alpha cDNA from Suberites domuncula was expressed in the oligohistidine expression vector pQ30 A by UMC-Mainz. For large-scale production of the recombinant silicatein-alpha, E. coli Host Strains M15 was transformed with the plasmid and cultured in bioreactor BIOSTAT A plus. Induction was performed with IPTG. The lysate was purified and refolded using the Profinia Protein Purification System.
Glu-tagged silicatein, carrying an additional C-terminal His tag, was prepared in bacterial expression vector pBAD/gIII A. The construct was used to transform TOP10 E. coli cells. The expression of the recombinant protein was induced with arabinose. Then, proteins were extracted and purified by Ni-NTA affinity chromatography. In addition, silicatein alpha was expressed in the yeast Pichia pastoris. The purity of the expressed proteins was checked by SDS-PAGE.
The expression and purification of S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 was performed by BOKU. For the production of biomass, the mesophilic, gram-positive bacterium Ly. sphaericus CCM 2177 was grown in a fermenter in continuous culture in nutrient broth medium for 7 days. Cell wall fragments from whole cells were prepared by ultrasonication of Ly. sphaericus cells, extraction of the plasma membran with Triton X-100, and washing with Tris/HCl buffer.
Isolation of S-layer protein from the peptidoglycan-containing sacculi (PGS) was performed by separation of SbpA from PGS by addition of 5 M GHCl in Tris/HCl buffer. Purification of S-layer protein SbpA was performed by gel permeation chromatography (GPC), followed by lyophilisation. The quality control of purified S-layer protein SbpA was performed by SDS-PAGE.
The obtained S-layer material was used for in vitro recrystallization studies for the functionalization of different solid supports and for recrystallization at the air water interface.
The expression and purification of fusion proteins was performed as follows (UMC-Mainz and BOKU). S-layer fusion protein carrying a C-terminally fused core-streptavidin (rSbpA31-1068/STAV40-157): Cloning of the recombinant S-layer fusion protein rSbpA/STAV in E. coli TG1 was performed using the pET system. Heterologous expression of the fusion protein between the S-layer protein SbpA of Ly. sphaericus CCM 2177 and streptavidin in E. coli BL21(DE3)star was carried out following the instructions described in the pET System Manual (Novagen). Purification of fusion protein was done by gel permeation chromatography in presence of the chaotrophic reagent 2 M guanidinehydrochloride.
The gene encoding the fusion protein rSbpA/Silicatein was amplified with PCR and cloned (in two steps) into the restriction sites NcoI, BamHI as well as XhoI of the plasmid pET28a following the instructions of the pET System Manual. The plasmid construct was cloned into E. coli TG1. Positive clones were isolated from selective agar plates containing kanamycin. Determination of the occurrence of the chimaeric insert encoding the rSbpA/Silicatein fusion protein was done by restriction analysis.
The plasmid carrying the chimeric gene encoding the fusion protein was isolated from E. coli TG1 by using a Plasmid midiprep kit (Qiagen). The CaCl2-competent expression stain E. coli BL21(DE3)star was chemically transformed with the purified plasmid. Heterologous expression of the fusion protein in E. coli BL21(DE3)star was carried out as described in the pET System Manual (Novagen).
Production of the fusion protein rSbpA/Silicatein by heterologous expression in E. coli was checked by SDS-PAGE and Western-blot analysis using a polyclonal rabbit antibody raised against the S-layer portion of the fusion protein. Purification of the S-layer fusion protein rSbpA31-1068/Silicatein was done by exploiting the binding of the His-tag to a Ni-NTA-column in the presence of the chaotropic reagent guanidinehydrochloride.
UMC-Mainz could propose a new model for the catalytic mechanism of silicatein. This mechanism leads to the formation of reactive, cyclic silicic acid species (trisiloxane rings and higher-membered siloxane rings) which easily promote the silica polycondensation reaction. The modeling studies revealed that the synthetic substrate, TEOS, fits very well into the substrate pocket of the silicatein-alpha molecule, which comprises the amino acids of the catalytic center, Ser, His and Asn.
In addition, UMC-Mainz studied the different stages of the process termed bio-sintering. UMC-Mainz addressed for the first time sintering as component of a multi-stage process (including polymerization, gelation, aging, and solidification) within a biological system, ultimately giving rise to the synthesis of (fused) siliceous spicules. UMC-Mainz could present evidence that the solidification of the axial cylinder is mediated by a silicatein-driven, bio-sintering process.
With the scaffold protein silintaphin-1, a first silicatein interactor that facilitates the formation of the axial filament and, consequently, the formation of the growing spicule was discovered by UMC-Mainz. Silintaphin-1 facilitates and controls not only the assembly of silicatein as filaments but also the assembly of nanoparticles (gamma-Fe2O3, titania, or silica) as ordered structures. Silintaphin-1 was discovered during a yeast two-hybrid library screening.
The silintaphin-1 interaction with silicatein during the structure-guiding bio-silica formation was studied. Recombinant silicatein and recombinant silintaphin-1 were used at different stoichiometric ratios to form axial filaments and to synthesize biosilica. UMC-Mainz could demonstrate that the enzymatic activity of silicatein-alpha strongly increased by 5.3-fold (substrate: TEOS), leading to significantly enhanced synthesis of biosilica.
Furthermore, UMC-Mainz could elucidate the process of hardening / ageing of biosilica. UMC-Mainz could show that in sponge primmorphs (three-dimensional [3D]-cell culture system of sponges), manganese (Mn-sulfate) strongly affects the morphology and the hardness of the skeletal elements. During incubation of primmorphs with Mn-sulfate, spicules were formed that have lost their regular morphology and their smooth surfaces. The spicules formed in the presence of Mn-sulfate were porous and less rigid. Micro-hardness properties of the spicules were determined applying the nanoindentation method. In contrast to spicules that developed in primmorphs in the absence of Mn-sulfate and which displayed an almost plane surface in the fracture areas, the fracture sites of spicules isolated from primmorphs from Mn-sulfate containing cultures showed a granular appearance. The average hardness of the spicules developed in the absence of Mn-sulfate is about 1.5 GPa, while that of spicules from Mn-sulfate cultures was much lower in the range of 0.35 GPa.
First attempts to mimic that process in vitro by using recombinant silicatein-alpha were successfully performed by addition of poly(ethylene glycol) (PEG) during the enzymatic silicatein mediated reaction. PEG has been shown to facilitate sol–gel processes during silica formation starting from TEOS and forming solid silica particles by interconnected spheroidal silica nanoparticles. At neutral pH values and at room temperature PEG with its ether oxygen groups [acting as hydrogen-bonding acceptor] interacts readily with silanol groups [hydrogen-bonding donor] of the silica species via hydrogen bonds
For these experiments recombinant silicatein-alpha was added to an assay system containing prehydrolyzed TEOS as substrate. In the absence of PEG, 55 microgram of bio-silica per ml assay was synthesized by 6 microgram of enzyme under the incubations conditions used. After addition of PEG to the substrate, prehydrolyzed TEOS, the amount of biosilica formed increased and reached at molar ratios between prehydrolyzed TEOS and PEG (between 1:0.1 and 1:1) significantly higher values.
Highlights:
• Successful expression and purification of S-layer - silicatein fusion protein (rSbpA/Silicatein)
• New model of silicatein reaction mechanism
• Discovery of the process of bio-sintering
• Isolation of two new silicatein interactors: silintaphin-1 and silintaphin-2
• Elucidation of mechanism of hardening /ageing of biosilica
WP2: Self-assembly of these modules to nanoporous membranes
The objective of this work package was the fabrication of the functionalized membranes / matrices from their basic building blocks (S-layer proteins, silicateins, fusion proteins).
The specific objectives were: (1) Preparation of biotinylated recombinant silicatein and / or biotinylated S-layer proteins; (2) Self-assembly of the S-layer protein monomers on various surfaces, including gas/water interface; (3) Functionalization of the S-layer-coated surface by binding of streptavidin; (4) Binding of the biotinylated silicatein to the functionalized S-protein layer; and (5) Functionalisation of micro-array waveguides with membranes.
Biotinylated S-layer proteins were produced by BOKU. In SPR studies performed for the functionalization of solid support with S-layer poteins, biotinylation of wild-type S-layer protein SbpA was done using the heterobifunctional cross-linker EZ-Link Sulfo-NHS-Biotin.
The recrystallization of wild-type S-layer protein as well as of the recombinant S-layer fusion proteins was performed after solubilization of S-layer protein/recombinant fusion protein by addition of 5 M GHCl. S-layer monomers were produced by removal of the GHCl by dialysis against A. dest. SbpA recrystallization is a Ca2+-dependent process and occurs by the addition of 10 mM CaCl2.
The S-layer protein SbpA of Ly. sphaericus CCM 2177 which was produced and purified in WP1 could successfully recrystallized a) as self-assembly products in suspension, b) as closed monolayers on silicon chips, c) as monolayers on positively charged liposomes, and d) as recrystallized monolayers on lipid films. The recrystallization ability was checked by using transmission electron microscopy as well as atomic force microscopy.
The purified lyophilized rSbpA/STAV fusion protein showed excellent recrystallization properties, resulting in self-assembly products having an average size of 1 micrometer clearly showed the square lattice pattern.
In TEM pictures of self-assembly products of rSbpA/Silicatein formed in suspension, the square lattice symmetry of the SbpA portion in the fusion protein was clearly visible. Lattices showed a center-to-center spacing of the morphological unit of 13.1 nm, which is typical for SbpA. Results indicated that the fusion of silicatein to the C-terminal end of the S-layer protein SbpA did not interfere with the recrystallization properties of the S-layer protein.
In SPR studies, stabilization of the obtained crystalline monolayer of the S-layer protein SbpA on the gold chips was done by crosslinking with glutaraldehyde (amine-reactive homobifunctional crosslinker) which reacts with free amino groups of the S-layer protein.
In a BIACORE 2000 SPR apparatus, the S-layer protein SbpA of Ly. sphaericus CCM 2177 was recrystallized on a SPR-gold-chip. In a second step, the S-layer lattice was stabilized by cross-linking with glutaraldehyde. After washing step, the S-layer was biotinylated with NHS-Biotin and subsequently, active streptavidin tetramers could be bound on the S-layer lattice in a density of 1 ng/mm2. The result was an S-layer-based sensor chip with the ability to bind biotinylated silicatein. This set up can be used to bind biotinylated silicatein, resulting in an S-layer-based sensor system.
In an alternative approach, after recrystallization of SbpA on the chip surface and formation of a closed crystalline SbpA monolayer and cross-linking, free carboxyl groups on the surface of the S-layer lattice were activated with EDC/NHS 1-ethyl-3-[3-(dimethylamino)propyl carbodiimide/N-hydroxy-succinimide]. In a further step, stable peptide bounds were formed between the activated carboxyl groups of the S-layer matrix and free amino groups of strepatavidin molecules. As a result streptavidin could be bound on the chip surface in a density of 1 ng/mm2.
Surprisingly, binding of a biotinylated macromolecule to the active streptavidin on the sensor chip revealed a leakage of the S-layer after some minutes of incubation indicating that the very strong interaction between streptavidin and the biotinylated ligand led to a removal of the S-layer from the gold surface. Due to this fact, a new approach based on SbpA-cysteine mutants was developed.
In SPR experiments, recombinant SbpA cysteine mutants carrying one single cysteine residue at the N-terminus of the S-layer protein were covalently linked to the surface of the SPR-gold-chip. Then, the development of the sensor chip (crosslinking of the S-layer, EDC activation and binding of streptavidin was done as described before. In this approach, no leakage of the S-layer lattice from the gold chip during incubation with biotinylated peroxidase could be observed (model system). The developed sensor chip can be used for the binding of biotinylated silicatein.
Functionalisation of micro-array waveguides with membranes was studied using two different substrates: Si3N4 and SiO2. AFM investigations revealed that both substrates could be coated with the recombinant S-layer as the square lattice structure could be easily seen, but only the SiO2 surface exhibited a closed monolayer while on the substrate having the Si3N4 surface chemistry only patches of recrystallized monolayer could be detected. As the Si3N4 surface corresponds to the real sensor chip surface the coating conditions were changed; washing was only conducted with Milli Q water and a pre-coating step with Poly-l-lysine (PLL) was performed. With these changes also on the Si3N4 substrate a closed monolayer of the rSpbA31-1068ZZ could be easily detected.
The properties of rSpbA31-1068ZZ to form a complete crystalline monolayer onto the surfaces were used within the sensor chip from LX.
Further the rSpbA31-1068ZZ S-layer protein was recrystallized onto wafers from Surfix supplied by LX using the epoxy surface chemistry for covalent binding of the S-layer protein replacing the crosslinking steps with dimethyl pimelimidate (DMP). Therefore, no additional crosslinking with DMP was needed and the recrystallization of the S-layer could be easily performed at LX after the rSpbA31-1068ZZ monomeric solution.
To confirm the IgG binding and the stability of the covalently, via epoxy groups, bound rSpbA31-1068ZZ the coated epoxy wafers were incubated with human IgG. Subsequently a pH shift was performed and the amount of IgG that could be eluted of the rSpbA31-1068ZZ coated epoxy wafers was measured with alternative techniques using ELISA, as UV measurements did not exhibit the required sensitivity. The stability was checked by treating the coated wafers with a 0.1 M NaOH solution for 20 minutes and subsequent IgG recovery studies.
As ELISA system rSpbA31-1068ZZ coated GREINER ELISA plates were incubated with the eluate obtained from the S-layer coated epoxy wafers. Bound IgG was detected with anti human IgG peroxidase labelled antibodies and TMB as substrate.
The stability of the rSpbA31-1068ZZ monolayer that should be obtained by the epoxy activated surfaces was investigated by incubation of the wafers with 0.1 M NaOH.
The ELISA system showed that more than 80 ng human IgG could be bound per 1 cm2 rSpbA31-1068ZZ coated epoxy activated surfaces. After the treatment with 0.1M NaOH, the binding capacity decreased for approximately 50 % which confirmed the increased stability of the S-layer cross-linked to the wafer surface exploiting the epoxy chemistry.
Highlights:
• Self-assembly / recrystallization of S-layer - silicatein fusion protein (rSbpA/Silicatein) on various surfaces
• Functionalization of the S-layer-coated surfaces by binding of streptavidin
• Binding of protein to functionalized S-protein layers
• Functionalisation of micro-array waveguides (Si3N4)
• Confirmation of the stability of S-layer cross-linked to the wafer surface
WP3: Reinforcement of the generated membranes by silica (metal oxide) nanoparticle formation / deposition
The objective of this work package was the reinforcement of the functionalized membranes generated in WP2 by biocatalytic (via silicatein) formation of silica or other metal oxides, as well as by sol-gel processes.
The specific objectives were: (1) Biocatalytic formation of silica or other metal oxides (via silicatein) on the S-layer-based membranes; (2) Formation of silica on the S-layer-based membranes via the sol-gel process; (3) Design of membranes having organic polymers or natural polymers as a component; and (4) Application of the new technology for preparation of adsorbers for drinking water systems.
The biocatalytic formation of silica on S-layer-based membranes was measured with in situ QCMD by using a Q-Sense E4 instrument (Q-Sense) (BOKU).
One major objective of UPMC was to design novel robust inorganic nanocoatings compatible with the immobilization of biomolecules for the design of biosensors and nanofactories. From the state-of-the-art in this area, this was a challenging task as (i) ultra-thin sol-gel layers are usually prepared using solvent-based processes that are not always compatible with the preservation of the biomolecule conformation and, hence, activity, and (ii) these layers are very prone to dissolution in biologically-relevant conditions.
In this context, a first major progress made during this project was the development of a novel technology for the deposition of multi-layers of silica thin films in purely aqueous media. Interestingly this process was found compatible with the immobilization of one specific enzyme extracted from valuable foodstuff (truffles) that may be further used for sensor design. Another important outcome was that the formation of a thin silica layer on substrates coated with S-layers was possible and induces some organization of the silica network, without impacting the activity of entrapped enzymes. However, it was found that these silica layers were highly porous, leading to rapid leaching of small enzymes. Such an effect was not observed for antibodies that showed preserved immuno-affinity over several days. Despite these interesting performances, it was found to be difficult to use the newly-developed protocol for suitable coating of complex micro-chips as used by some partners (LX). However it is worth mentioning that this protocol was recently shown to be compatible with the immobilization of whole bacteria while allowing their in silica division, providing an interesting tool for more fundamental biological studies.
This led to a second important development, the design of titania thin films. One particular originality of the proposed approach was to use a specific precursor that is water soluble, thus avoiding the use of organic solvent. The protocol was difficult to set up due to its sensitivity to precursor concentration, coating conditions, ionic strength and buffer composition, drying place and time, among other important parameters. However, the final protocol is very simple and should be easily reproduced by others. It was also possible to demonstrate the first deposition of multi-layered titania films and their compatibility with enzyme and antibody immobilization. The robustness of these films could be ascertained over two months and showed comparable performances as commercial immuno-microplates. Finally, they could be deposited in a suitable manner on LX substrates. These data raised an important range of questions especially as the titania formulation was highly sensitive to the presence of proteins. This unfortunately made the interaction with S-layers unfavourable but, on the other hand, opens an important area of investigations at the biomineral interface.
A third development related to material science was achieved during the preparation of hybrid membranes. UPMC showed the possibility to associate two liquid crystalline systems, hydroxypropylcellulose and vanadium oxides, to create soft materials with unprecedented organization. In parallel, UPMC could design thin films associating silica and collagen, two very common, fully biocompatible components. Although these materials have some drawbacks in terms of optical properties that have limited their further use for sensing in this project, the first system should be studied further due to their possible application for energy (batteries) whereas the second is currently being investigated for biomedical applications.
Various partners were involved in the application of the new technology for preparation of absorbers for drinking water systems (IWW, UMC-Mainz, BOKU, UPMC). The aim was to build absorbers based on S-layer technology functionalized with selected antibodies against Legionella surface antigens, which reduce the number of Legionella possibly present in the water systems of e.g. homes, nursing homes, hospitals and hotels.
To bind antibodies against Legionella pneumophila to a filtration unit in an oriented way the S-layer fusion protein rSbpA31-1068ZZ comprising the IgG binding domain of Protein A was chosen. The IgG directed against Legionella pneumophila will bind via their Fc moiety and should capture Legionella pneumophila if present in the fluids passing the membrane.
To allow unhindered flow through a membrane a pore size of 3 micrometers was chosen. To achieve filtration membranes, capable of binding IgG, the S-layer fusion protein had to be recrystallized to and within the filter structure. This was done by inserting the nitrocellulose membrane into a filtration unit and by filter the fusion protein solution in the presence of CaCl2 through the nitrocellulose membrane. This allows the recrystallization of the fusion protein onto and within the filter structure. UV280nm measurements of the S-layer protein solution before and after the filter allowed a semi quantitative determination of the binding success. Subsequently the S-layer protein coating was stabilized by crosslinking with DMP and antibodies raised against Legionella pneumophilia were applied to the filter discs. The so activated filter discs were sent to IWW for evaluation.
Highlights:
• Development of a novel technology for the deposition of multi-layers of silica thin films in purely aqueous media
• Method for biocatalytic reinforcement of functionalized membranes with silica or other metal oxides
• Formation of silica on the S-layer-based membranes via the sol-gel process
• Design of membranes having organic polymers or natural polymers as a component (V2O5 - hydroxypropyl cellulose and collagen - silica)
• Design of titania thin films
• Successul adsorption with a high occupancy and release under controlled conditions of Legionella pneumophila from an S-layer/protein A/polyclonal antibody membrane
WP4: Physical-chemical characterization of the generated functional membranes
The objective of this work package was the physical-chemical characterization of the nanoporous membranes and nanosieves formed by biocatalytic silica deposition on S-layer protein – silicatein matrices or matrices formed by fusion proteins, and or sol-gel technology.
The specific objectives were: (1) Determination of morphology; (2) Determination of the stability of the membranes; and (3) Capability of self-repair.
The tasks of WU involved the preparation and characterisation of well-defined layers on surfaces and membranes. The main goal was to come to well defined membranes and process design for novel membrane processes. In their studies, WU focused on accumulation of particles on membranes under flow conditions. This is a logical complementary activity to get the overall membrane process to work optimally, since using flow to keep particles as much as possible away from the surface may prevent many interaction problems ab initio.
WU used a well-defined nickel sieve onto which in a later stage S-layers and silicatein were deposited, and tested for the separation of yeast cells. WU found that the size of the particles in the permeate could be controlled by the applied transmembrane pressure and cross-flow velocity (which induces particle migration away from the membrane), at high flux and without notable fouling taking place. All results could be summarized into one master curve based on the typical times in the system. WU was also successful to control the process conditions in such a way that they could specifically deposit small cells while removing the large ones, leading to new options for process design. A proof-of-principle of this latter mechanism has been given. When testing microsieves with deposited S-layers WU found that they were lacking mechanical stability, therefore, they focused on new membrane processes, and defined windows of operation for them, which defines to working space for any membrane or sieve, and that can be applied as soon as modified sieves are available.
The capability of self-repair was investigated by UMC-Mainz. The most used structural materials are vulnerable to cracking and are not repaired if not special supplements are added to those materials. This is different to bio-materials which are formed and maintained in organisms, where the integrity of the
The self-healing property of sponge biosilica can be utilized to engineer novel hybrid materials, with silicatein as a functional template, which are more resistant towards physical stress and fracture. In sponge biosilica, the organic template remains embedded into the mineralic phase even after completion of the mineral formation. This finding implies that the functionality of the template remains unaffected because the organic macromolecules forming this template are protected against denaturation and degradation after becoming encased within the mineral structures. As a consequence the organic mineral-forming polymers become functionally active again, after uncovering from their mineralic envelope. As a consequence, the organic template has a self-healing function after destruction of the inorganic shell.
High resolution SEM investigations of sponge spicule lamellae revealed that they are composed of 5-7 nm large nanoparticles. If those samples are exposed to HF, resulting in dissolution of the silica matrix, the organic scaffold, surrounding the nanoparticles becomes visible. The dimension of the holes, formed by the organic material, varies between 5-7 nm, supporting the view that organic proteins are surrounding the silica nanoparticles.
After treatment of the spicules with HF, the proteinaceous filaments can be recovered. With progressing time, the silica is dissolved, leaving behind only the organic axial filament. It is surprising that the proteinaceous axial filament remains preserved in those spicules. The silicatein in these axial filaments remain functionally active as silica-binding protein or as enzyme after dissolution of the biosilica shell.
Highlights:
• Accumulation of particles on membranes under flow conditions: Control of size of particles by the applied transmembrane pressure and cross-flow velocity
• Master curve based on the typical times in the system
• Demonstration of the self-healing property of biosilica for engineering novel hybrid materials
WP5: Applying encapsulation technology for the design of nanoporous membranes used as biocatalysts and / or sensors
The objective of this work package was the integration of silica-encapsulated biocatalysts and antibodies into the fortified functionalized membranes, obtained biocatalytically (via silicatein) or via the sol-gel process. The integrated biocatalysts and antibodies will serve as constituents of novel, membrane integrated nanofactories and sensors.
The specific objectives of this work package were: (1) Entrapment of biocatalysts (enzymes) in the silica matrix; (2) Entrapment of antibodies directed against small molecules in the silica matrix; (3) Construction of a nanofactory based on entrapped enzyme; (4) Construction of a biosensor based on entrapped antibody; (5) Construction of a bacterial antigen biosensor; and (6) Construction of the capsules based on S-layers with well-defined properties for the controlled release of entrapped molecules.
It was possible to use these novel titania layers for designing biosensors. In the case of enzymes, UPMC demonstrated the feasibility of performing a tri-enzyme cascade within a single material, a fact that, to our knowledge, was not reported so far in sol-gel thin films. UPMC could even show that these systems could be used to titrate lactose in milk, suggesting their possible application for food monitoring. In the case of antibodies, environmental-relevant surfaces could also be designed by entrapping antibodies that can recognize some component of bacterial cell membranes. Our studies suggest that the antibodies are partially inside the titania layer while the other part is pointing out at the film surface: hence not only the antibodies are immobilized and stabilized but they also remain able to interact with antigens. In this context, first tests made by IWW do indicate the higher cell adhesion on titania-containing anti-LPS antibodies compared to pure titania films. It is unfortunate that time has missed to associate the titania films with the LioniX chips for detection by IWW.
The design and construction of a bacterial antigen (L. pneumophila) biosensor was performed in close cooperation of IWW, BOKU and LX.
Within WP2 BOKU and LX showed the successful functionalization of the stoichiometric nitride waveguide material with the rSbpa ZZ S-layer protein. A bacterial antigen biosensor can be constructed by the binding of a specific antibody to the S-layer functionalized nitride surface of a micro ring resonator sensor connected to a read-out system (WP7).
For this purpose BOKU supplied LX with the rSbpa ZZ S-layer protein and CaCl2 recrystallization buffer. IWW supplied the antibody for pseudomonas fluorescens and the corresponding bacteria. The choice for this bacterium was based on its similarities with L. pneumophila whereas it is harmless. This allowed LX to work with the bacteria in a normal lab, gain experience and optimize the detection system before a final test could be performed at IWW with L. pneumophila.
Highlights:
• Design of biosensors using novel titania layers
• Demonstration of the feasibility of performing a tri-enzyme cascade within a single material
• Use of these systems to titrate lactose in milk (possible application for food monitoring)
• Entrapment of antibodies recognizing component of bacterial cell membranes
• Design and construction of a bacterial antigen (L. pneumophila) biosensor
• Construction of a bacterial antigen biosensor by binding of a specific antibody to the S-layer functionalized nitride surface of a micro ring resonator sensor connected to a read-out system
• Demonstration that the sensing system works very accurate and with a very high sensitivity comparable to SPR equipment
• Improvement of the performance by a factor of 10 – 100 in the case a reference MRR sensor is applied in the system
WP6: Exploiting the newly developed technique for the manufacture of novel micro/nanosieves
The aim of this work package was the generation of hydrophobic to hydrophilic or protein repelling surfaces on microsieves, as well as the generation of microsieves with smaller pore sizes using the developed technology.
The specific objectives of this work package were: (1) Development of S-layers-coated microsieves to prevent undesired interactions between components of the liquid and the microsieve surface and (2) Development of membranes with smaller pore sizes on microsieves.
The task of AM was the exploition of the newly developed technique for the manufacture of novel micro/nanosieves. The aim was the generation of hydrophobic to hydrophilic or protein repelling surfaces on microsieves, as well as the generation of microsieves with smaller pore sizes using the developed technology.
The S-layer technology was used for the generation of microsieves with additional functionality, such as protein repellence and smaller pore sizes. This work has been done by AM in collaboration with WU and BOKU. In the first project period, AM explored an innovative zwitterionic coating to prevent protein interaction with the microsieve surface. In order to obtain nanoporous membranes, AM supplied microsieves of 450 nm pore size dimensions for coating with S-layers.
With current lithographic tools the production of microsieves with sub-micron pore is feasible. However creating nanopores in the order of 10 nm or smaller is still challenging and difficult to achieve with lithographic techniques. Our idea was that the combination of conventional procedures for the manufacture of microsieves and the new technique developed in this project will allow the generation of membranes with smaller pore sizes by formation of nanoporous S-layers spanning the existing pores on the microsieves. We expected that the new developed techniques for surface functionalization with self-assembled S-layer proteins might be a way to create such nanoporous membranes.
Several attempts to produce S-layer coated microsieves failed to give satisfactory results. Therefore an alternative approach was developed for biofunctionalization of microsieves. A highly reproducible and facile method to covalently attach antibodies on the SixN4 surface of microsieves was achieved by a two-step procedure: attachment of an epoxide-functionalized monolayer by a photochemical reaction, followed by immobilization of antibodies.
Silicon nitride (SixN4, x>3) surfaces were functionalized with an epoxide-terminated monolayer by a UV-induced reaction. Hydrogen-terminated SixN4 substrates were obtained through etching with HF solution, and employed in the photochemical (lambda = 254 nm) attachment of 1,2-epoxy-9-decene.
The stability of the epoxide-terminated monolayers on SixN4 surfaces as well as on microsieves was studied in argon atmosphere for 1 month. No significant changes in both water contact angle and XPS spectra of the surfaces were found after 1-month storage, indicating long-term stability.
Anti-Salmonella antibodies-coated SixN4 surfaces were obtained via coupling of lysine residues at the outside of the antibodies to the epoxide-coated surface.
Our results revealed that the biofunctionalized microsieves can be used for the removal of potential pathogens from house-hold drinking water. Instead of removing the pathogens by size-based membrane filtration, the pathogens can also be removed by selective capture on the surface of the membrane. This is possible by functionalizing the microsieves with antibodies specific for the pathogens. A prototype membrane was further developed and tested.
Highlights:
• Development of microsieves with an innovative zwitterionic coating to prevent protein interaction with the microsieve surface
• Application of microsieves of 450 nm pore size dimensions for coating with S-layers in order to obtain nanoporous membranes
• Development of a reproducible method to covalently attach antibodies on the SixN4 surface of microsieves by a two-step procedure
• Demonstration of the stability of the epoxide-terminated monolayers on SixN4 surfaces and microsieves
• Preparation of anti-Salmonella antibodies-coated SixN4 surfaces
• Application of the biofunctionalized microsieves for the removal of potential pathogens from house-hold drinking water
• Development of a prototype membrane
WP7: Exploiting the newly developed technique in microfluidics based sample processing and micro-array development
The objective was to build and test an ‘end-to-end’ desk-top system with functions / steps miniaturized and integrated which are essential to demonstrate the feasibility of a future development; an evanescent wave excited micro-array platform will be developed.
The specific objectives of this work package were: (1) Design of integrated system and (2) Realization of integrated system.
Optical biosensors present several advantages with respect to, for example, the more established electrochemical or piezoelectric principles, because they are insensitive to electromagnetic interferences (enabling extremely high sensitivity) and can potentially be used in aggressive environments. Thanks to the progress in planar waveguide technology in the last decade, including the excellent miniaturization capabilities based on optoelectronic integration with for example VCSELs, these platforms are emerging in both applications as a sensor as well as screening instruments for molecule interactions and functionalization procedures.
In the original description of the work package, integration of pre-sampling functionality in the end-to-end demonstration system was proposed. At the beginning of the project it has been decided to focus only on the detection of bacteria using the MRR sensing system, which in principle, does not require pre-sampling on-chip.
There are three basic detection schemes using the evanescent field of an optical wave propagating in a waveguide:
1) Fluorescence based optical detection
2) Refractive index based optical detection
3) Absorbance based optical detection
The fabrication scheme and associated technology for each is similar. Therefore in the first two years of the project, LX worked successfully on both the development of new fabrication routes as well as on improvements of existing TriPleX based technologies. The first approach increased the design and fabrication flexibility, resulting in a higher compatibility with the proposed on chip optical characterization and possible future pre-treatment functionalities. The second approach resulted in improved sensitivity of the opto-fluidic chip and higher yield values. As a result, basic building blocks have been formed which can be used to rapidly design and fabricate opto-fluidic microchips modelled to a specific task.
Highlight:
This approach led to two mature fabrication routes for TriPleX based opto-fluidic devices including the possibility to integrate metal pattering. One uses a stack of silicon and borofloat substrates, the other makes use of only fused silica glass. The additional advantage of the latter is its (UV) transparency and non-conductive behaviour opening up the possibility to incorporate additional conductive sensing elements.
Initially the absorbance based detection approach had been chosen as detection platform due to its simplicity. Although its resolution and sensitivity would not be sufficient to function as suitable biosensor for this project, it allowed basic and quick functionalization of waveguide surfaces to test the compatibility with the evanescent field sensing detection principle. After the optimization of different process and functionalization steps, the step to the refractive index based micro ring resonator (MRR) sensing platform could be made.
Highlight:
During the project the fabricated absorbance based detection chips have been used in multiple project tasks and the developed platform resulted in a working and often used demonstration kit. The detection principle is concurrently being optimized in the Dutch NanoNextNL 10C.01 project in which it is prone to become an integrated system for online micro reactor synthesis monitoring and analysis. The system is also adapted to function as educational tool in the Dutch department of nanotechnology at Saxion Hogeschool Enschede.
Highlight:
With use of the developed absorbance based detection chips, the innovative concept of on chip filtering for fluorescence based detection platforms was explored. The filtering principle is based on the deposition of a dye, stabilized in PMMA, in the detection window. If the dye (based on its absorption properties) is chosen correct, the excitation signal can be filtered out whereas the fluorescent signal is left unimpaired, resulting in a simple, compact and low cost on chip filter system with high sensitivity. The chosen dye has an absorbance peak around 642 nm and is transparent around 665 nm. The first is the wavelength of excitation of the chosen example fluophore Alexa fluor 647 and the latter the wavelength of emission. Using a white light source and spectrometer, a suppression of >40 dB could be measured within a transmission window of 30-50 nm!
Highlight:
Within this project two approaches were explored for the immobilization of antibodies on the nitride surface of waveguides. These are functionalization using a sol-gel layer (UPMC technology) and functionalization using the S-layer (BOKU technology). Antibodies would be entrapped first in the 3D matrix and second bind to the 2D single layer. Material samples, absorbance based chip and even MRR chip have been prepared and supplied to the different partners to optimize the processes.
Highlight:
In addition LX supplied epoxy activated surfaces to BOKU based on alkene/alkyne chemistry, in collaboration with sister company and subcontractor Surfix (www.surfix.nl). These resulted in a covalent bond between surface and S-layer.
After the second year of the project, electronics for the detection system based on refractive index changes, e.g. the micro ring resonator (MRR) system became available through developments within the FP7 BIOMONAR project. The modular build-up of the electronics allows an easy upgraded to a sensor array readout system. The current electronics allow the read-out of up to 4 sensors (array) simultaneously.
Highlight:
With the use of the developed and optimized fabrication processes, different series of high performance micro ring resonator chips have been fabricated. The final series included an integrated, ‘on-board’, light splitter allowing an 2 x 1 array of MRR sensors of which one could be used as reference. Fluidic handling is being supported by two types of manufactured glass covers, one with microfluidic channels for controlled fluidic handling and one with open top, allowing direct access to the MRR surface, simplifying local functionalization.
Highlight:
The performances of these TriPleX MRR chips were very impressive, showing a sensitivity of 110 nm/RIU and a resolution of 1 pm in wavelength. This allows the detection of a change in refractive index of less than 9•10-6 RIU which is comparable with standard SPR systems. This was more than sufficient for the intended biosensor and therefore the sensing principle and system was accepted as the detection platform technology for the objective of this work package. Currently optimization activities are aiming on a sensitivity of 10-7 – 10-8 RIU, among others allowed by the application of a reference MRR sensor on the same chip.
The TriPleX based MRR biosensor chips are economically feasible in lower volume (niche) markets, as relatively cheap contact lithography is allowed. Moreover, low-cost hybrid integration of inexpensive VCSELs and detectors is enabled by the high coupling efficiency to TriPleX waveguides.
Highlight:
Functionalization experiments of the surface of the micro ring resonator with the S-layer and binding tests of the antibody have successfully been performed by LX. The final shift in response was 5032 RU which is comparable with the values obtained with SPR measurements.
A suitable bio-detection model for Pseudomonas fluorescens for testing was chosen by IWW. The choice for this bacterium is based on its similarities with L. pneumophila (rod-shaped, gram negative), the specified bacteria to be detected in the project, and its harmlessness. This allowed LX to work with the bacteria in a normal lab and gain experience and optimize the detection system before the final test could be performed at IWW using L. pneumophila. Final tests of functionality and sensitivity of the MRR detection system for the detection of P. fluorescens have been performed at the lab of IWW in corporation with LX. These clearly show the potential of the MRR sensor platform by presenting the full end-to-end result from build-up of sensor functionality, S-layer recrystallization on the surface, antibody binding and the attempt to detect bacteria.
WP8: Up-scaling, development of prototypes, and evaluation by endusers
The objective of this work package was to investigate options to adapt the developed techniques for an industrial production. This work package also involved the evaluation of the developed techniques by the endusers being part of the consortium.
The specific objectives of this work package were: (1) Up-scaling of production; (2) Optimization of yield; (3) Development of prototypes of the functionalized membranes; and (4) Evaluation of the newly developed functionalized membranes.
For large-scale production of the recombinant silicatein-alpha, a bioreactor was used. Fermentation proceeded in a culture volume of 3 liters with additional aeration and stirring at 37°C with controlled pH until reaching mid-log phase. The expression of the recombinant protein was induced by addition of IPTG. The lysate was purified with Profinia Protein Purification System. The protein concentration was measured with 2-D Quant Kit and verified by densitometric analysis with Odyssey Infrared Imaging system.
To obtain an active biotin binding S-layer protein the recombinant S-layer fusion protein rSbpA STAV, where a monomer of streptavidin was fused to the S-Layer protein moiety, the addition of three monomeric streptavidin units, followed by a refolding process, was necessary. Beside the fact that two recombinant proteins had to be expressed (rSbpA ST and streptavidin) the active rSbpA STAV had to be generated by refolding processes and labor-intensive purification steps. A rapid dilution step and two chromatographic steps were necessary as already described previously.
Two additional S-layer ST fusion proteins where constructed: His–rSbpA Strep 16 and rSbpA Strep 12 having 16 or 12 amino acids as linkers respectively. The new S-layer fusion proteins were cloned and expressed by exploiting the pET system.
With the new purification method of rSbpA ST an active S-layer fusion protein could be obtained without refolding and additional purification steps. Additionally the functional biotin binding S-layer protein could be obtained at higher yields providing S-layer coated substrates for binding of biotinylated proteins at a larger scale.
AM developed prototypes of the functionalized membranes.
1. A new method combining selective capture, microfiltration and automated fluorescence imaging for the rapid detection of microorganisms has been explored. Selective capture of Salmonella bacteria was demonstrated with highly porous micro-engineered silicon nitride membranes (microsieves) with uniform pore sizes in the range of 0.45 to 5.0 micrometers. We have explored a two-step method to obtain antibody coating on microsieves: 1,2-Epoxy-9-decene was photochemically attached to the silicon nitride microsieve surface, subsequently epoxide-terminated microsieves were bio-functionalized with anti-Salmonella antibodies.
2. The influence of flow rate in capture efficiency of antibody-coated 3.5-micrometer microsieves was investigated. The capture percentage increased from ~30 % to ~70 % when the flow rate decreased from 100 microliter/min to 5 microliter/min, respectively.
3. The use of antibody-coated microsieves as microbial selective capture devices was shown to be promising for the direct detection of microorganisms, giving a strong impulse to the further development of rapid diagnostics.
The conclusions based on the results are: Antibody-coated microsieves with pore sizes larger than the size of Salmonella displayed a significant increase in the capture efficiency as compared to uncoated microsieves. The capture efficiency can be obtained up to 66 % at the flow rate of 5 microliter/min. The detection protocol can be performed within few hours, which is significantly faster than diagnostic techniques such as agar-plating, and magnetic microspheres. The results exhibit the great potential of combining bioselective capture and microfiltration for the direct and rapid detection of microorganisms in crude biological samples.
The evaluation of the newly developed functionalized membranes in removing potential pathogens in household drinking water was performed by IWW.
Different types of functionalized surfaces have been developed within this project and have been evaluated by IWW in cooperation with the other partners in Mem-S project. The surfaces which have been developed had the aim to use them as filtration membranes, nanosieves and biosensors. Surfaces having specific functions are of great interest because they have the potential to be used e. g. as diagnostic tools to detect specific microorganisms or as a treatment step and to reduce human exposure e. g. to Legionella pneumophila. As target organisms L. pneumophila was chosen within this project because this species is water born and a human pathogen often found in house hold and technical water systems.
Several techniques and testing methods have been applied such as encapsulated and crystallization technology for the design of nanoporous membranes used as biocatalysts and/or sensors. Evaluation and testing of the developed functionalized surfaces have been performed by IWW in cooperation with the other partners.
Functionalized S-layer lattices with immobilized antibodies had been coated on commercial available filters with pore size 5 micrometers by BOKU together with the producer (Pall Corporation, Germany). Evaluation of this functionalized filtration membrane by IWW and the target organisms L. pneumophila showed a good retention. Regeneration by controlled release via pH shift to 3.5 was performed also the rate of yield varied from approximately 10% to 50%.
A further functionalized surface was developed and produced based on sol-gel titania with immobilized antibodies to L. pneumphila by UPMC. Testing showed a high selectivity for L. pneumophila, therefore this surface might be applicable as diagnostic tool also because of the low yield further improvement is necessary.
IWW and LX could demonstrate functionalization of a developed biosensor based on micro ring resonator system with crystallized S-layer and the immobilization of an antibody on this layer by a shift in resonance wavelength, comparable to results obtained by SPR analysis. No positive demonstration could be performed with a functionalized and integrated membrane biosensor platform based on a micro ring resonator as detection tool for bacteria. One reason might be an insufficient binding of the antibody to catch specifically bacterial like L. pneumophila. Another reason might be that the active sensing area on the ring resonator is too small for detection of bacterial without pre-sampling steps.
A proof of concept has been performed by IWW for an integrated membrane biosensor platform based on functionalized silicon nitride microsieves together with AM. This surface was developed with the aim to analyze specific bacteria such as e. g. Salmonella species or Legionella species. Nanosieves made of silicon nitride coated with epoxy material were coupled with antibodies. Testing by IWW showed a high potential for these surfaces as diagnostic tools. The microsieves can be applied as filtration units e.g. with a pore size ranging from 0.45 micrometer to 5.0 micrometer. Automatic quantification of trapped bacteria is possible by enumeration by an automated microscopic system. Duration of one assay will be in the range of 1-2 hours. Improvement is necessary in reproducibility of the effectiveness of capture ability.
Highlights:
• Successful up-scaling of silicatein production
• Method for preparation of active S-layer fusion protein without refolding and additional purification steps
• Development of prototypes of the functionalized membranes
• Antibody-coated microsieves as microbial selective capture devices turned out to be promising for the direct detection of microorganisms
• Evaluation of S-layer functionalized filtration membrane with immobilized antibodies revealed good retention of target organisms L. pneumophila
• Testing of sol-gel titania functionalized surface with immobilized antibodies: high selectivity for L. pneumophila
• Proof of concept for integrated membrane biosensor platform based on functionalized silicon nitride microsieves: high potential as diagnostic tool
Potential Impact:
Technical and scientific impact – In this project, a novel type of functionalized membranes have been developed, which offers a variety of potential applications. Our approach was new because it combined for the first time two important discoveries in the field of nano(bio)technology – (1) “paracrystalline“ structures made of S-layer proteins, and (2) enzyme/silicatein-mediated formation of biosilica. The enzymatic, structure-directed formation of inorganic sieve-like structures based on functionalized membranes and/or linked with proteinaceous membrane-like structures cannot be realized by other approaches. Therefore this project brought much progress beyond the state of the art, both in the field of S-layer proteins and silicatein-based silica nanobiotechnology. This project markedly extended the range of possible applications of S-layers and silicatein, in particular towards applications with significant economic impact. The generated functionalized membranes and coatings of nanosieves can be used for the fabrication of highly-ordered inorganic-organic composite structures. We focused on their application in the production of nanosieves used in food technology, Lab-on-a-Chip systems and in removal / detection of pathogens in drinking water system.
Economic impact – The market volume is estimated to be extremely large, in view of the fact that the developed technique can be used in a wide range of applications, reaching from food industry and water purification to biosensor applications and microelectronics. To date, there are no products on the market which make use of the fascinating property of S-layer proteins to form nanoporous “paracrystalline” structures as well as the unique ability of silicateins to synthesize silica – via an enzymatic mechanism - under mild, environmentally compatible conditions (low temperatures and pressures). In this project unknown scientific territory has been entered, whereby the chances of success of this project have been considered as very high.
Social impact – The achieved innovations are also important from the ecological point of view: Silicateins are able to synthesize silica or other metal oxides under mild, energy-saving and environmentally friendly conditions. This and the fact that procedures using Nature as a model (“bionics”) are applied, are also of benefit for the acceptance of (nano)biotechnology in the public. Products which function following the principles of bionics possess increasing interest to potential customers/users.
Main dissemination activities of the project and the exploitation of results:
The scientific results of this project have been included in a number of publications in peer-reviewed international journals.
Publications in refereed scientific journals: about 30.
Presentations at scientific meetings: about 30
Distribution of leaflets, brochures, and CD-Roms: UMC-Mainz and NTM produced a number of leaflets, brochures, and CD-roms concerning the research topic of the project (sponge biosilica - nanotechnology).
Contributions to press, TV, broadcast: UMC-Mainz (W.E.G. Müller) made several contributions to TV (channels ARD, ZDF, 3sat, arte, and others) and the press about the topic “Silicatein and biosilica”; e.g. Mare, Feb 2010, No. 78 (http://www.mare.de/index.php?article_id=2087(öffnet in neuem Fenster)) Spiegel, 21.03.2010 (http://www.spiegel.de/wissenschaft/natur/0,1518,682663,00.html(öffnet in neuem Fenster)) and FAZ, 20. June 2010 (Meeresschwämme – Langweiler als Lehrer).
Organisation of a workshop: The 2-day Workshop in Vienna at the institute of BOKU was organized from 17-18 November 2011. This workshop was also open for guests from universities and industry from outside the project. Laboratory work in the frame of the Workshop in Vienna: The basic production methods for recombinant and wild type monomeric S-layer proteins solutions were shown and recrystallization of S-layer proteins on solid substrates and the generation of self assembly products in solution were performed. Visualization was done by microscopic techniques (AFM and TEM). The binding properties towards human IgG of recombinant S-layer protein rSpbA31-1068ZZ were investigated after recrystallization onto gold wafers by SPR.
Organisation of a Summer school: The Summer school 2012 was organised at the Rudjer Boskovic Institute in Zagreb, Croatia, together with Marie Curie ITN BIOMINTEC from August 22 until August 25, 2012. Some participants of Mem-S project (UMC-Mainz, NTM, and WU) had already participated in the Summer school 2010 of BIOMINTEC in Rovinj from August 16 - 20, 2010.
Activities to improve public acceptance of nanotechnology: UMC-Mainz and NTM participated in the national competition “Germany – Land of Ideas”, initiative “365 sites” – and became winners. This opened a number of additional opportunities to disseminate the results of this project to a broader audience.
UMC-Mainz and NTM organized or participated in the following public events:
1. Mainz: “City of Science 2011“ - “Science Market”: Mainz has become the German “City of Science 2011“. In this frame, various events of the city of Mainz and the University / University Medical Center had been organized such as the “Science Market”. In frame of this event, UMC-Mainz had the possibility to present the topic “Biomaterials from the deep-sea“ to a broader public (4.-5. June 2011) – Exhibition of public presentation/lecture by W.E.G. Müller. In the frame of “Mainz - City of Science”, the Coordinator gave a further public presentation about marine biominerals and their technical application on 18th October 2011.
2. Spektrale 2011 (Exhibition): The partner at UMC-Mainz (W.E.G. Müller) was one of the main exhibitors in the event “Spektrale 2011” in Mainz. This exhibition took place in the Rheingoldhalle in Mainz from 15 July until 14 August 2011. W.E.G. Müller was responsible for the design of a complete room during this exhibition in the thematic area “Deep-sea sponges and the application of sponge biosilica and the biosilica-forming enzymes in nanobiotechnology”.
3. Exhibition: Transfercafé in the frame of “Mainz - German City of Science 2011”: The aim of the Transfercafé is to support the cooperation between business, especially small and medium-sized enterprises, and science. NTM and UMC-Mainz are presented as a best practice example of successful cooperation between companies and academic institutions (March to November 2011).
4. Expo 2012 in Yeosu, Korea: UMC-Mainz had been selected in a competitive call as exhibitor in the German Pavilion at the Expo 2012 in Yeosu, Korea (“The Living Ocean and Coast”). One central topic of this Expo was novel technologies / applications of materials from marine organisms. Therefore, we had the opportunity to communicate the results of this project to a broader public.
The theme "Nanotechnology from the deep sea" was treated in a series of articles in magazines, in the Internet and in interviews.
By the fact that the Mem-S Coordinator, W.E.G. Müller, holds an ERC Advanced Investigator Grants in the field "biosilica", the effectiveness of the dissemination measures further increased. In addition, he succeeded to establish a German-Chinese Joint Center for Bio-inspired Materials (from 2013 until 2022).
Protection of intellectual property: 10 patents
Assessment of the marketing potential of the techniques / products developed in the project: The assessment of the marketing potential of the techniques and products developed in the frame of the project was performed by the SME partners of this project (NTM, AM, LX). The SME partners were also exploring potential user groups interested in the products. The marketing analysis revealed that the products/techniques developed in the frame of this project are of interest for various sectors, more than originally envisaged. These sectors comprise, among others, the food sector (biosilica encapsulation), biomedical sector (orthopaedic implants), diagnostics sector (application in bioassays), pharmaceutical sector (nanomembranes in drug delivery), dental applications (silica membranes), white biotechnology sector (biocatalysts, encapsulation technology), cleaning agents / washing powders (enzyme encapsulation), dental care (tooth paste: enzyme encapsulation), and recombinant protein technology (silicateins).
Application fields Lab-on-a-Chip: The development of miniaturized and automated instrumentation for (bio-)analysis (Lab-on-a-Chip) has a large potential in all kinds of application fields. The interferometric sensor principles such as the MRR have a good position to enter this application field. This technology can be used as portable, battery operated field instrumentation or can be incorporated into larger (monitoring) systems. Another area is the R&D market in which screening of molecular interactions in widely applied.
List of Websites:
Project website address: http://www.eu-mem-s.de/(öffnet in neuem Fenster)
Contact details:
Prof. Dr. Werner E. G. Müller; ERC Advanced Investigator Group, Institut für Physiologische Chemie, Universitätsmedizin der Johannes Gutenberg-Universität, Duesbergweg 6, D-55128 Mainz, Germany; Phone: 06131-39-25910; Fax: 06131-39-25243; E-mail: wmueller@uni-mainz.de
There is strong interest in the development of novel functionalized membranes which can be used as microsieves, as a component of integrated analytical systems, in food processing, drug discovery and diagnostic applications. This project was based on a combination of three break-through technologies, developed by the applicants in the past, with high impact for nano(bio)technological application: (i) the S-layer technology allowing the construction of nanoporous protein lattices, (ii) the biocatalytic formation of inorganic materials by silicatein, a group of unique enzymes capable to catalyze the formation of porous silica from soluble precursors, and (iii) the sol-gel technique for encapsulation (immobilization) of biomolecules serving as biocatalyst or as a component of sensors. The goal of this project was to design and fabricate - based on molecular biology inspired approaches - nano-porous bio-inorganic membranes with novel functionalities for industrial application. These membranes were formed by S-layer proteins, which are able to assemble to highly ordered structures of defined pore-size, and recombinant silicateins or silicatein fusion proteins. The innovative functionalized membranes developed in this project exploit two principles: (i) protein self-assembly and (ii) enzymatic (silicatein-mediated) deposition of inorganic material used for reinforcement of the membranes as well as for encasing biomolecules, providing the membranes with new functionalities. The S-layer-coated surfaces could be functionalized by binding of streptavidin and used for binding of biotinylated silicatein. The developed functionalized membranes could be immobilized on inert silicon-nitride waveguide core material. The methods developed were used for encapsulation of biocatalysts (enzymes) and antibodies directed against small molecules in optically transparent silica matrices. Using this approach, we could design an enzymatic biosensor within silica thin films through the sol-gel technology. The silica matrix was formed either enzymatically (via silicatein) or via sol-gel techniques. The new technologies were applied for the preparation of absorbers for drinking water systems. The aim was to build absorbers based on S-layer technology functionalized with selected antibodies against Legionella surface antigens, which reduce the number of Legionella possibly present in the water systems of e.g. homes, nursing homes, hospitals and hotels. Moreover, a bacterial antigen (L. pneumophila) biosensor was constructed by the binding of a specific antibody to the S-layer functionalized nitride surface of a micro ring resonator sensor connected to a read-out system. The S-layer technology was also used for the generation of microsieves with additional functionality, such as protein repellence and smaller pore sizes. Our results revealed that the biofunctionalized microsieves can be used for the removal of potential pathogens from house-hold drinking water. A proof of concept has been performed for an integrated membrane biosensor platform based on functionalised silicon nitride microsieves. The dissemination and communication activities in this project also included activities to improve the public acceptance of nanotechnology. The new techniques were exploited by three research-based SMEs and the enduser involved in the project, in microfluidics based sample processing and micro-array development, in industrial nanosieves, as well as in sensors in drinking water systems.
Project Context and Objectives:
The production of new micro/nanoporous membranes, which can be functionalised, is in rapid development and has been shown to have a high potential in a wide range of industrial applications. The immobilization of biomolecules serving as biocatalysts or as a component of sensors into organised structures presents many advantages in terms of cost reduction, high-throughput sample processing, integration, and automation. Bioactive molecules can be oriented onto a nanostructured surface or encased in an inorganic matrix. This project aimed to develop novel functionalised inorganic-organic membranes, with selective and defined physico-chemical properties, which can be used as nanosieves, as a component of integrated analytical systems, in food processing, drug discovery and diagnostic applications. Bioactive molecules such as enzymes and antibodies, immobilized within the inorganic-organic hybrid architecture of the membrane, maintain their biological activity and are more stable, and/or are only accessible for a selected (size) range of substrates compared to their freely soluble counterparts. The manufacture of nanoporous membranes reinforced by an inorganic skeleton and combined with bioactive biomolecules is a highly innovative field. This project aimed to combine two parallel approaches for the formation of functional inorganic-organic membranes. The first one regarded the formation of a single-layered structure derived from bacteria and archaea, named S-layer, in which the self-assembling property of the subunits into a porous “paracrystalline” structure is exploited as a template on which other molecules, such as silicateins, can bind. The second approach regarded the encapsulation of biomolecules into the porous silica glass formed either biocatalytically (via silicateins, a special group of enzymes catalyzing the formation of inorganic metal oxides) or by sol-gel process. While the first approach was completely biological, being the S-layer derived from living organisms, the second one resulted in an inorganic-organic hybrid composition of the membrane, in which organic molecules can be encapsulated. Both nanobiotechnology approaches are inspired by molecular biology and are based on processes of self-organisation at mild environmental conditions.
The specific objectives of this project were:
1. Design and synthesis of the basic building blocks (modules) of the new membranes by molecular biology methods: S-layer proteins, silicateins, fusion proteins
2. Self-assembly of these modules to nanoporous membranes
3. Reinforcement of the generated membranes by silica (metal oxide) nanoparticle formation / deposition
4. Physical-chemical characterization of the generated functional membranes
5. Applying encapsulation technology for the design of nanoporous membranes used as biocatalysts and / or sensors
6. Exploiting the newly developed technique for the manufacture of novel micro/nanosieves and capsules
Project Results:
The experimental part of this project was performed in eight work packages (WP1 – WP8) concerning S & T activities. The results can be summarized as follows.
WP1: Design and synthesis of the basic building blocks (modules) of the new membranes by molecular biology methods: S-layer proteins, silicateins, fusion proteins
The objective of this work package was the expression, purification and characterization of the building blocks (modules) used for the construction of the functional membranes. The specific objectives were: (1) Expression and purification of recombinant silicateins; (2) Expression and purification of S-layer proteins; (3) Expression and purification of fusion proteins; and (3) Characterization of the recombinant proteins.
The silicatein-alpha cDNA from Suberites domuncula was expressed in the oligohistidine expression vector pQ30 A by UMC-Mainz. For large-scale production of the recombinant silicatein-alpha, E. coli Host Strains M15 was transformed with the plasmid and cultured in bioreactor BIOSTAT A plus. Induction was performed with IPTG. The lysate was purified and refolded using the Profinia Protein Purification System.
Glu-tagged silicatein, carrying an additional C-terminal His tag, was prepared in bacterial expression vector pBAD/gIII A. The construct was used to transform TOP10 E. coli cells. The expression of the recombinant protein was induced with arabinose. Then, proteins were extracted and purified by Ni-NTA affinity chromatography. In addition, silicatein alpha was expressed in the yeast Pichia pastoris. The purity of the expressed proteins was checked by SDS-PAGE.
The expression and purification of S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 was performed by BOKU. For the production of biomass, the mesophilic, gram-positive bacterium Ly. sphaericus CCM 2177 was grown in a fermenter in continuous culture in nutrient broth medium for 7 days. Cell wall fragments from whole cells were prepared by ultrasonication of Ly. sphaericus cells, extraction of the plasma membran with Triton X-100, and washing with Tris/HCl buffer.
Isolation of S-layer protein from the peptidoglycan-containing sacculi (PGS) was performed by separation of SbpA from PGS by addition of 5 M GHCl in Tris/HCl buffer. Purification of S-layer protein SbpA was performed by gel permeation chromatography (GPC), followed by lyophilisation. The quality control of purified S-layer protein SbpA was performed by SDS-PAGE.
The obtained S-layer material was used for in vitro recrystallization studies for the functionalization of different solid supports and for recrystallization at the air water interface.
The expression and purification of fusion proteins was performed as follows (UMC-Mainz and BOKU). S-layer fusion protein carrying a C-terminally fused core-streptavidin (rSbpA31-1068/STAV40-157): Cloning of the recombinant S-layer fusion protein rSbpA/STAV in E. coli TG1 was performed using the pET system. Heterologous expression of the fusion protein between the S-layer protein SbpA of Ly. sphaericus CCM 2177 and streptavidin in E. coli BL21(DE3)star was carried out following the instructions described in the pET System Manual (Novagen). Purification of fusion protein was done by gel permeation chromatography in presence of the chaotrophic reagent 2 M guanidinehydrochloride.
The gene encoding the fusion protein rSbpA/Silicatein was amplified with PCR and cloned (in two steps) into the restriction sites NcoI, BamHI as well as XhoI of the plasmid pET28a following the instructions of the pET System Manual. The plasmid construct was cloned into E. coli TG1. Positive clones were isolated from selective agar plates containing kanamycin. Determination of the occurrence of the chimaeric insert encoding the rSbpA/Silicatein fusion protein was done by restriction analysis.
The plasmid carrying the chimeric gene encoding the fusion protein was isolated from E. coli TG1 by using a Plasmid midiprep kit (Qiagen). The CaCl2-competent expression stain E. coli BL21(DE3)star was chemically transformed with the purified plasmid. Heterologous expression of the fusion protein in E. coli BL21(DE3)star was carried out as described in the pET System Manual (Novagen).
Production of the fusion protein rSbpA/Silicatein by heterologous expression in E. coli was checked by SDS-PAGE and Western-blot analysis using a polyclonal rabbit antibody raised against the S-layer portion of the fusion protein. Purification of the S-layer fusion protein rSbpA31-1068/Silicatein was done by exploiting the binding of the His-tag to a Ni-NTA-column in the presence of the chaotropic reagent guanidinehydrochloride.
UMC-Mainz could propose a new model for the catalytic mechanism of silicatein. This mechanism leads to the formation of reactive, cyclic silicic acid species (trisiloxane rings and higher-membered siloxane rings) which easily promote the silica polycondensation reaction. The modeling studies revealed that the synthetic substrate, TEOS, fits very well into the substrate pocket of the silicatein-alpha molecule, which comprises the amino acids of the catalytic center, Ser, His and Asn.
In addition, UMC-Mainz studied the different stages of the process termed bio-sintering. UMC-Mainz addressed for the first time sintering as component of a multi-stage process (including polymerization, gelation, aging, and solidification) within a biological system, ultimately giving rise to the synthesis of (fused) siliceous spicules. UMC-Mainz could present evidence that the solidification of the axial cylinder is mediated by a silicatein-driven, bio-sintering process.
With the scaffold protein silintaphin-1, a first silicatein interactor that facilitates the formation of the axial filament and, consequently, the formation of the growing spicule was discovered by UMC-Mainz. Silintaphin-1 facilitates and controls not only the assembly of silicatein as filaments but also the assembly of nanoparticles (gamma-Fe2O3, titania, or silica) as ordered structures. Silintaphin-1 was discovered during a yeast two-hybrid library screening.
The silintaphin-1 interaction with silicatein during the structure-guiding bio-silica formation was studied. Recombinant silicatein and recombinant silintaphin-1 were used at different stoichiometric ratios to form axial filaments and to synthesize biosilica. UMC-Mainz could demonstrate that the enzymatic activity of silicatein-alpha strongly increased by 5.3-fold (substrate: TEOS), leading to significantly enhanced synthesis of biosilica.
Furthermore, UMC-Mainz could elucidate the process of hardening / ageing of biosilica. UMC-Mainz could show that in sponge primmorphs (three-dimensional [3D]-cell culture system of sponges), manganese (Mn-sulfate) strongly affects the morphology and the hardness of the skeletal elements. During incubation of primmorphs with Mn-sulfate, spicules were formed that have lost their regular morphology and their smooth surfaces. The spicules formed in the presence of Mn-sulfate were porous and less rigid. Micro-hardness properties of the spicules were determined applying the nanoindentation method. In contrast to spicules that developed in primmorphs in the absence of Mn-sulfate and which displayed an almost plane surface in the fracture areas, the fracture sites of spicules isolated from primmorphs from Mn-sulfate containing cultures showed a granular appearance. The average hardness of the spicules developed in the absence of Mn-sulfate is about 1.5 GPa, while that of spicules from Mn-sulfate cultures was much lower in the range of 0.35 GPa.
First attempts to mimic that process in vitro by using recombinant silicatein-alpha were successfully performed by addition of poly(ethylene glycol) (PEG) during the enzymatic silicatein mediated reaction. PEG has been shown to facilitate sol–gel processes during silica formation starting from TEOS and forming solid silica particles by interconnected spheroidal silica nanoparticles. At neutral pH values and at room temperature PEG with its ether oxygen groups [acting as hydrogen-bonding acceptor] interacts readily with silanol groups [hydrogen-bonding donor] of the silica species via hydrogen bonds
For these experiments recombinant silicatein-alpha was added to an assay system containing prehydrolyzed TEOS as substrate. In the absence of PEG, 55 microgram of bio-silica per ml assay was synthesized by 6 microgram of enzyme under the incubations conditions used. After addition of PEG to the substrate, prehydrolyzed TEOS, the amount of biosilica formed increased and reached at molar ratios between prehydrolyzed TEOS and PEG (between 1:0.1 and 1:1) significantly higher values.
Highlights:
• Successful expression and purification of S-layer - silicatein fusion protein (rSbpA/Silicatein)
• New model of silicatein reaction mechanism
• Discovery of the process of bio-sintering
• Isolation of two new silicatein interactors: silintaphin-1 and silintaphin-2
• Elucidation of mechanism of hardening /ageing of biosilica
WP2: Self-assembly of these modules to nanoporous membranes
The objective of this work package was the fabrication of the functionalized membranes / matrices from their basic building blocks (S-layer proteins, silicateins, fusion proteins).
The specific objectives were: (1) Preparation of biotinylated recombinant silicatein and / or biotinylated S-layer proteins; (2) Self-assembly of the S-layer protein monomers on various surfaces, including gas/water interface; (3) Functionalization of the S-layer-coated surface by binding of streptavidin; (4) Binding of the biotinylated silicatein to the functionalized S-protein layer; and (5) Functionalisation of micro-array waveguides with membranes.
Biotinylated S-layer proteins were produced by BOKU. In SPR studies performed for the functionalization of solid support with S-layer poteins, biotinylation of wild-type S-layer protein SbpA was done using the heterobifunctional cross-linker EZ-Link Sulfo-NHS-Biotin.
The recrystallization of wild-type S-layer protein as well as of the recombinant S-layer fusion proteins was performed after solubilization of S-layer protein/recombinant fusion protein by addition of 5 M GHCl. S-layer monomers were produced by removal of the GHCl by dialysis against A. dest. SbpA recrystallization is a Ca2+-dependent process and occurs by the addition of 10 mM CaCl2.
The S-layer protein SbpA of Ly. sphaericus CCM 2177 which was produced and purified in WP1 could successfully recrystallized a) as self-assembly products in suspension, b) as closed monolayers on silicon chips, c) as monolayers on positively charged liposomes, and d) as recrystallized monolayers on lipid films. The recrystallization ability was checked by using transmission electron microscopy as well as atomic force microscopy.
The purified lyophilized rSbpA/STAV fusion protein showed excellent recrystallization properties, resulting in self-assembly products having an average size of 1 micrometer clearly showed the square lattice pattern.
In TEM pictures of self-assembly products of rSbpA/Silicatein formed in suspension, the square lattice symmetry of the SbpA portion in the fusion protein was clearly visible. Lattices showed a center-to-center spacing of the morphological unit of 13.1 nm, which is typical for SbpA. Results indicated that the fusion of silicatein to the C-terminal end of the S-layer protein SbpA did not interfere with the recrystallization properties of the S-layer protein.
In SPR studies, stabilization of the obtained crystalline monolayer of the S-layer protein SbpA on the gold chips was done by crosslinking with glutaraldehyde (amine-reactive homobifunctional crosslinker) which reacts with free amino groups of the S-layer protein.
In a BIACORE 2000 SPR apparatus, the S-layer protein SbpA of Ly. sphaericus CCM 2177 was recrystallized on a SPR-gold-chip. In a second step, the S-layer lattice was stabilized by cross-linking with glutaraldehyde. After washing step, the S-layer was biotinylated with NHS-Biotin and subsequently, active streptavidin tetramers could be bound on the S-layer lattice in a density of 1 ng/mm2. The result was an S-layer-based sensor chip with the ability to bind biotinylated silicatein. This set up can be used to bind biotinylated silicatein, resulting in an S-layer-based sensor system.
In an alternative approach, after recrystallization of SbpA on the chip surface and formation of a closed crystalline SbpA monolayer and cross-linking, free carboxyl groups on the surface of the S-layer lattice were activated with EDC/NHS 1-ethyl-3-[3-(dimethylamino)propyl carbodiimide/N-hydroxy-succinimide]. In a further step, stable peptide bounds were formed between the activated carboxyl groups of the S-layer matrix and free amino groups of strepatavidin molecules. As a result streptavidin could be bound on the chip surface in a density of 1 ng/mm2.
Surprisingly, binding of a biotinylated macromolecule to the active streptavidin on the sensor chip revealed a leakage of the S-layer after some minutes of incubation indicating that the very strong interaction between streptavidin and the biotinylated ligand led to a removal of the S-layer from the gold surface. Due to this fact, a new approach based on SbpA-cysteine mutants was developed.
In SPR experiments, recombinant SbpA cysteine mutants carrying one single cysteine residue at the N-terminus of the S-layer protein were covalently linked to the surface of the SPR-gold-chip. Then, the development of the sensor chip (crosslinking of the S-layer, EDC activation and binding of streptavidin was done as described before. In this approach, no leakage of the S-layer lattice from the gold chip during incubation with biotinylated peroxidase could be observed (model system). The developed sensor chip can be used for the binding of biotinylated silicatein.
Functionalisation of micro-array waveguides with membranes was studied using two different substrates: Si3N4 and SiO2. AFM investigations revealed that both substrates could be coated with the recombinant S-layer as the square lattice structure could be easily seen, but only the SiO2 surface exhibited a closed monolayer while on the substrate having the Si3N4 surface chemistry only patches of recrystallized monolayer could be detected. As the Si3N4 surface corresponds to the real sensor chip surface the coating conditions were changed; washing was only conducted with Milli Q water and a pre-coating step with Poly-l-lysine (PLL) was performed. With these changes also on the Si3N4 substrate a closed monolayer of the rSpbA31-1068ZZ could be easily detected.
The properties of rSpbA31-1068ZZ to form a complete crystalline monolayer onto the surfaces were used within the sensor chip from LX.
Further the rSpbA31-1068ZZ S-layer protein was recrystallized onto wafers from Surfix supplied by LX using the epoxy surface chemistry for covalent binding of the S-layer protein replacing the crosslinking steps with dimethyl pimelimidate (DMP). Therefore, no additional crosslinking with DMP was needed and the recrystallization of the S-layer could be easily performed at LX after the rSpbA31-1068ZZ monomeric solution.
To confirm the IgG binding and the stability of the covalently, via epoxy groups, bound rSpbA31-1068ZZ the coated epoxy wafers were incubated with human IgG. Subsequently a pH shift was performed and the amount of IgG that could be eluted of the rSpbA31-1068ZZ coated epoxy wafers was measured with alternative techniques using ELISA, as UV measurements did not exhibit the required sensitivity. The stability was checked by treating the coated wafers with a 0.1 M NaOH solution for 20 minutes and subsequent IgG recovery studies.
As ELISA system rSpbA31-1068ZZ coated GREINER ELISA plates were incubated with the eluate obtained from the S-layer coated epoxy wafers. Bound IgG was detected with anti human IgG peroxidase labelled antibodies and TMB as substrate.
The stability of the rSpbA31-1068ZZ monolayer that should be obtained by the epoxy activated surfaces was investigated by incubation of the wafers with 0.1 M NaOH.
The ELISA system showed that more than 80 ng human IgG could be bound per 1 cm2 rSpbA31-1068ZZ coated epoxy activated surfaces. After the treatment with 0.1M NaOH, the binding capacity decreased for approximately 50 % which confirmed the increased stability of the S-layer cross-linked to the wafer surface exploiting the epoxy chemistry.
Highlights:
• Self-assembly / recrystallization of S-layer - silicatein fusion protein (rSbpA/Silicatein) on various surfaces
• Functionalization of the S-layer-coated surfaces by binding of streptavidin
• Binding of protein to functionalized S-protein layers
• Functionalisation of micro-array waveguides (Si3N4)
• Confirmation of the stability of S-layer cross-linked to the wafer surface
WP3: Reinforcement of the generated membranes by silica (metal oxide) nanoparticle formation / deposition
The objective of this work package was the reinforcement of the functionalized membranes generated in WP2 by biocatalytic (via silicatein) formation of silica or other metal oxides, as well as by sol-gel processes.
The specific objectives were: (1) Biocatalytic formation of silica or other metal oxides (via silicatein) on the S-layer-based membranes; (2) Formation of silica on the S-layer-based membranes via the sol-gel process; (3) Design of membranes having organic polymers or natural polymers as a component; and (4) Application of the new technology for preparation of adsorbers for drinking water systems.
The biocatalytic formation of silica on S-layer-based membranes was measured with in situ QCMD by using a Q-Sense E4 instrument (Q-Sense) (BOKU).
One major objective of UPMC was to design novel robust inorganic nanocoatings compatible with the immobilization of biomolecules for the design of biosensors and nanofactories. From the state-of-the-art in this area, this was a challenging task as (i) ultra-thin sol-gel layers are usually prepared using solvent-based processes that are not always compatible with the preservation of the biomolecule conformation and, hence, activity, and (ii) these layers are very prone to dissolution in biologically-relevant conditions.
In this context, a first major progress made during this project was the development of a novel technology for the deposition of multi-layers of silica thin films in purely aqueous media. Interestingly this process was found compatible with the immobilization of one specific enzyme extracted from valuable foodstuff (truffles) that may be further used for sensor design. Another important outcome was that the formation of a thin silica layer on substrates coated with S-layers was possible and induces some organization of the silica network, without impacting the activity of entrapped enzymes. However, it was found that these silica layers were highly porous, leading to rapid leaching of small enzymes. Such an effect was not observed for antibodies that showed preserved immuno-affinity over several days. Despite these interesting performances, it was found to be difficult to use the newly-developed protocol for suitable coating of complex micro-chips as used by some partners (LX). However it is worth mentioning that this protocol was recently shown to be compatible with the immobilization of whole bacteria while allowing their in silica division, providing an interesting tool for more fundamental biological studies.
This led to a second important development, the design of titania thin films. One particular originality of the proposed approach was to use a specific precursor that is water soluble, thus avoiding the use of organic solvent. The protocol was difficult to set up due to its sensitivity to precursor concentration, coating conditions, ionic strength and buffer composition, drying place and time, among other important parameters. However, the final protocol is very simple and should be easily reproduced by others. It was also possible to demonstrate the first deposition of multi-layered titania films and their compatibility with enzyme and antibody immobilization. The robustness of these films could be ascertained over two months and showed comparable performances as commercial immuno-microplates. Finally, they could be deposited in a suitable manner on LX substrates. These data raised an important range of questions especially as the titania formulation was highly sensitive to the presence of proteins. This unfortunately made the interaction with S-layers unfavourable but, on the other hand, opens an important area of investigations at the biomineral interface.
A third development related to material science was achieved during the preparation of hybrid membranes. UPMC showed the possibility to associate two liquid crystalline systems, hydroxypropylcellulose and vanadium oxides, to create soft materials with unprecedented organization. In parallel, UPMC could design thin films associating silica and collagen, two very common, fully biocompatible components. Although these materials have some drawbacks in terms of optical properties that have limited their further use for sensing in this project, the first system should be studied further due to their possible application for energy (batteries) whereas the second is currently being investigated for biomedical applications.
Various partners were involved in the application of the new technology for preparation of absorbers for drinking water systems (IWW, UMC-Mainz, BOKU, UPMC). The aim was to build absorbers based on S-layer technology functionalized with selected antibodies against Legionella surface antigens, which reduce the number of Legionella possibly present in the water systems of e.g. homes, nursing homes, hospitals and hotels.
To bind antibodies against Legionella pneumophila to a filtration unit in an oriented way the S-layer fusion protein rSbpA31-1068ZZ comprising the IgG binding domain of Protein A was chosen. The IgG directed against Legionella pneumophila will bind via their Fc moiety and should capture Legionella pneumophila if present in the fluids passing the membrane.
To allow unhindered flow through a membrane a pore size of 3 micrometers was chosen. To achieve filtration membranes, capable of binding IgG, the S-layer fusion protein had to be recrystallized to and within the filter structure. This was done by inserting the nitrocellulose membrane into a filtration unit and by filter the fusion protein solution in the presence of CaCl2 through the nitrocellulose membrane. This allows the recrystallization of the fusion protein onto and within the filter structure. UV280nm measurements of the S-layer protein solution before and after the filter allowed a semi quantitative determination of the binding success. Subsequently the S-layer protein coating was stabilized by crosslinking with DMP and antibodies raised against Legionella pneumophilia were applied to the filter discs. The so activated filter discs were sent to IWW for evaluation.
Highlights:
• Development of a novel technology for the deposition of multi-layers of silica thin films in purely aqueous media
• Method for biocatalytic reinforcement of functionalized membranes with silica or other metal oxides
• Formation of silica on the S-layer-based membranes via the sol-gel process
• Design of membranes having organic polymers or natural polymers as a component (V2O5 - hydroxypropyl cellulose and collagen - silica)
• Design of titania thin films
• Successul adsorption with a high occupancy and release under controlled conditions of Legionella pneumophila from an S-layer/protein A/polyclonal antibody membrane
WP4: Physical-chemical characterization of the generated functional membranes
The objective of this work package was the physical-chemical characterization of the nanoporous membranes and nanosieves formed by biocatalytic silica deposition on S-layer protein – silicatein matrices or matrices formed by fusion proteins, and or sol-gel technology.
The specific objectives were: (1) Determination of morphology; (2) Determination of the stability of the membranes; and (3) Capability of self-repair.
The tasks of WU involved the preparation and characterisation of well-defined layers on surfaces and membranes. The main goal was to come to well defined membranes and process design for novel membrane processes. In their studies, WU focused on accumulation of particles on membranes under flow conditions. This is a logical complementary activity to get the overall membrane process to work optimally, since using flow to keep particles as much as possible away from the surface may prevent many interaction problems ab initio.
WU used a well-defined nickel sieve onto which in a later stage S-layers and silicatein were deposited, and tested for the separation of yeast cells. WU found that the size of the particles in the permeate could be controlled by the applied transmembrane pressure and cross-flow velocity (which induces particle migration away from the membrane), at high flux and without notable fouling taking place. All results could be summarized into one master curve based on the typical times in the system. WU was also successful to control the process conditions in such a way that they could specifically deposit small cells while removing the large ones, leading to new options for process design. A proof-of-principle of this latter mechanism has been given. When testing microsieves with deposited S-layers WU found that they were lacking mechanical stability, therefore, they focused on new membrane processes, and defined windows of operation for them, which defines to working space for any membrane or sieve, and that can be applied as soon as modified sieves are available.
The capability of self-repair was investigated by UMC-Mainz. The most used structural materials are vulnerable to cracking and are not repaired if not special supplements are added to those materials. This is different to bio-materials which are formed and maintained in organisms, where the integrity of the
The self-healing property of sponge biosilica can be utilized to engineer novel hybrid materials, with silicatein as a functional template, which are more resistant towards physical stress and fracture. In sponge biosilica, the organic template remains embedded into the mineralic phase even after completion of the mineral formation. This finding implies that the functionality of the template remains unaffected because the organic macromolecules forming this template are protected against denaturation and degradation after becoming encased within the mineral structures. As a consequence the organic mineral-forming polymers become functionally active again, after uncovering from their mineralic envelope. As a consequence, the organic template has a self-healing function after destruction of the inorganic shell.
High resolution SEM investigations of sponge spicule lamellae revealed that they are composed of 5-7 nm large nanoparticles. If those samples are exposed to HF, resulting in dissolution of the silica matrix, the organic scaffold, surrounding the nanoparticles becomes visible. The dimension of the holes, formed by the organic material, varies between 5-7 nm, supporting the view that organic proteins are surrounding the silica nanoparticles.
After treatment of the spicules with HF, the proteinaceous filaments can be recovered. With progressing time, the silica is dissolved, leaving behind only the organic axial filament. It is surprising that the proteinaceous axial filament remains preserved in those spicules. The silicatein in these axial filaments remain functionally active as silica-binding protein or as enzyme after dissolution of the biosilica shell.
Highlights:
• Accumulation of particles on membranes under flow conditions: Control of size of particles by the applied transmembrane pressure and cross-flow velocity
• Master curve based on the typical times in the system
• Demonstration of the self-healing property of biosilica for engineering novel hybrid materials
WP5: Applying encapsulation technology for the design of nanoporous membranes used as biocatalysts and / or sensors
The objective of this work package was the integration of silica-encapsulated biocatalysts and antibodies into the fortified functionalized membranes, obtained biocatalytically (via silicatein) or via the sol-gel process. The integrated biocatalysts and antibodies will serve as constituents of novel, membrane integrated nanofactories and sensors.
The specific objectives of this work package were: (1) Entrapment of biocatalysts (enzymes) in the silica matrix; (2) Entrapment of antibodies directed against small molecules in the silica matrix; (3) Construction of a nanofactory based on entrapped enzyme; (4) Construction of a biosensor based on entrapped antibody; (5) Construction of a bacterial antigen biosensor; and (6) Construction of the capsules based on S-layers with well-defined properties for the controlled release of entrapped molecules.
It was possible to use these novel titania layers for designing biosensors. In the case of enzymes, UPMC demonstrated the feasibility of performing a tri-enzyme cascade within a single material, a fact that, to our knowledge, was not reported so far in sol-gel thin films. UPMC could even show that these systems could be used to titrate lactose in milk, suggesting their possible application for food monitoring. In the case of antibodies, environmental-relevant surfaces could also be designed by entrapping antibodies that can recognize some component of bacterial cell membranes. Our studies suggest that the antibodies are partially inside the titania layer while the other part is pointing out at the film surface: hence not only the antibodies are immobilized and stabilized but they also remain able to interact with antigens. In this context, first tests made by IWW do indicate the higher cell adhesion on titania-containing anti-LPS antibodies compared to pure titania films. It is unfortunate that time has missed to associate the titania films with the LioniX chips for detection by IWW.
The design and construction of a bacterial antigen (L. pneumophila) biosensor was performed in close cooperation of IWW, BOKU and LX.
Within WP2 BOKU and LX showed the successful functionalization of the stoichiometric nitride waveguide material with the rSbpa ZZ S-layer protein. A bacterial antigen biosensor can be constructed by the binding of a specific antibody to the S-layer functionalized nitride surface of a micro ring resonator sensor connected to a read-out system (WP7).
For this purpose BOKU supplied LX with the rSbpa ZZ S-layer protein and CaCl2 recrystallization buffer. IWW supplied the antibody for pseudomonas fluorescens and the corresponding bacteria. The choice for this bacterium was based on its similarities with L. pneumophila whereas it is harmless. This allowed LX to work with the bacteria in a normal lab, gain experience and optimize the detection system before a final test could be performed at IWW with L. pneumophila.
Highlights:
• Design of biosensors using novel titania layers
• Demonstration of the feasibility of performing a tri-enzyme cascade within a single material
• Use of these systems to titrate lactose in milk (possible application for food monitoring)
• Entrapment of antibodies recognizing component of bacterial cell membranes
• Design and construction of a bacterial antigen (L. pneumophila) biosensor
• Construction of a bacterial antigen biosensor by binding of a specific antibody to the S-layer functionalized nitride surface of a micro ring resonator sensor connected to a read-out system
• Demonstration that the sensing system works very accurate and with a very high sensitivity comparable to SPR equipment
• Improvement of the performance by a factor of 10 – 100 in the case a reference MRR sensor is applied in the system
WP6: Exploiting the newly developed technique for the manufacture of novel micro/nanosieves
The aim of this work package was the generation of hydrophobic to hydrophilic or protein repelling surfaces on microsieves, as well as the generation of microsieves with smaller pore sizes using the developed technology.
The specific objectives of this work package were: (1) Development of S-layers-coated microsieves to prevent undesired interactions between components of the liquid and the microsieve surface and (2) Development of membranes with smaller pore sizes on microsieves.
The task of AM was the exploition of the newly developed technique for the manufacture of novel micro/nanosieves. The aim was the generation of hydrophobic to hydrophilic or protein repelling surfaces on microsieves, as well as the generation of microsieves with smaller pore sizes using the developed technology.
The S-layer technology was used for the generation of microsieves with additional functionality, such as protein repellence and smaller pore sizes. This work has been done by AM in collaboration with WU and BOKU. In the first project period, AM explored an innovative zwitterionic coating to prevent protein interaction with the microsieve surface. In order to obtain nanoporous membranes, AM supplied microsieves of 450 nm pore size dimensions for coating with S-layers.
With current lithographic tools the production of microsieves with sub-micron pore is feasible. However creating nanopores in the order of 10 nm or smaller is still challenging and difficult to achieve with lithographic techniques. Our idea was that the combination of conventional procedures for the manufacture of microsieves and the new technique developed in this project will allow the generation of membranes with smaller pore sizes by formation of nanoporous S-layers spanning the existing pores on the microsieves. We expected that the new developed techniques for surface functionalization with self-assembled S-layer proteins might be a way to create such nanoporous membranes.
Several attempts to produce S-layer coated microsieves failed to give satisfactory results. Therefore an alternative approach was developed for biofunctionalization of microsieves. A highly reproducible and facile method to covalently attach antibodies on the SixN4 surface of microsieves was achieved by a two-step procedure: attachment of an epoxide-functionalized monolayer by a photochemical reaction, followed by immobilization of antibodies.
Silicon nitride (SixN4, x>3) surfaces were functionalized with an epoxide-terminated monolayer by a UV-induced reaction. Hydrogen-terminated SixN4 substrates were obtained through etching with HF solution, and employed in the photochemical (lambda = 254 nm) attachment of 1,2-epoxy-9-decene.
The stability of the epoxide-terminated monolayers on SixN4 surfaces as well as on microsieves was studied in argon atmosphere for 1 month. No significant changes in both water contact angle and XPS spectra of the surfaces were found after 1-month storage, indicating long-term stability.
Anti-Salmonella antibodies-coated SixN4 surfaces were obtained via coupling of lysine residues at the outside of the antibodies to the epoxide-coated surface.
Our results revealed that the biofunctionalized microsieves can be used for the removal of potential pathogens from house-hold drinking water. Instead of removing the pathogens by size-based membrane filtration, the pathogens can also be removed by selective capture on the surface of the membrane. This is possible by functionalizing the microsieves with antibodies specific for the pathogens. A prototype membrane was further developed and tested.
Highlights:
• Development of microsieves with an innovative zwitterionic coating to prevent protein interaction with the microsieve surface
• Application of microsieves of 450 nm pore size dimensions for coating with S-layers in order to obtain nanoporous membranes
• Development of a reproducible method to covalently attach antibodies on the SixN4 surface of microsieves by a two-step procedure
• Demonstration of the stability of the epoxide-terminated monolayers on SixN4 surfaces and microsieves
• Preparation of anti-Salmonella antibodies-coated SixN4 surfaces
• Application of the biofunctionalized microsieves for the removal of potential pathogens from house-hold drinking water
• Development of a prototype membrane
WP7: Exploiting the newly developed technique in microfluidics based sample processing and micro-array development
The objective was to build and test an ‘end-to-end’ desk-top system with functions / steps miniaturized and integrated which are essential to demonstrate the feasibility of a future development; an evanescent wave excited micro-array platform will be developed.
The specific objectives of this work package were: (1) Design of integrated system and (2) Realization of integrated system.
Optical biosensors present several advantages with respect to, for example, the more established electrochemical or piezoelectric principles, because they are insensitive to electromagnetic interferences (enabling extremely high sensitivity) and can potentially be used in aggressive environments. Thanks to the progress in planar waveguide technology in the last decade, including the excellent miniaturization capabilities based on optoelectronic integration with for example VCSELs, these platforms are emerging in both applications as a sensor as well as screening instruments for molecule interactions and functionalization procedures.
In the original description of the work package, integration of pre-sampling functionality in the end-to-end demonstration system was proposed. At the beginning of the project it has been decided to focus only on the detection of bacteria using the MRR sensing system, which in principle, does not require pre-sampling on-chip.
There are three basic detection schemes using the evanescent field of an optical wave propagating in a waveguide:
1) Fluorescence based optical detection
2) Refractive index based optical detection
3) Absorbance based optical detection
The fabrication scheme and associated technology for each is similar. Therefore in the first two years of the project, LX worked successfully on both the development of new fabrication routes as well as on improvements of existing TriPleX based technologies. The first approach increased the design and fabrication flexibility, resulting in a higher compatibility with the proposed on chip optical characterization and possible future pre-treatment functionalities. The second approach resulted in improved sensitivity of the opto-fluidic chip and higher yield values. As a result, basic building blocks have been formed which can be used to rapidly design and fabricate opto-fluidic microchips modelled to a specific task.
Highlight:
This approach led to two mature fabrication routes for TriPleX based opto-fluidic devices including the possibility to integrate metal pattering. One uses a stack of silicon and borofloat substrates, the other makes use of only fused silica glass. The additional advantage of the latter is its (UV) transparency and non-conductive behaviour opening up the possibility to incorporate additional conductive sensing elements.
Initially the absorbance based detection approach had been chosen as detection platform due to its simplicity. Although its resolution and sensitivity would not be sufficient to function as suitable biosensor for this project, it allowed basic and quick functionalization of waveguide surfaces to test the compatibility with the evanescent field sensing detection principle. After the optimization of different process and functionalization steps, the step to the refractive index based micro ring resonator (MRR) sensing platform could be made.
Highlight:
During the project the fabricated absorbance based detection chips have been used in multiple project tasks and the developed platform resulted in a working and often used demonstration kit. The detection principle is concurrently being optimized in the Dutch NanoNextNL 10C.01 project in which it is prone to become an integrated system for online micro reactor synthesis monitoring and analysis. The system is also adapted to function as educational tool in the Dutch department of nanotechnology at Saxion Hogeschool Enschede.
Highlight:
With use of the developed absorbance based detection chips, the innovative concept of on chip filtering for fluorescence based detection platforms was explored. The filtering principle is based on the deposition of a dye, stabilized in PMMA, in the detection window. If the dye (based on its absorption properties) is chosen correct, the excitation signal can be filtered out whereas the fluorescent signal is left unimpaired, resulting in a simple, compact and low cost on chip filter system with high sensitivity. The chosen dye has an absorbance peak around 642 nm and is transparent around 665 nm. The first is the wavelength of excitation of the chosen example fluophore Alexa fluor 647 and the latter the wavelength of emission. Using a white light source and spectrometer, a suppression of >40 dB could be measured within a transmission window of 30-50 nm!
Highlight:
Within this project two approaches were explored for the immobilization of antibodies on the nitride surface of waveguides. These are functionalization using a sol-gel layer (UPMC technology) and functionalization using the S-layer (BOKU technology). Antibodies would be entrapped first in the 3D matrix and second bind to the 2D single layer. Material samples, absorbance based chip and even MRR chip have been prepared and supplied to the different partners to optimize the processes.
Highlight:
In addition LX supplied epoxy activated surfaces to BOKU based on alkene/alkyne chemistry, in collaboration with sister company and subcontractor Surfix (www.surfix.nl). These resulted in a covalent bond between surface and S-layer.
After the second year of the project, electronics for the detection system based on refractive index changes, e.g. the micro ring resonator (MRR) system became available through developments within the FP7 BIOMONAR project. The modular build-up of the electronics allows an easy upgraded to a sensor array readout system. The current electronics allow the read-out of up to 4 sensors (array) simultaneously.
Highlight:
With the use of the developed and optimized fabrication processes, different series of high performance micro ring resonator chips have been fabricated. The final series included an integrated, ‘on-board’, light splitter allowing an 2 x 1 array of MRR sensors of which one could be used as reference. Fluidic handling is being supported by two types of manufactured glass covers, one with microfluidic channels for controlled fluidic handling and one with open top, allowing direct access to the MRR surface, simplifying local functionalization.
Highlight:
The performances of these TriPleX MRR chips were very impressive, showing a sensitivity of 110 nm/RIU and a resolution of 1 pm in wavelength. This allows the detection of a change in refractive index of less than 9•10-6 RIU which is comparable with standard SPR systems. This was more than sufficient for the intended biosensor and therefore the sensing principle and system was accepted as the detection platform technology for the objective of this work package. Currently optimization activities are aiming on a sensitivity of 10-7 – 10-8 RIU, among others allowed by the application of a reference MRR sensor on the same chip.
The TriPleX based MRR biosensor chips are economically feasible in lower volume (niche) markets, as relatively cheap contact lithography is allowed. Moreover, low-cost hybrid integration of inexpensive VCSELs and detectors is enabled by the high coupling efficiency to TriPleX waveguides.
Highlight:
Functionalization experiments of the surface of the micro ring resonator with the S-layer and binding tests of the antibody have successfully been performed by LX. The final shift in response was 5032 RU which is comparable with the values obtained with SPR measurements.
A suitable bio-detection model for Pseudomonas fluorescens for testing was chosen by IWW. The choice for this bacterium is based on its similarities with L. pneumophila (rod-shaped, gram negative), the specified bacteria to be detected in the project, and its harmlessness. This allowed LX to work with the bacteria in a normal lab and gain experience and optimize the detection system before the final test could be performed at IWW using L. pneumophila. Final tests of functionality and sensitivity of the MRR detection system for the detection of P. fluorescens have been performed at the lab of IWW in corporation with LX. These clearly show the potential of the MRR sensor platform by presenting the full end-to-end result from build-up of sensor functionality, S-layer recrystallization on the surface, antibody binding and the attempt to detect bacteria.
WP8: Up-scaling, development of prototypes, and evaluation by endusers
The objective of this work package was to investigate options to adapt the developed techniques for an industrial production. This work package also involved the evaluation of the developed techniques by the endusers being part of the consortium.
The specific objectives of this work package were: (1) Up-scaling of production; (2) Optimization of yield; (3) Development of prototypes of the functionalized membranes; and (4) Evaluation of the newly developed functionalized membranes.
For large-scale production of the recombinant silicatein-alpha, a bioreactor was used. Fermentation proceeded in a culture volume of 3 liters with additional aeration and stirring at 37°C with controlled pH until reaching mid-log phase. The expression of the recombinant protein was induced by addition of IPTG. The lysate was purified with Profinia Protein Purification System. The protein concentration was measured with 2-D Quant Kit and verified by densitometric analysis with Odyssey Infrared Imaging system.
To obtain an active biotin binding S-layer protein the recombinant S-layer fusion protein rSbpA STAV, where a monomer of streptavidin was fused to the S-Layer protein moiety, the addition of three monomeric streptavidin units, followed by a refolding process, was necessary. Beside the fact that two recombinant proteins had to be expressed (rSbpA ST and streptavidin) the active rSbpA STAV had to be generated by refolding processes and labor-intensive purification steps. A rapid dilution step and two chromatographic steps were necessary as already described previously.
Two additional S-layer ST fusion proteins where constructed: His–rSbpA Strep 16 and rSbpA Strep 12 having 16 or 12 amino acids as linkers respectively. The new S-layer fusion proteins were cloned and expressed by exploiting the pET system.
With the new purification method of rSbpA ST an active S-layer fusion protein could be obtained without refolding and additional purification steps. Additionally the functional biotin binding S-layer protein could be obtained at higher yields providing S-layer coated substrates for binding of biotinylated proteins at a larger scale.
AM developed prototypes of the functionalized membranes.
1. A new method combining selective capture, microfiltration and automated fluorescence imaging for the rapid detection of microorganisms has been explored. Selective capture of Salmonella bacteria was demonstrated with highly porous micro-engineered silicon nitride membranes (microsieves) with uniform pore sizes in the range of 0.45 to 5.0 micrometers. We have explored a two-step method to obtain antibody coating on microsieves: 1,2-Epoxy-9-decene was photochemically attached to the silicon nitride microsieve surface, subsequently epoxide-terminated microsieves were bio-functionalized with anti-Salmonella antibodies.
2. The influence of flow rate in capture efficiency of antibody-coated 3.5-micrometer microsieves was investigated. The capture percentage increased from ~30 % to ~70 % when the flow rate decreased from 100 microliter/min to 5 microliter/min, respectively.
3. The use of antibody-coated microsieves as microbial selective capture devices was shown to be promising for the direct detection of microorganisms, giving a strong impulse to the further development of rapid diagnostics.
The conclusions based on the results are: Antibody-coated microsieves with pore sizes larger than the size of Salmonella displayed a significant increase in the capture efficiency as compared to uncoated microsieves. The capture efficiency can be obtained up to 66 % at the flow rate of 5 microliter/min. The detection protocol can be performed within few hours, which is significantly faster than diagnostic techniques such as agar-plating, and magnetic microspheres. The results exhibit the great potential of combining bioselective capture and microfiltration for the direct and rapid detection of microorganisms in crude biological samples.
The evaluation of the newly developed functionalized membranes in removing potential pathogens in household drinking water was performed by IWW.
Different types of functionalized surfaces have been developed within this project and have been evaluated by IWW in cooperation with the other partners in Mem-S project. The surfaces which have been developed had the aim to use them as filtration membranes, nanosieves and biosensors. Surfaces having specific functions are of great interest because they have the potential to be used e. g. as diagnostic tools to detect specific microorganisms or as a treatment step and to reduce human exposure e. g. to Legionella pneumophila. As target organisms L. pneumophila was chosen within this project because this species is water born and a human pathogen often found in house hold and technical water systems.
Several techniques and testing methods have been applied such as encapsulated and crystallization technology for the design of nanoporous membranes used as biocatalysts and/or sensors. Evaluation and testing of the developed functionalized surfaces have been performed by IWW in cooperation with the other partners.
Functionalized S-layer lattices with immobilized antibodies had been coated on commercial available filters with pore size 5 micrometers by BOKU together with the producer (Pall Corporation, Germany). Evaluation of this functionalized filtration membrane by IWW and the target organisms L. pneumophila showed a good retention. Regeneration by controlled release via pH shift to 3.5 was performed also the rate of yield varied from approximately 10% to 50%.
A further functionalized surface was developed and produced based on sol-gel titania with immobilized antibodies to L. pneumphila by UPMC. Testing showed a high selectivity for L. pneumophila, therefore this surface might be applicable as diagnostic tool also because of the low yield further improvement is necessary.
IWW and LX could demonstrate functionalization of a developed biosensor based on micro ring resonator system with crystallized S-layer and the immobilization of an antibody on this layer by a shift in resonance wavelength, comparable to results obtained by SPR analysis. No positive demonstration could be performed with a functionalized and integrated membrane biosensor platform based on a micro ring resonator as detection tool for bacteria. One reason might be an insufficient binding of the antibody to catch specifically bacterial like L. pneumophila. Another reason might be that the active sensing area on the ring resonator is too small for detection of bacterial without pre-sampling steps.
A proof of concept has been performed by IWW for an integrated membrane biosensor platform based on functionalized silicon nitride microsieves together with AM. This surface was developed with the aim to analyze specific bacteria such as e. g. Salmonella species or Legionella species. Nanosieves made of silicon nitride coated with epoxy material were coupled with antibodies. Testing by IWW showed a high potential for these surfaces as diagnostic tools. The microsieves can be applied as filtration units e.g. with a pore size ranging from 0.45 micrometer to 5.0 micrometer. Automatic quantification of trapped bacteria is possible by enumeration by an automated microscopic system. Duration of one assay will be in the range of 1-2 hours. Improvement is necessary in reproducibility of the effectiveness of capture ability.
Highlights:
• Successful up-scaling of silicatein production
• Method for preparation of active S-layer fusion protein without refolding and additional purification steps
• Development of prototypes of the functionalized membranes
• Antibody-coated microsieves as microbial selective capture devices turned out to be promising for the direct detection of microorganisms
• Evaluation of S-layer functionalized filtration membrane with immobilized antibodies revealed good retention of target organisms L. pneumophila
• Testing of sol-gel titania functionalized surface with immobilized antibodies: high selectivity for L. pneumophila
• Proof of concept for integrated membrane biosensor platform based on functionalized silicon nitride microsieves: high potential as diagnostic tool
Potential Impact:
Technical and scientific impact – In this project, a novel type of functionalized membranes have been developed, which offers a variety of potential applications. Our approach was new because it combined for the first time two important discoveries in the field of nano(bio)technology – (1) “paracrystalline“ structures made of S-layer proteins, and (2) enzyme/silicatein-mediated formation of biosilica. The enzymatic, structure-directed formation of inorganic sieve-like structures based on functionalized membranes and/or linked with proteinaceous membrane-like structures cannot be realized by other approaches. Therefore this project brought much progress beyond the state of the art, both in the field of S-layer proteins and silicatein-based silica nanobiotechnology. This project markedly extended the range of possible applications of S-layers and silicatein, in particular towards applications with significant economic impact. The generated functionalized membranes and coatings of nanosieves can be used for the fabrication of highly-ordered inorganic-organic composite structures. We focused on their application in the production of nanosieves used in food technology, Lab-on-a-Chip systems and in removal / detection of pathogens in drinking water system.
Economic impact – The market volume is estimated to be extremely large, in view of the fact that the developed technique can be used in a wide range of applications, reaching from food industry and water purification to biosensor applications and microelectronics. To date, there are no products on the market which make use of the fascinating property of S-layer proteins to form nanoporous “paracrystalline” structures as well as the unique ability of silicateins to synthesize silica – via an enzymatic mechanism - under mild, environmentally compatible conditions (low temperatures and pressures). In this project unknown scientific territory has been entered, whereby the chances of success of this project have been considered as very high.
Social impact – The achieved innovations are also important from the ecological point of view: Silicateins are able to synthesize silica or other metal oxides under mild, energy-saving and environmentally friendly conditions. This and the fact that procedures using Nature as a model (“bionics”) are applied, are also of benefit for the acceptance of (nano)biotechnology in the public. Products which function following the principles of bionics possess increasing interest to potential customers/users.
Main dissemination activities of the project and the exploitation of results:
The scientific results of this project have been included in a number of publications in peer-reviewed international journals.
Publications in refereed scientific journals: about 30.
Presentations at scientific meetings: about 30
Distribution of leaflets, brochures, and CD-Roms: UMC-Mainz and NTM produced a number of leaflets, brochures, and CD-roms concerning the research topic of the project (sponge biosilica - nanotechnology).
Contributions to press, TV, broadcast: UMC-Mainz (W.E.G. Müller) made several contributions to TV (channels ARD, ZDF, 3sat, arte, and others) and the press about the topic “Silicatein and biosilica”; e.g. Mare, Feb 2010, No. 78 (http://www.mare.de/index.php?article_id=2087(öffnet in neuem Fenster)) Spiegel, 21.03.2010 (http://www.spiegel.de/wissenschaft/natur/0,1518,682663,00.html(öffnet in neuem Fenster)) and FAZ, 20. June 2010 (Meeresschwämme – Langweiler als Lehrer).
Organisation of a workshop: The 2-day Workshop in Vienna at the institute of BOKU was organized from 17-18 November 2011. This workshop was also open for guests from universities and industry from outside the project. Laboratory work in the frame of the Workshop in Vienna: The basic production methods for recombinant and wild type monomeric S-layer proteins solutions were shown and recrystallization of S-layer proteins on solid substrates and the generation of self assembly products in solution were performed. Visualization was done by microscopic techniques (AFM and TEM). The binding properties towards human IgG of recombinant S-layer protein rSpbA31-1068ZZ were investigated after recrystallization onto gold wafers by SPR.
Organisation of a Summer school: The Summer school 2012 was organised at the Rudjer Boskovic Institute in Zagreb, Croatia, together with Marie Curie ITN BIOMINTEC from August 22 until August 25, 2012. Some participants of Mem-S project (UMC-Mainz, NTM, and WU) had already participated in the Summer school 2010 of BIOMINTEC in Rovinj from August 16 - 20, 2010.
Activities to improve public acceptance of nanotechnology: UMC-Mainz and NTM participated in the national competition “Germany – Land of Ideas”, initiative “365 sites” – and became winners. This opened a number of additional opportunities to disseminate the results of this project to a broader audience.
UMC-Mainz and NTM organized or participated in the following public events:
1. Mainz: “City of Science 2011“ - “Science Market”: Mainz has become the German “City of Science 2011“. In this frame, various events of the city of Mainz and the University / University Medical Center had been organized such as the “Science Market”. In frame of this event, UMC-Mainz had the possibility to present the topic “Biomaterials from the deep-sea“ to a broader public (4.-5. June 2011) – Exhibition of public presentation/lecture by W.E.G. Müller. In the frame of “Mainz - City of Science”, the Coordinator gave a further public presentation about marine biominerals and their technical application on 18th October 2011.
2. Spektrale 2011 (Exhibition): The partner at UMC-Mainz (W.E.G. Müller) was one of the main exhibitors in the event “Spektrale 2011” in Mainz. This exhibition took place in the Rheingoldhalle in Mainz from 15 July until 14 August 2011. W.E.G. Müller was responsible for the design of a complete room during this exhibition in the thematic area “Deep-sea sponges and the application of sponge biosilica and the biosilica-forming enzymes in nanobiotechnology”.
3. Exhibition: Transfercafé in the frame of “Mainz - German City of Science 2011”: The aim of the Transfercafé is to support the cooperation between business, especially small and medium-sized enterprises, and science. NTM and UMC-Mainz are presented as a best practice example of successful cooperation between companies and academic institutions (March to November 2011).
4. Expo 2012 in Yeosu, Korea: UMC-Mainz had been selected in a competitive call as exhibitor in the German Pavilion at the Expo 2012 in Yeosu, Korea (“The Living Ocean and Coast”). One central topic of this Expo was novel technologies / applications of materials from marine organisms. Therefore, we had the opportunity to communicate the results of this project to a broader public.
The theme "Nanotechnology from the deep sea" was treated in a series of articles in magazines, in the Internet and in interviews.
By the fact that the Mem-S Coordinator, W.E.G. Müller, holds an ERC Advanced Investigator Grants in the field "biosilica", the effectiveness of the dissemination measures further increased. In addition, he succeeded to establish a German-Chinese Joint Center for Bio-inspired Materials (from 2013 until 2022).
Protection of intellectual property: 10 patents
Assessment of the marketing potential of the techniques / products developed in the project: The assessment of the marketing potential of the techniques and products developed in the frame of the project was performed by the SME partners of this project (NTM, AM, LX). The SME partners were also exploring potential user groups interested in the products. The marketing analysis revealed that the products/techniques developed in the frame of this project are of interest for various sectors, more than originally envisaged. These sectors comprise, among others, the food sector (biosilica encapsulation), biomedical sector (orthopaedic implants), diagnostics sector (application in bioassays), pharmaceutical sector (nanomembranes in drug delivery), dental applications (silica membranes), white biotechnology sector (biocatalysts, encapsulation technology), cleaning agents / washing powders (enzyme encapsulation), dental care (tooth paste: enzyme encapsulation), and recombinant protein technology (silicateins).
Application fields Lab-on-a-Chip: The development of miniaturized and automated instrumentation for (bio-)analysis (Lab-on-a-Chip) has a large potential in all kinds of application fields. The interferometric sensor principles such as the MRR have a good position to enter this application field. This technology can be used as portable, battery operated field instrumentation or can be incorporated into larger (monitoring) systems. Another area is the R&D market in which screening of molecular interactions in widely applied.
List of Websites:
Project website address: http://www.eu-mem-s.de/(öffnet in neuem Fenster)
Contact details:
Prof. Dr. Werner E. G. Müller; ERC Advanced Investigator Group, Institut für Physiologische Chemie, Universitätsmedizin der Johannes Gutenberg-Universität, Duesbergweg 6, D-55128 Mainz, Germany; Phone: 06131-39-25910; Fax: 06131-39-25243; E-mail: wmueller@uni-mainz.de