WO2017017628A1 - Synthesis of tridimensional graphene oxide based materials - Google Patents

Abstract

The present invention relates to a three-dimensional structure made of graphene comprising at least two layers substantially constituted by graphene oxide therebetween one intermediate layer is interposed comprising protein rings or protein tubes of nanometric size as well as a process for implementing said structure. The present invention also relates to the use as adhesive, for implementing three-dimensional structures based upon hydrophilic materials, a protein capable of forming rings or tubes of nanometric dimensions having two surfaces with annular shape above and below a plane of the ring, not necessarily identical surfaces, at least partially hydrophobic and equipped with amino acids active from a redox point of view. Moreover, the present invention further relates to the manufacturing of 3D materials based upon graphene oxide functionalized with different metal species. The mineralisation of such materials is possible thanks to the engineering of the inner cavity of ring-like proteins with peptides equipped with amino acids capable of linking fixedly several metals.

Description

SYNTHESIS OF TRIDIMENSIONAL GRAPHENE OXIDE BASED MATERIALS

DESCRIPTION

The present invention relates to a three-dimensional hydrogel or aerogel structure made of graphene comprising at least two layers substantially constituted by graphene oxide (GO) spaced apart by one protein intermediate layer, wherein the proteins are assembled in ring-like or nano-tube-like nanometric structures. The invention also relates to a process for implementing said structure and the uses thereof.

The present invention also relates to the use, as adhesive for manufacturing three-dimensional structures based upon at least hydrophobic materials, a protein capable of assembling in structures having two opposite annular, at least partially hydrophobic, surfaces equipped with amino acids active from a redox point of view, said protein structures being able to trap or produce in situ metal nanoparticles.

STATE OF PRIOR ART

Graphene (Novoselov et al., 2004), a carbon allotrope with 2D structure, has resulted to be a material alternative to silicon thanks to excellent features thereof such as mechanical resistance (Lee et al., 2008), high electrical (Novoselov et al., 2004; Bolotin et al., 2008) and thermal (Balandin et al., 2008) conductivity. The derived graphene oxide (GO) (Huang et al., 201 1), showing additional properties such as the possibility of varying the electro-optical and chemical features of the graphene itself (Eda and Chhowalla, 2010), has added to further contribute to the graphene applicative potential.

GO and partially-reduced GO (rGO) properties have already demonstrated useful in the production of composite materials (Zhu et al., 2010; Huang et al., 2011) with several applications ranging from gas detection and sensors (Ratinac et al., 2010) to the use in nanobiology (Bianco, 2013). However, often GO uses for practical applications require to transform the 2D structure into more accessible 3D complexes, which can be manipulated. Among the mostly widespread 3D materials made of graphene hydrogels and aerogels can be found (Li et al., 2013; Xu et al., 2013), porous complexes with low weight comprising a net of carbon layers with high surface area with interstitial spaces full of liquid (hydrogel) or air (aerogel), respectively, the advantages thereof have resulted in applications such as regenerative medicine, energy storing and opto-electronics (Adhikari et al., 2012).

From what is known in literature (Chabot et al., 2014) the formation of 3D materials made of graphene and graphene oxide is based upon treatments of these materials with chemical and/or physical methods, such as exposure to high temperatures and high pressures (ex. autoclave), exposure to reducing agents (ex. hydrazine or ascorbic acid), exposure to chemical reticulating agents (ex. polyacrylamide) (Xu et al., 2010; Qin et al., 2012). Generally, with the methods described in the known state of art the GO initial suspension has a critical concentration of 3-5 mg/ml and the experimental conditions are very drastic either due to the use of high pressures and temperatures or as one has recourse to toxic materials (ex. hydrazine, polyacrylamide).

The object of the present invention is to provide a process for manufacturing three-dimensional structures made of graphene allowing to overcome the drawbacks present in the known state of art.

SUMMARY OF THE INVENTION

The present invention is based upon the surprising finding that proteins capable of assembling in oligomeric shapes, by forming rings or tubes of nanometric dimensions, can be used to promote the self-assembling of three-dimensional structures.

Such oligomeric shapes have peculiar chemical-structural features, linked to the presence of two surfaces.

A feature of such surfaces is the presence of hydrophobic amino acids which provide in the complex an at least partially hydrophobic character to the same surfaces and, consequently, "adhesive" properties with respect to an equally hydrophobic surface. An additional feature is the reduction activity of such surfaces, mainly linked to the presence of amino acids such as cysteine and/or methionine.

The present inventors have found that by mixing graphene oxide (GO) with the above-mentioned proteins in form of nano-rings or nano-tubes the formation of three- dimensional structures made of graphene itself is promoted, through a self-assembling mechanism, which provides the contemporary reduction of graphene oxide (GO) to rGO.

Therefore, firstly a three-dimensional structure is set forth in the present application, which structure comprises:

- at least two layers substantially constituted by graphene oxide in a reduced or partially reduced form spaced by

-one intermediate layer comprising protein rings or protein tubes of nanometric size, wherein each ring or nanotube has two base surfaces of annular form, opposite one another, each surface being in contact with one of said two layers; and

optionally wherein said rings or tubes comprise metal nanoparticles.

Secondly, a hydrogel or an aerogel, formed by the three-dimensional structures of the invention in aqueous dissolvent, is set forth in the present application.

Thirdly, a process for manufacturing a hydrogel, formed by said three-dimensional structures made of graphene, is set forth in the application, which process comprises a step of mixing in aqueous solution graphene oxide with already assembled proteins, or proteins capable of assembling in aqueous solution in structures with rings or tubes of nanometric dimensions having two surfaces with annular shape, the surfaces being at least partially hydrophobic and having redox capabilities; The following is further set forth in the application: a three-dimensional structure obtainable by means of the process of the invention; the use as adhesive in manufacturing three-dimensional structures based upon substantially hydrophilic materials, of the above-described proteins capable of assembling in oligomeric shapes by forming rings or tubes of nanometric dimensions; a material or device comprising the three-dimensional structure of the invention.

In particular, the process of the invention characterizes for a series of advantages with respect to known processes having the same purposes (manufacturing of 3D structures made of graphene) which can be summarized as follows:

1) the application of drastic experimental conditions (temperature and pressure) or the recourse to the use of toxic substances (such as hydrazines, polyacrylate) or cross-linking agents is not requested;

2) the initial concentrations of graphene oxide are about 10 times lower than those normally used with known chemical/physical methods (Chabot et al., 2014);

3) the formation of the 3D structure takes place contemporary to the reduction of GO graphene oxide which is brought to a state of partial reduction then suitable to manufacture conductive materials;

3) the possibility of functionalizing the 3D structures, by inserting on the proteins which form the rings or the nanotubes, by means, for example, genetic engineering, binding sites for various materials/molecules such as, for example, metal ions or metal nanoparticles. Other advantages and features of the present invention will result evident from the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Chemical structure of graphene oxide (GO) and reduced graphene oxide (rGO). As evident from the figure the reduction of the GO causes increase of unsaturation of carbon atoms and a consequent decrease of the hydrophilic character of the material itself, which acquires at least partially hydrophobic character and possibly partial conductivity.

Figure 2. Diagram of the hydrogel formation of GO in the presence of Prx (Peroxiredoxin). The figure describes the methodology of preparation of the hydrogel, consisting of layers of superimposed GO interspersed with layers of protein.

Figure 3. Representation in "cartoon" of the 3D structure of a decamer of Prx. a) the 2-fold symmetry axes of the dimers and the 5-fold axis of the ring are shown. b) in grey it is shown one of the two base surfaces of the annular shape exposed to the solvent and it is also indicated the equivalence of the opposite surfaces above and below the transverse plane of the ring.

c) the hydrophobic surfaces are shown in black, while the sulfur-containing amino acids which are redox active , i.e. methionine and cysteine exposed on the ring surfaces, are highlighted in white.

d) the 10 polypeptide tales with amino acids capable of binding various metal ions, that extend from the N-terminal of each monomer in the central cavity of the decamer (represented in "cartoon") are shown.

Figure 4. AFM images and height profiles of GO molecules deposited by spin- coating on a Si02 substrate before and after the deposition of the Prx protein by drop- casting.

Figure 5. a) Formation of GO hydrogel induced by Prx in a solution of sodium phosphate and b) spectrophotometric analysis of the process, c-e) AFM images and height profiles of the GO-Prx complexes formed in solution immediately after mixing of the reagents.

Figure 6. STEM micrographies at different enlargements of the GO-Prx multi- layered complexes formed in solution soon after mixing.

Figure 7. a) SEM image and EDS microanalysis after lyophilisation by means of freeze-drying GO. b) SEM image and EDS microanalysis after lyophilisation by means of freeze-drying GO-Prx hydrogel. The scale corresponds to 10 μηι. c) XPS analysis related to the carbon of GO before and after reaction with Prx; d) XPS analysis of sulphur of Prx before and after reaction with GO.

Figure 8. a) Strategy used for internalizing gold nanoparticles (AuNP) inside 3D hydrogel. b) Formation of palladium nanoparticles (PdNP) in situ. Depending upon the order for adding the reagents in solution (1 : (Prx+Pd)+GO or 2: (Prx+GO)+Pd) two strategies can be followed for synthesizing metal nanoparticles inside the Prx cavity and internalizing them in the 3D material. Figure 9. a) STEM Images of GO-AuNPs conjugated in the presence of Prx (scale from left to right: 2 · m, 50 nm, 60 nm) and their EDS analysis show the presence of gold and sulfur that can be attributed to the presence of AuNPs and the protein, respectively; b) Images of STEM GO-conjugated AuNPs in the absence of Prx (scale from left to right: 90nm and 15nm).

Figure 10. TEM micrographs and EDS analysis of GO-Pd conjugates in the presence of Prx. a) 1 micron scale; b) 200nm scale; c) EDS analysis in which the presence of palladium is evidenced.

Figure 1 1. TEM micrographs and EDS analysis of GO-Pd conjugates in the absence of Prx. a) 1 micron scale; b) 200nm scale; c) EDS analysis in which the absence of palladium is evident.

DETAILED DESCRIPTION OF THE INVENTION A detailed description of the different aspects of the present invention is shown hereinafter.

THREE-DIMENSIONAL HYDROGEL/AEROGEL BASED ON GRAPHENE OXIDE (GO)

Hydrogels or aerogels of the present invention mainly consist, or comprise, a three-dimensional structure based on graphene oxide and protein material. In particular the three-dimensional structures of the invention, as base constituent element, comprise a sandwich-like structure constituted by two almost parallel layers or sheets of GO therebetween a spacing adhesive layer of protein character is interposed.

Several sandwich-like constituent elements can pile up through self-assembling in order to create as a whole complex multilayer structures which, by trapping the aqueous solvent in their pores or among their meshes, create the hydrogel of the invention. The multilayer material can comprise at least two structures of the invention. In particular, when the protein state is constituted, as better detailed hereinafter by peroxiredoxin protein (Prx), the base structures obtained soon after mixing graphene oxide (GO) and Prx have sizes of about 7 nm up to about 1 -2μηι, as it can be estimated from measurements of AFM shown in figure 5, and then constituted by 1 single layer of GO (1 nm) - Prx (5nm) - GO (1 nm) up to about 250 layers of the same.

Following drying after 1 hour as from mixing the reagents, the hydrogel produces an aerogel which concretely will have the shape of porous, spongy composite material, which is relatively elastic and resistant in time.

As illustrated in figure 1 , graphene oxide is a material with planar structure and mainly hydrophilic character, formed by one monoatomic layer of carbon atoms with high level of replacement by hydrophilic groups such as the hydroxyl, epoxide, carbonyl and carboxyl groups. To the purpose of the present invention under "graphene oxide (GO)" both a monolayer and a multilayer is meant, physically under the shape, for example, of flocks that is fragments of graphene oxide in the order of square microns.

Still as illustrated in figure 1 , it is known that the reduced graphene oxide (rGO) keeps the same planar structure of GO, but it is characterized by an increase in the non-saturation of the bonds between the carbon atoms with consequent increase in the hydrophilic character of the material itself, and acquisition of the at least partially hydrophobic character. Moreover the increase in the level of non-saturation of rGO (tending to restore the high level of conjugated non-saturation typical of graphene), gives the material property of high electrical conductivity particularly useful for manufacturing conductive materials and other applications.

As a consequence of the contact with the protein, having reducing activity, the graphene oxide constituting the two layers, is partially brought to the reduced state thereof (rGO). This feature, as already indicated above, modifies the properties of the material by giving it, on one side, an at least partially hydrophobic character, which eases both the interaction with the hydrophobic surfaces of the protein rings and the interaction with itself by means of the "pi stacking" interaction by further stabilizing the resulting structure, on the other side by providing a potential conductive capability.

The intermediate layer of protein nature is formed by a protein selected among the proteins capable of aggregating in oligomeric shapes by generating rings (or toroids) or tubes with nanometric sizes.

Examples of such proteins are:

-the mutein of peroxiredoxin I of Schistosoma mansoni (SmPrxl), functionalized, or not, at N-terminal with peptides capable of binding metals (as described in Angelucci F et al. 2013 and Ardini et al., 2014);

-C48S mutein of peroxiredoxin I of Schistosoma mansoni (SmPrxl -C48S) functionalized, or not, at N-terminal with peptides capable of binding metals (as described in Angelucci F et al. 2013);

- mutein of human peroxiredoxin III functionalized, or not, at N-terminal with peptides capable of binding metals (as described in Phillips et al., 2014);

-C48S mutein of human peroxiredoxin III functionalized, or not, at N-terminal with peptides capable of binding metals (as described in Phillips et al., 2014); -mutein of peroxiredoxin III of cattle (SP22- of wild type) functionalized at N- terminal with peptides capable of binding metals (as described in Gourlay et al., 2003);

- C47S mutein of peroxiredoxin III of cattle (SP22-C47S), functionalized, or not, at N-terminal with peptides capable of binding metals (as described in Gourlay et al., 2003);

- mutein of thermosoma, also called HSP60 or rosettasoma, functionalized, or not, at N-terminal with peptides capable of binding metals (described in McMillan et al., 2005);

-mutein of chaperone GroEL of Escherichia coli (described in Mendoza et al., 1991)

-mutein of Hcp1 protein (described in Ballister et al., 2008);

-mutein of SP1 protein functionalized at N-terminal with peptides capable of binding metals (as described in Medalsy et al., 2008);

-mutein of TRAP protein (described in Miranda et al., 2009).

By way of example, the protein rings of the intermediate layer formed by said proteins have nanometric sizes, in particular an outer diameter in the order of about 10 - 25 nm, for example, of about 13 nm, an inner diameter in the order of 5-7 nm, for example 6 and a thickness in the order of about 5 nm. Obviously the ring sizes are influence even by the features of the specific protein capable of oligomerizing.

As far as the protein nanotubes are concerned, they are obtained from the self- assembling of the single rings in aqueous environment. Each nanotube is constituted by a number of rings of 2 to 40, usually 4 to 20. The nanotube length usually is lower than 250 nm or 100 nm, preferably it is comprised between about 20 nm and about 100 nm, for example, of about 70 nm. The outer and inner diameter of the nanotubes corresponds to the one indicated above for the nanometric rings.

In the specific case, the rings and the tubes with nanometric sizes forming the intermediate protein layer have two opposite base surfaces, with annular shape, above and below the plane of the single ring or the rings forming the nanotube, which can be identical or not. A feature of such surfaces is the presence of hydrophobic amino acids providing, as a whole, an at least partially hydrophobic character to the same surfaces and consequently, "sticky" properties with respect to a partially hydrophobic surface such as the GO surface.

An additional feature is the reducing activity of such surfaces, mainly linked to the presence of amino acids such as cysteine and/or methionine. In an embodiment of the invention the selected protein is the mutein of wild type peroxiredoxin I of Schistosoma mansoni (SmPrxl) functionalized at N-terminal with peptides capable of binding metal ions.

In another embodiment a SmPrxl mutein is used, for example C48S mutein of peroxiredoxin I of Schistosoma mansoni (SmPrxl -C48S) and/or C47S mutein of peroxiredoxin III of cattle (SP22-C47S) both functionalized at N-terminals with similar peptides for metal ions.

As shown in literature, in aqueous environment, the single sub-units of SmPrxl (-25 kDa) assemble to form decamers (-250 kDa), characterized by a structure like a ring or toroid having the already shown sizes (Saccoccia et al., 2012; PDB code: 3ZTL; Figure 2). Said structure has two base surfaces, with annular shape, above and below a plane of the ring. It has been observed that the two annular surfaces are chemically and physically identical (Angelucci et al., 2015). This is due to the fact that the single sub-units firstly associate in dimers characterized by axes of symmetry of 180°, which subsequently assemble by forming the decamer (or pentamer of dimers) so that the binary axis of symmetry thereof is perpendicular to the quinary axis of symmetry of the ring by creating a D5 dihedral symmetry (Figure 3a, Angelucci et al., 2015). In particular, the two opposite annular surfaces above and below a plane of the ring include amino acids active from a redox point of view, such as cysteines and methionines, and they have (partially) "sticky" or hydrophobic surface portions. The presence of these features is directly correlated to the double activity of redox enzyme and of molecular chaperone in vivo explicated by SmPrxl and by its peculiar structure (Angelucci et al., 2013; Figuere 3b and 3c ). The features of the two opposite surfaces are kept even if the single toroidal structures pile up to form nanotubes with above shown sizes. Therefore each nanotube will have equally two base surfaces, with annular shape, opposite with respect to the cross axis and having redox properties and hydrophobic and sticky surface areas.

The Gro-EL proteins from Escherichia coli, SmPrxl (C48S) mutant, the mitochondrial Prx from cattle and human type, C47S mutant of mitochondrial Prx from cattle, C48S mutant of mitochondrial Prx from human type, mutein of Hcp1 protein, mutein of chaperone GroEL of Escherichia coli, mutein of SP1 protein and mutein of TRAP protein are all ring-like structures equally equipped with cysteines/methionines exposed externally that is at the annular surfaces and capable both of forming nanotubular structures and of being sticky.

Experimentally, the inventors have demonstrated that GO placed on a hydrophilic support of silicon dioxide shows as single 2D molecules with height of -0.9- 1 nm, as expected. After the contact with peroxiredoxin (SmPrxl) the GO layers appear to be covered homogeneously with uniform round particles with average height of ~5 nm and width of -25 nm, in sufficient agreement with the sizes of the ring of SmPrxl. Furthermore, it has been noted that most part of the detected rings show a similar height (~5 nm) by suggesting that they likely arrange in similar manner on the underneath GO layers, reasonably placed flat on the carbon surface (Figure 4).

The proteins forming the nanometric structures of the intermediate layer can even be mutagenized by means of genetic engineering techniques, so as to be functionalized with the purpose of inserting molecules, substances, particles of interest inside said rings or nanotubes.

By way of example and not for limitative purpose, the proteins can be functionalized for the complexation of metal nanoparticles (as described in Ardini et al., 2014).

In particular, such functionalization can provide the insertion in the proteic molecule of binding sites for metal ions such as for example silver (I), nickel (II), zinc (II), iron (ll/lll), ruthenium (II), cobalt, (II), palladium (II), platinum, (II) and cadmium (II) by adding to the N-terminal end the protein of residues of histidine, aspartate, cysteine, methionine and glutamate.

Such functionalization allows the complexation with gold nanoparticles, in turn functionalized with bivalent metallic cations or with nanoparticles of silver, palladium, zinc, ruthenium, platinum, copper, nickel, iron, cobalt, cadmium.

The three-dimensional structure can be either in the form of hydrate hydrogel, thus with water molecules present among the layers, or in the form of the dried aerogel.

PREPARATION PROCESS.

The process for preparing hydrogel containing the 3D structures of the invention provides, as main passage, mixing graphene oxide (GO) in aqueous solution with the protein material. Through a self-assembling mechanism, the two reagents produce three-dimensional structures, which, upon coagulating, create a relatively compact colloid which traps the aqueous medium among the meshes thereof, that is forming the hydrogel.

As starting protein material the above-shown proteins can be used in oligomeric states with lower order. In fact, once placed in aqueous solution and under particular experimental conditions, they aggregate to form autonomously rings or tubes of nanometric dimensions, as described in the previous section.

Alternatively, already aggregated proteins in form of nano-rings or preformed nanotubes can be used.

In the latter case, the process could provide a preliminary passage for forming nano-rings and nanotubes. By way of example and not for limitative purpose, this preliminary step can be performed by putting in buffer solution the protein at room temperature, until the formation of the rings or nanometric tubes is observed.

In the contact between the GO layers and the protein material, the reducing capability of the two opposite surfaces of the nano-rings or nanotubes causes the at least partial reduction of GO to reduced graphene oxide (rGO) by increasing consequently the hydrophobic feature and the conductive capability. Then the hydrophobic interaction between the opposite annular surfaces of the protein nanostructures and the hydrophobized surfaces of rGO allows the self-assembling of the different chemical species with formation of the 3D structures of the invention.

The mixing of reagents can take place by using a weight ratio between GO and protein comprised between 1 and 4. By way of example, GO at a concentration of 0.2- 0.3 mg/ml in aqueous solution can be mixed with anyone of the above-mentioned proteins at a concentration of 0.1 mg/ml.

The experimental conditions of the process provide that the mixing between protein and GO is performed in solution having a substantially neutral pH value, at room temperature, preferably comprised between about 18 and 24°C.

Optionally the process of the invention can even provide an additional passage of integrating or internalizing in the three-dimensional structure of materials, molecules, compounds, additional elements of interest, such as for example metal nanoparticles.

In exemplifying way, such internalization can take place according to the three different schemes shown hereinafter and in figure 8.

A first scheme provides a preliminary passage consisting in the complexation of the protein with the metal nanoparticles before the step of mixing with GO (as described in Ardini et al., 2014).

A second scheme provides the addition of metal ions before the step of mixing the protein material and the graphene oxide followed by a reducing passage for the formation of the nanoparticles of metal in the elementary state.

A third scheme, instead, provides a step of adding metal ions to the gel formed by GO and protein material and then downwards the mixing step, the subsequent treatment with reducing agent for the formation of the nanoparticles of metal in the elementary state.

The metal nanoparticles which can incorporate in a 3D structure and useful to the purposes of the present invention, for example, are nanoparticles made of metal in the elementary state, both preformed and in situ generated nanoparticles of gold, palladium, platinum, iron, cobalt, nickel, copper, ruthenium, zinc, cadmium.

In order to promote the bond between the metal ions and the protein, the protein can be suitably modified. By way of example, SmPrxl was used both to convey gold preformed nanoparticles inside the 3D hydrogel (Figure 8a) and as scaffolding for the in-sity synthesis of palladium nanoparticles inside the same hydrogel (Figure 8b). The SmPrxl engineering at the N-terminal end allows to provide the protein molecule with binding sites for metal ions (Figure 3d). In case of SmPrxl, several residues of aspartate and histidine were added for each monomer, which forms the decamer (described in Ardini et al., 2014). These residues can bind bivalent cations by means of ionic bonds or bonds coordinating to high affinity, such as for example nickel (II), zinc (II), iron (ll/lll), ruthenium (II), cobalt, (II), palladium (II), platinum, (II) and cadmium (III). For example, by means of this modification SmPrxl is capable of interacting with preformed AuNP, functionalized with Ni(ll), as shown in a previous study (Ardini et al., 2014). Notwithstanding the bond with gold, SmPrxl keeps sticky properties and it is capable of interacting with GO by forming the colloid which, in this case, is functionalized with AuNP.

Moreover, by way of example, the capability of engineered SmPrxl to bind metal cations further allows the in-situ synthesis of palladium nanoparticles. By mixing SmPrxl and Pd²⁺ ions in buffer solution and by adding GO after 5 min to the mixture the formation of hydrogel takes place again. The treatment of SmPrxl-Pd²⁺-GO precipitate with sodium borohydride allows to obtain GO multilayer structures covered with NP clearly visible by means of Transmission Electron Microscopy (TEM) (Figure 10a and 10b).

The process of the invention can further provide a step of drying/dehydrating the so-obtained hydrogel by obtaining the corresponding aerogel. The drying can be performed with any suitable technique, for example, by means of lyophilisation.

By way of example, the 3D structure obtained from mixing is subsequently recovered by centrifugation and subsequently dried by means of freeze-drying. The so-obtained aerogel results to be spongy, relatively elastic and it does not collapse on itself even after weeks of preservation.

APPLICATION

The use of hydrogels and aerogels based on three-dimensional structures of the invention is widely documented in the state of art. In particular, such structures find application in the field of the biological engineering, electronics, energy, water treatment, chemical catalysis, etc. In particular, different types of materials/devices based on the three-dimensional structure of the invention can be developed, such as, for example, biocompatible scaffolds for regenerative medicine, devices and sensors for the detection of biological molecules and/or gas, transparent materials, touch screens, light emitters, conductive electrodes, solar cells, super capacitors, devices for ultrafiltration for the recovery of heavy metals, catalysts.

An additional application of the proteins of the invention is the use thereof as adhesive through weak interactions. In fact proteins capable of aggregating in structures having two opposite surfaces, with respect to a plane of transversal symmetry, at least partially hydrophobic surfaces, can be generally applied as adhesives/aggregating agents or nanoparticles by allowing the formation of 3D structures. By way of example, proteins usable for this purpose are selected from the group comprising: peroxiredoxin I of Schistosoma mansoni of wild type; SmPrxl-C48S mutein of peroxiredoxin of Schistosoma mansoni; SP22 of peroxiredoxin III of cattle of wild type; human peroxiredoxin III, SP22-C47S mutein of peroxiredoxin III of cattle; C48S mutein of human peroxiredoxin III; mutein of chaperone GroEL of Escherichia coli; mutein of chaperone GroEL of Escherichia coli; mutein of Hcp1 protein; mutein of SP1 protein and mutein of TRAP protein.

Preferred embodiments of the invention

3D GO, defined by the invention, can be implemented according to several modes to obtain the wished effect.

Experiments of conjugation between GO (0.3mg/ml_) and SmPrxl (0.15mg/ml_) show that in the mixture between both substances in aqueous solution buffered by sodium phosphate (20mM) at pH 7.5 and room temperature (22 °C) the GO coagulation in discrete colloidal particles triggers quickly. These particles, in turn, tend to aggregate within ~1 hour to form an apparently porous material which can be re- suspended in solution as relatively compact material. It has been also found that the material breaks down in particles after stirring and re-forms again showing a process of reversible self-assembling (Figure 5a). The process can be followed by means of measurements for "scattering" the light by recording the optical absorbance of the GO- SmPrxl mixture in the Uv-Vis range. According to the material formation and precipitation, the absorbance of soluble GO tends to decrease in time as well as the protein absorbance by suggesting a co-precipitation event (Figure 5b). AFM images of GO aggregates formed after mixing with SmPrxl (see Figure 5a) and deposited by drop-casting show a heterogeneous population of hybrid GO-Prx complexes with various sizes. As expected, the smallest complexes appear to be formed by single layers of GO wholly covered by protein rings by assuming a total height of -11 nm and then suggesting a SmPrxl-GO-SmPrxl "sandwich-like" model (5+1+5 = 11 nm) (Figure 5c). More complex structures show a multilayer architecture with several overlapped layers of GO and, at high enlargements, the presence of Prx is noted among the several layers. Consequently, the height of these structures increases up to several tens of nanometers (Figure 4d). Data of Scanning Transmission Electron Microscope (STEM) confirm the multilayer organization of the GO molecules after interaction with SmPrxl (Fig. 6). GO-SmPrxl hydrogel can be easily lyophilised by means of freeze- drying while keeping a defined 3D architecture. The material deriving therefrom is a spongy dehydrated and relatively elastic aerogel which does not collapse on itself even after weeks of preservation. Analyses of Scanning Electron Microscopy (SEM) performed on material sections show a GO inner network delimiting porous cavities with diameters of 5-15 μηι. At high enlargements, it is noted that the matrix is constituted by thick multilayer sheets of GO according to the structures observed from AFM and STEM analyses (see Figure 5 and Figure 6). Elementary analyses of Energy Dispersive Spectroscopy (EDS) demonstrate the presence of SmPrxl inside the inner network of aerogel by detecting considerable amounts of nitrogen (N) and sulphur (S), two typical elements of the protein. In fact, by lyophilising a GO solution without SmPrxl, which so is not capable to coagulate, a thick 2D film is produced which does not have cavities and the elementary analysis thereof does not highlight the presence of N and S (Figure 7a and 7b).

By means of X-ray Photoelectron Spectroscopy (XPS) it is noted that the compound formation is accompanied by a redox process GO is partially reduced probably with loss of epoxy groups (Figure 7c) whereas SmPrxl is oxidized on thiols of cysteines and on thioethers of methionines, as highlighted by a decrease in the peak which can be ascribed to the C-0 bonds (about 287 eV) in graphene and to the appearance of a signal which can be ascribed to the oxidation of cysteines and methionines with sulfonic acid and sulphone, respectively (about 169.3 and 170.4 eV).

In an additional example 3DGO is functionalized with metal species by exploiting the presence of amino acidic ends at the N-terminals capable of binding the metals (for example palladium) of mutein of SmPrxl (Figure 3d) so as to favour the in- situ formations of nanoparticles with discrete and uniform dimension (about 3 nm) after treatment with reducing agents or to allow the bond with highly efficient preformed nanoparticles (ex. Au-Nps, Nanoprobes inc., NY, USA.). In this same solution, SmPrxl was used both to convey gold preformed nanoparticles inside the 3D hydrogel (Figure 8a) and as scaffolding for the in-situ synthesis of palladium nanoparticles of palladium inside the same hydrogel (Figure 8b). SmPrxl engineering at the N-terminal end allows to provide the protein molecule with binding sites for metal ions. In this case, 4 residues of aspartate and 6 residues of histidine were added for each monomer, equalling to a total of 40 aspartates and 60 histidines for each decamer. These residues can bind bivalent cations by means of ionic bonds or bond coordinating to high affinity. By means of this modification, SmPrxl is capable of interacting with preformed AuNP, functionalized with ions Ni(ll), as shown in a previous study (Ardini et al., 2014). Notwithstanding the bond with gold, SmPrxl keeps sticky properties and it is capable of interacting with GO by forming the colloid which, in this case, is functionalized with AuNP. This can be checked by UV-Vis spectroscopy by recording the optical absorbance of AuNP apart from that of GO and of SmPrxl. The optical spectra show that in presence of SmPrxl the GO colloid absorbs AuNP almost fully, which particles presumably remain trapped between the GO layers. On the contrary, GO alone is not capable of binding significant amounts of AuNP if these are not bound to SmPrxl (not shown data). The analyses of scanning TEM (STEM) verify the spectroscopy data by highlighting the presence of AuNP covering the multilayer structures of GO (Figure 9a). The gold presence is further highlighted by EDS spectroscopy showing a typical signal of the metal (Figure 9a). On the contrary, GO not functionalized with SmPrxl does not show significant amounts of AuNP as also verified by means of EDS (Figure 9b). The capability of engineered SmPrxl to bind metal cations further allows the in-situ synthesis of nanoparticles of palladium. By mixing SmPrxl and Pd²⁺ ions in buffer solution and by adding GO to the mixture after 5 min the formation of hydrogel takes place again. The treatment of the SmPrxl-Pd²⁺-GO precipitate with sodium borohydride allows to obtain GO multilayer structures covered with NP clearly visible by Transmission Electron Microscopy (TEM) (Figure 10a and 10b). The particles appear to be well dispersed approximatively on the whole GO surface and they show average sizes of 3.3 nm (standard deviation= 2 nm) according to the diameter of the inner cavity of SmPrxl (Figure 3). EDS measurements confirm the presence of palladium (Figure 10c). On the contrary, the same treatment carried out on the sample without protein does not highlight the presence of defined PdNP as confirmed by EDS (Figure 11). The internalization of metal nanoparticles is obtained even by changing the order for adding the reagents (Figure 8b), that is at first forming the 3D hydrogel in presence of Prx and then adding Pd(ll) in solution (not shown results). This synthesis method demonstrates the capability of hydrogel formed by means of the "sticky" SmPrxl to absorb palladium ions free in solution, and then to act as adsorbing material to be used for the environment rehabilitation.

EXPERIMENTAL AND ANALYTICAL SECTION

The preparation of wild type peroxiredoxin I of Schistosoma mansoni, of its C48S mutein and of SP22 C47S mutein of peroxiredoxin III of cattle, all functionalized at N- terminal with the metal binding peptides are described in Saved & Williams 2004, in Angelucci et al. 2013, and in Gourlay et al. 2003, respectively. By way of example herebelow the preparation of C48S mutein of SmPrxl is shown.

Mutagenesis. Competent cells of E.coli belonging to the DH5a strain are transformed with the plasmid carrier containing the wt-SmPrxl gene. The plasmid is then purified by transformed cells made to grow over night in liquid medium LB (Luria Bertani) with addition of antibiotic ampicillin (50 μg/ml).

In order to perform the C48S mutagenesis a pair of primers known in literature is used (Angelucci F et al.2013): A Quick change mutagenesis was then performed by means of PCR, by using Pfu Ultra High Fidelity DNA Polimerase (Agilent Technologies). At the end, the samples were treated directly with Dpnl (Thermo Scientific) enzyme, specific for methylated DNA, to degrade the copy plasmid, as originally methylated.

Such samples subjected to treatment with Dpnl are used directly to transform competent cells of E.coli (DH5a). From these cells the SmPrxl(C48S) mutant plasmid was purified and used for subsequent transformation of strains of E.coli BL21 (DE3)pLysS Singles for the protein expression.

Bacterial transformation. Competent bacterial cells BL21 (DE3)pLysS Singles (Novagen) are transformed by heat-shock under sterility conditions with the recombinant plasmids. In the specific case, 1 of plasmid is mixed with 50 of cell stock which has been thawed in ice in advance. The suspension is incubated 5 minutes in ice, 45 seconds at 42 °C and again 5 minutes in ice before adding 500 of liquid medium LB 25 g L-1 (Sigma-Aldrich) and incubation at 37°C for 1 hour under stirring at 130 rpm. At the end of incubation, 100 of suspension are plated by means of suitable "L"-like (Becton Dickinson) loop on selective solid medium LB and Agar 15 g L-1 additioned with antibiotics ampicillin 0.1 g L-1 and chloramphenicol 0.034 g L-1 (Sigma-Aldrich) and included in specific Petri (Becton Dickinson) capsule. The capsule is subsequently incubated at 37°C for 16 hours until obtaining single bacterial colonies. Bacterial culture and protein expression. By means of suitable point-like loop (Becton Dickinson) one single colony is taken from the plate to transfer it into 10 mL of liquid medium additioned with the antibiotics before incubating at 37°C for 16 hours under stirring at 130 rpm. The suspension is subsequently mixed in 1 L of liquid medium additioned with antibiotics and incubated at 37°C under stirring at 130 rpm until reaching a value of optical absorbance at 600 nm equal to 0.4. Under these conditions the cells are induced to express the protein by means of adding IPTG 1 mM (Sigma- Aldrich) and additional incubation for 3 hours. The so-obtained bacterial culture is centrifuged for 1 hour at 3500 rpm and 10°C and the whole cell pellet deriving therefrom is re-suspended in 30 mL of lysis buffer TRIS/HCI 30 mM (Euroclone), NaCI 0.5 M (Sigma-Aldrich), EDTA 2 mM (Euroclone), β-mercaptoethanol 2 mM (Sigma- Aldrich), protease inhibitors 1X (Calbiochem) pH 8.0. The suspension is exhaustively stirred to break down possible cell aggregates and subsequently put in ice before being sonicated for extracting the bacterial cytosolic content. The sonication is performed for 45 min with 3 s-pulses with width equal to 30% at intervals of 9 s by using a small probe. The solution deriving therefrom is subsequently centrifuged at 12000 rpm for 1 hour at 10°C and the supernatant is filtered manually by using membranes of cellulose acetate with porosity of 0.45 μηι (mdi).

Protein purification. The purification is obtained by means of I MAC affinity chromatography by using a column 5-mL Sepharose HisTrap connected to a chromatograph FPLC AKTAprime plus (GE Healthcare). The column is equilibrated in advance in water and NiS04 50 mM (Fisher Scientific) and subsequently in buffer TRIS/HCI 30 mM, NaCI 0.5 M, imidazole 20 mM (Sigma-Aldrich) pH 8.0. The filtered supernatant is loaded manually in column and eluted by chromatograph by applying with 2 mL min-1 flow increasing amounts of imidazole in the following order: I) 4 column volumes of imidazole 20 mM; II) 4 column volumes of imidazole 50 mM; III) gradient of 8 column volumes up to imidazole 0.5 M; IV) 4 column volumes of imidazole 0.5 M. The elution of proteins is followed by measuring in time the variation in absorbance at 280 nm typical of aromatic residues. All proteins elute in the interval 350-500 mM of imidazole. The purity of collected fractions is assayed by means of not native electrophoresis SDS-PAGE by using 3% polyacrilamide loading gel (Sigma- Aldrich) in buffer TRIS/HCI 0,5 M pH 6.8 and 12% running gel in TRIS/HCI 1 M pH 8.3. The electrophoresis assays are all performed in running buffer TRIS/HCI 0.3 M pH 8.3. The pure fractions are subsequently incubated at 4°C for 16 hours with β- mercaptoethanol 2 mM so that the proteins keep in a reduced chemical state. In case of wt-SmPrxl, an additional incubation at 4°C for 16 hours with EDTA 2 mM is requested to remove possible nickel ions linked to the histidine ends, which nickel ions cause protein disordered aggregation (Ardini et al., 2014).

Preparation of samples. The proteins are dialyzed against buffer 20mM NaH₂P04/Na₂l-IP04, pH 7.4 by using filtering devices with molecular cut of 30kDa (Millipore). The dyalisis was repeated until reducing the amounts of TRIS, imidazole, EDTA and β-mercaptoethanol at nM concentrations. The end protein preparations are filtered sterilely and kept at 4°C until a maximum of 4 months. The concentration of proteins is determined by means of UV-Vis spectrophotometry based upon a coefficient of molar extinction at 280 nm equal to 1 mg-1 ml cm-1 and a molecular weight of 25 kDa in case of wt-SmPrxl and SmPrxl(C48S) (Saccoccia et al. Structure. 2012 Mar 7;20(3):429-39); an extinction coefficient equal to 0.73 mg-1 ml cm-1 and a molecular weight of 22 kDa are instead considered for SP22(C47S) (Gourlay et al. J Biol Chem. 2003 Aug 29;278(35):32631-7.).

Assembling of GO-based hvdrogels. The colloidal hydrogels were obtained with a mixing technique as described hereinafter. In order to obtain the composite material GO-SmPrxl, 0.3 mg/mlbGO (Graphene Supermarket) was dissolved in 20 mM sodium phosphate buffer (Panreac AppliChem) pH 7.5 inside a quartz cuvette with 0.5 cm of optical path. The solution was vigorously mixed with a vortex apparatus (Velp Scientifica) in order to melt GO uniformly before adding 0.6 μΜ SmPrxl. After adding protein, the resulting mixture was mixed again before incubation at room temperature (23°C) by using an orbital stirrer (Ika) rotating at 320 rpm. Parallelly, a GO sample without control proteins was prepared and treated.

Functionalization of 3DGO with preformed gold nanoparticles (AuNP). In this case, 0.6 μΜ di AuNPs (diameter of 1.8 nm) functionalized with Ni²⁺-nta (Nanoprobes inc., NY, USA.) were pre-mixed for 10 minutes with 0.6 μΜ SmPrxl (1AuNP per protein ring) in phosphate buffer including imidazole 20 mM (Sigma). This strategy was exploited successfully to obtain SmPrxl-AUNP adducts by taking advantage of the strong interaction between the histidine residues placed in the ring cavity and the Ni²⁺ ions existing on the nanoparticle surface. The presence of imidazole was demonstrated being fundamental to modulate such interaction, thus by avoiding the formation of amorphous insoluble aggregates (Ardini et al., 2014). The SmPrxl-AUNP mixture was stirred with a vortex and centrifuged for 5 min at 12000 rpm, before mixing the resulting supernatant with 0.3 mg ml^"1 of GO, the reaction was then left to stir for 10 min. As control sample, a solution of GO-AUNP without proteins was prepared. After 0, 10, 30 and 60 min of incubation the absorption spectra of the relative samples were collected in the range 230-600 nm.

Experiments of absorption in Pd²⁺ batches measured with thioglvcolic acid (TGA). By considering that the inner surface of the SmPrxl ring includes repeated sequences of polyHis, the capability of absorbing Pd²⁺ ions was evaluated. This was carried out by using TGA organic acid capable of binding to Pd²⁺ by forming a coloured TGA-Pd²⁺ complex (molar ratio 2: 1) the amount thereof can be estimated by spectroscopy (Mathew e Innocenzo, 2010). A buffered solution of 1 mM (NH₄)₂PdCI₄ (Sigma-Aldrich) was mixed with 0.6 μΜ SmPrxl (> 1600Pd²⁺ per protein ring) and the so-obtained mixture was left to react 5 min under magnetic stirring at room temperature before being ultra-centrifuged 10 min at 10000 rpm. Then, the sample supernatant was diluted 4 times in borate buffer pH 9.5 (Fluka) before mixing with TGA 3 mM (Sigma-Aldrich). The solution was analyzed immediately by recording the optical absorbance in the range 230-600 nm in a quartz cuvette with optical path having a length of 1 cm to detect the presence of not associated Pd²⁺. Afterwards, the retention capability of Pd²⁺ ions by GO-SmPrxl hydrogel was also checked. To this purpose, Pd²⁺ was added to hydrogel and the mixture was left under stirring for about 1 h at room temperature. The precipitate was extensively washed by suspending it uniformly in fresh phosphate buffer and, after ultra-centrifugation, the resulting supernatant each time was assayed with TGA. At the same time a control sample without proteins was prepared by mixing 1 mM (NH₄)₂PdCI₄ and 0.3 mg/ml GO and analogously treated to evaluate with TGA the binding and retention capability of Pd²⁺ ions by GO.

In situ synthesis of palladium nanoparticles (PdNPs). Based upon the capability of SmPrxl ring to interact with bivalent metals, the growth in the cavity of PdNP protein was carried out after chemical reduction of the GO-SmPrxl-Pd²⁺ resulting material with NaBH₄. In this case, 1 mM (NH₄) ₂PdCI₄ in phosphate buffer including imidazole 40mM was mixed with 0.6 μΜ SmPrxl (> 1600Pd²⁺ per SmPrxl ring) by stirring for 5 minutes at room temperature before adding 0.3 mg ml^"1 of GO. The so-obtained GO-SmPrxl- Pd²⁺ mixture was ultra-centrifuged for 10 min at 10000 rpm. The resulting precipitate was re-suspended in phosphate buffer without imidazole and the supernatant was essayed with the spectroscopic method based upon TGA to determine the Pd²⁺ content (see above). The precipitate was widely washed with buffer containing imidazole 40 mM to wash out Pd²⁺ not bound specifically to the protein and/or to GO and, after centrifugation, again the supernatant was analyzed by means of assay based upon TGA. The re-suspended precipitate was left to incubate for 1 hour at room temperature under stirring before reducing it by adding 50 mM of NaBH₄ (Sigma-Aldrich) for 1 h. Even the material deriving from incubation of GO-SmPrxl preformed hydrogel with Pd²⁺ (see above) was reduced under the same conditions. The resulting materials were washed twice with phosphate buffer to remove the excess in not reacted NaBH₄.

Atomic force microscopy (AFM). The surface properties of the GO-based composite materials were scanned by means of AFM. Depending upon the sought features, the samples were prepared in different way as follows. In a first series of analyses, very thin samples were prepared by making 30 μΙ_ of 0.2 mg/ml GO in 20 mM phosphate buffer of sodium pH 7.5 to drop on a 1-cm² Si0₂ based hydrophilic substrate pretreated with piranha solution, followed by 1 min of spin-coating at 2200 revolutions per minute. Then, 3 μΙ_ of a 0.08 μΜ solution of SmPrxl in the same buffer were deposited by drop- casting and quickly dried up with a light flow of nitrogen. The sample was exhaustively rinsed with distilled water before drying again. In a second series of analyses, thicker samples were produced to study the morphological features thereof. In this case, 0.02 mg/ml GO and 0.04 μΜ SmPrxl were mixed, stirred by means of vortex and 3 microlitres of the mixture immediately were made to drop on the silica medium, rinsed and dried up in the previous way. The images were taken by using a microscope with scanning probe D5000 Digital (Veeco) placed in dry environment and provided with a e NCHV silicon point covered with antimonium with about 9 nm radius of curvature, with resonance frequency between 344 and 371 kHz and constant spring between 20 and 80 N/m (Bruker). The scanning tip was shifted in air in "tapping" mode on an area of 5 μηι² of sample with scanning speed of 1 Hz, with a set point width of 1.1-1.3 (proportional and integral gains were adequately syntonized for each sample). All captured images, at last, were displayed and processed by using Gwyddion v2.37 modular software. Scanning Electron Microscopy (SEM) and Energy Dispersive Spectrometry (EDS). The 3D morphological features of the GO-based composite materials were observed by means of SEM analysis by using a LEO 1530 electron microscope (Zeiss-Gemini). Before observations, all samples were subjected to a treatment of 3 h of lyophilisation by freeze-drying by using a VirTis Bench Top 2k apparatus (SP Industries, Inc). This procedure was performed to guarantee the complete removal of water in the samples without altering significantly the architecture thereof. The so-obtained lyophilizated materials were cut then fixed with glue on Si0₂-based (1 cm²) substrate before doing the analysis. All images were acquired under-vacuum at 10^"6 Torr by using an accelerating voltage of 5-10 kV. A SEM XL-30-CP microscope provided with a DX4 EDAX thin window system for EDS microanalysis was used to detect the presence of the protein components. X-ray Photoelectron Spectroscopy (XPS). XPS analysis was performed on GO to verify the chemical-physical state thereof after SmPrxl bond. Under-vacuum spectra were obtained (10^"9 Torr) with a PHI 1257 spectrometer provided with a monocromatic source Al Ka (hv = 1486.6 eV) by using a let-through energy of 11.75 eV (93.9 eV Survey), corresponding to an overall experimental resolution of 0.25 eV. The acquired XPS spectra were interpolated by mean of Voigt contour and Shirley backgrounds.

Scanning/transmission (STEM) and transmission (TEM) electron microscopy, energy dispersive spectrometry (EDS) of 3DGO functionalized with AuNP and PdNP. The samples were observed by means of FEI Tecnai F20 ST microscope, with electronic beam at the accelerating voltage of 120 keV, by recording the images of a Gatan MSC794 camera. In the STEM operating mode, the images were acquired with a high-angle dark field annular detector FISCHIONE, whereas the compositional analysis was performed by using an energy loss spectrometer for X-rays INCA EDS. In case of 3DGO functionalized with PdNP the samples were observed even by means of a CM-100 (Philips) transmission electron microscope equipped with tungsten filament operating at 80 kV.

The 3DGO samples functionalized with AuNP were prepared as follows: 400 μΙ GO 0.3 mg/ml was washed in isopropanol and water, subsequently Prx 0.15 mg/ml and AuNPs 0.6 μΜ in phosphate buffer were added. One waited for the formation of hydrogel, which after centrifugation, was washed 3 times with distilled water and deposited on carbon grid (Agar Scientific) for the display at microscope and EDS analysis. The same identical sample was prepared even in absence of protein and analyzed. Both samples were vacuum-dried before subjecting to analysis.

In case of 3DGO functionalized with PdNP, after reduction with NaBH4 and subsequent washing (see above), an aliquot of hydrogel of 20uL was deposited on a carbon grid (Agar Scientific). After washing with distilled water the grid was put under vacuum and dried. At last, the samples were observed by means of TEM and STEM and analyzed by EDS. In the same way, a control sample without proteins constituted by 0.3 mg ml-1 GO and 1 mM (NH₄)₂PdCI₄ was equally treated and analyzed. BIBLIOGRAPHY

- Adhikari, B., Biswas, A. & Banerjee, A. Graphene oxide-based hydrogels to make metal nanoparticle-containing reduced graphene oxide-based functional hybrid hydrogels. ACS Appl. Mater. Interfaces 4, 5472-5482 (2012).

- Angelucci F, Saccoccia F, Ardini M, Boumis G, Brunori M, Di Leandro L, Ippoliti R, Miele AE, Natoli G, Scotti S, Bellelli A. Switching between the alternative structures and functions of a 2-Cys peroxiredoxin, by site-directed mutagenesis. J Mol Biol. 425, 4556-68 (2013).

- Angelucci F, Bellelli A, Ardini M, Ippoliti R, Saccoccia F, Morea V. One ring (or two) to hold them all - on the structure and function of protein nanotubes. FEBS J. doi: 10.1 11 1/febs.13336, (2015). - Ardini et al., Metal-induced self-assembly of peroxiredoxin as a tool for sorting ultrasmall gold nanoparticles into one-dimensional clusters. Nanoscale (2014).

- Balandin, A. A. et al. Superior thermal conductivity of single-layer graphene. Nano Lett. 8, 902-907 (2008).

- Bianco, A. Graphene: safe or toxic? The two faces of the medal. Angew. Chem., Int. Ed. 52, 4986-4997 (2013).

- Bolotin, K.I. et al. Ultrahigh electron mobility in suspended graphene. Solid State Commun. 146, 351-355 (2008).

- Chabot, V., Higgins, D., Yu, A., Xiao, X., Chen, Z. & Zhang, J. A review of graphene and graphene oxide sponge: material synthesis and applications to energy and the environment. Energy Environ. Sci. 7, 1564-1596 (2014).

- Ballister ER, Lai AH, Zuckermann RN, Cheng Y, Mougous JD. In vitro self-assembly of tailorable nanotubes from a simple protein building block. Proc Natl Acad Sci U S A. 105, 3733-8 (2008). - Eda, G. & Chhowalla, M. Chemically derived graphene oxide: towards large-area thin- film electronics and optoelectronics. Adv. Mater. 22, 2392-2415 (2010). - Huang, X. et al. Graphene-based materials: synthesis, characterization, properties, and applications. Small 7, 1876-1902 (201 1). - Gourlay LJ, Bhella D, Kelly SM, Price NC, Lindsay JG. Structure-function analysis of recombinant substrate protein 22 kDa (SP-22). A mitochondrial 2CYS peroxiredoxin organized as a decameric toroid. J Biol Chem. 278, 32631-7 (2003).

- Lee, C, Wei, X., Kysar, J.W. & Hone, J. Measurement of the elastic properties and intrinsic strength of monolayer graphene. Science 321 , 385-388 (2008)

- Li, W. et al. High-density three-dimension graphene macroscopic objects for high- capacity removal of heavy metal ions. Scie. Rep. 3, 2125 (2013). - Mathew, B. & Innocent, D. Spectrophotometric determination of palladium(ll) using thioglycolic acid. Asian J. Chem. 22, 7551-7556 (2010).

- McMillan, R.A., Howard, J., Zaluzec, N.J., Kagawa, H.K., Mogul, R., Li, Y.F., Paavola, CD., Trent, J.D. A self-assembling protein template for constrained synthesis and patterning of nanoparticle arrays. J Am Chem Soc.127, 2800-1 (2005).

- Medalsy I, Dgany O, Sowwan M, Cohen H, Yukashevska A, Wolf SG, Wolf A, Koster A, Almog O, Marton I, Pouny Y, Altman A, Shoseyov O, Porath D. SP1 protein-based nanostructures and arrays. Nano Lett. 8, 473-7 (2008).

- Mendoza JA, Rogers E, Lorimer GH, Horowitz PM. Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese. J Biol Chem. 266, 13044-9 (1991).

- Miranda FF, Iwasaki K, Akashi S, Sumitomo K, Kobayashi M, Yamashita I, Tame JR, Heddle JG. A self-assembled protein nanotube with high aspect ratio. Small. 5, 2077-

84 (2009).

- Novoselov, K.S. et al. Electric field effect in atomically thin carbon films. Science 306, 666-669 (2004).

- Phillips AJ, Littlejohn J, Yewdall NA, Zhu T, Valery C, Pearce FG, Mitra AK, Radjainia M, Gerrard JA. Peroxiredoxin is a versatile self-assembling tecton for protein nanotechnology. Biomacromolecules. 15, 1871-81 (2014).

- Qin, S., Liu, X., Zhuo, R. & Zhang, X. Microstructure-controllable graphene oxide hydrogel film based on a pH-responsive graphene oxide hydrogel. Macromol. Chem.

Phys. 213, 2044-2051 (2012).

- Ratinac, K.R., Yang, W., Ringer, S.P. & Braet, F. Toward ubiquitous environmental gas sensors capitalizing on the promise of graphene. Environ. Sci. Technol. 44, 1167- 1 176 (2010).

- Saccoccia F, Di Micco P, Boumis G, Brunori M, Koutris I, Miele AE, Morea V, Sriratana P, Williams DL, Bellelli A, Angelucci F. Moonlighting by different stressors: crystal structure of the chaperone species of a 2-Cys peroxiredoxin. Structure. 20, 429- 39 (2012).

- Sayed AA, Williams DL. Biochemical characterization of 2-cys peroxiredoxins from Schistosoma mansoni. J Biol Chem. 279, 26159-66 (2004). - Xu, Y., Lin, Z., Huang, X., Wang, Y., Huang, Y. & Duan, X. Functionalized graphene hydrogel-based high-performance supercapacitors. Adv. Mater. 25, 5779-5784 (2013).

- Zhu, Y. et al. Graphene and graphene oxide: synthesis, properties, and applications. Adv Mater. 22, 3906-3924 (2010).

Claims

1. A three-dimensional structure made of graphene comprising:

- at least two layers substantially constituted by graphene oxide in a reduced or partially reduced form spaced by

-one intermediate layer comprising protein rings or protein tubes of nanometric size, wherein each ring or nanotube has two base surfaces of annular form, opposite one another, each surface being in contact with one of said two layers; and

optionally wherein said rings or tubes comprise metal nanoparticles.

2. The structure according to claim 1 , wherein said two opposite surfaces above and below the plane of the ring are at least partially hydrophobic and equipped with amino acids active from a redox point of view.

3. The structure according to anyone of claims 1 to 2, wherein said rings or tubes of nanometric sizes are formed from proteins selected from the group comprising: the mutein of wildtype peroxiredoxin I of Schistosoma mansoni; SmPrxl- C48S mutein of peroxiredoxin of Schistosoma mansoni; SP22 mutein of wild type peroxiredoxin III bovine; SP22-C47S mutein of peroxiredoxin III of cattle; mutein of human wild type peroxiredoxin III; C48S mutein of human peroxiredoxin III; mutein of chaperone GroEL of Escherichia coli; mutein of chaperone GroEL of Escherichia coli; mutein of Hcp1 protein; mutein of SP1 protein and mutein of TRAP protein.

4. The structure according to claim 3, wherein said proteins are optionally functionalized at the N-terminal with aminoacid residues capable of binding bivalent ions.

5. The three-dimensional structure according to anyone of claims 1 to 4, wherein said metal nanoparticles are selected from the group comprising nanoparticles of gold, palladium, nickel, ruthenium, copper, zinc, platinum, iron, cobalt, cadium; and wherein said nanoparticles are optionally functionalized with metal ions.

6 The three-dimensional structure according to claim 4 or 5, wherein said ions are selected from the group comprising nickel (II), zinc (II), iron (ll/lll), ruthenium (II), cobalt, (II), palladium (II), platinum, (II) and cadmium (III).

7. The three-dimensional structure according to anyone of claims 1 to 6, comprising more than two layers of graphene oxide alternated by a protein layer.

8. A gel essentially consisting or comprising three-dimensional structures according to any one of claims 1 to 7.

9. The gel according to claim 8 which is a hydrogel or an aerogel.

10. A process for manufacturing a gel essentially consisting or comprising three-dimensional structures according to anyone of claims 1 to 7 comprising a step of:

-mixing in aqueous solution graphene oxide with proteins capable of self- assembling in aqueous solution, or already assembled, in rings or tubes of nanometric dimensions, wherein each ring or nanotube has two base surfaces of annular form, opposite one another, said surfaces being at least partially hydrophobic and with reductive capacity, up the observation of the formation of said gel.

1 1. The process according to claim 10, wherein said proteins that forms said rings or tubes of nanometric dimensions are selected from the group comprising: the mutein of wildtype peroxiredoxin I of Schistosoma mansoni; SmPrxl-C48S mutein of peroxiredoxin of Schistosoma mansoni; SP22 mutein of wild type peroxiredoxin III bovine; SP22-C47S mutein of peroxiredoxin III of cattle; mutein of human wild type peroxiredoxin III; C48S mutein of human peroxiredoxin III; mutein of chaperone GroEL of Escherichia coli; mutein of chaperone GroEL of Escherichia coli; mutein of Hcp1 protein; mutein of SP1 protein and mutein of TRAP protein, said proteins being optionally functionalized at N-terminal with amino acid residues capable of binding metal ions.

12. The process according to claim 10 or 1 1 , wherein said mixing step is carried out at a substantially neutral PH and room temperature, preferably for about 60 minutes.

13. The process according to anyone of claims 10 to 12, comprising a step of internalisation of metal nanoparticles in said gel.

14. The process according to claim 13, wherein said internalisation occurs by means of:

- prior to the mixing step, a step of complexation of said proteins with metal nanoparticles optionally complexed to metal ions; and/or

- prior to the mixing step, a step of complexation of said proteins with metal ions, followed by a reduction step; and/or - after the mixing step, a step of adding metal ions to the newly-formed gel, followed by a reduction step.

15. The process according to claim 14, wherein said metal ions are selected from the group comprising nickel (II), zinc (II), iron (ll/lll), ruthenium (II), cobalt, (II), palladium (II), platinum, (II) and cadmium (III).

16. The process according to anyone of claims 13 to 15, wherein said metal nanoparticles are selected from the group comprising nanoparticles of gold, palladium, nickel, ruthenium, copper, zinc, platinum, iron, cobalt, cadmium; and wherein said nanoparticles are optionally functionalized with metal ions.

17. The process according to anyone of claims 10 to 16, further comprising a step of separation of said gel from the aqueous solution, preferably by centrifugation.

18. The process according to claim 17, further comprising a step of drying said separated gel thus obtaining an aerogel.

19. A gel obtainable by means of the process according to anyone of claims 10 to 17 in the form of hydrate hydrogel or according to claim 18 in the form of the dried aerogel.

20. A material or device comprising the gel according to claim 8 or 9, or according to claim 19.

21. Use as adhesive in the production of three-dimensional structures made of substantially hydrophobic materials, of proteins capable of self-assembling in rings or tubes of nanometric dimensions having two surfaces of annular form, opposite one another, above and below a plane of the ring, which are equipped with amino acids active from a redox point of view and at least partially hydrophobic.

22. The use according to claim 21 , wherein said rings or tubes are formed from proteins selected from the group comprising: the mutein of wildtype peroxiredoxin I of Schistosoma mansoni; SmPrxl-C48S mutein of peroxiredoxin of Schistosoma mansoni; SP22 mutein of wild type peroxiredoxin III bovine; SP22-C47S mutein of peroxiredoxin III of cattle; mutein of human wild type peroxiredoxin III; C48S mutein of human peroxiredoxin III; mutein of chaperone GroEL of Escherichia coli; mutein of chaperone GroEL of Escherichia coli; mutein of Hcp1 protein; mutein of SP1 protein and mutein of TRAP protein.

23. The use of the structure according to anyone of claims 1-7 or the gel according to claim 8 or 19 as biocompatible scaffolds for regenerative medicine, devices and sensors for the detection of biological molecules and/or gas, transparent materials, touch screens, light emitters, fuel cells, conductive electrodes, solar cells, super capacitors, devices for ultrafiltration for the recovery of heavy metals, catalysts.