WO2016203388A1

WO2016203388A1 - Process for the production of functionalized graphene

Info

Publication number: WO2016203388A1
Application number: PCT/IB2016/053527
Authority: WO
Inventors: Federica VALENTINI
Original assignee: Graphene Nanotechnologies Hub S.R.L.
Priority date: 2015-06-15
Filing date: 2016-06-15
Publication date: 2016-12-22

Abstract

Process for the production of nanometric graphene sheets functionalized with at least one biomolecule, wherein an electrochemical cell (1) is provided, comprising two electrodes (2, 3) of graphite immersed in an electrolysis bath (6), wherein the electrolysis bath (6) comprises an electrolytic solution and said biomolecule, the process comprising a step in which a predetermined electric potential (E) is applied to the electrodes (2, 3), whereby the biomolecule covalently binds to a first one (2) of said two electrodes (2, 3) which acts as an anode, and said first electrode (2) exfoliates, thereby producing graphene sheets (7) functionalized with said at least one biomolecule.

Description

PROCESS FOR THE PRODUCTION OF FUNCTIONALIZED GRAPHENE

Field of the invention

The present invention relates to functionalized nano-graphene, in particular to graphene in the form of sheets, to a production process and to uses thereof.

Background art

Ancient manuscript papers are subject to acid and oxidizing attack, in particular due to the iron-gall inks. Over time, deep lacerations of the paper supports occur, with apparent gaps and detachment of material. In addition to such mechanical damages, there are also others of optical nature, since the paper supports are subject to undesired yellowing. Traditional restoration treatments aimed to combat the phenomena related to the acidity and oxidation of the paper have considerable drawbacks. In fact, the prior art involves the use of calcium propionate as a de- acidifying agent, which acts on the acid groups, and borane tert-butylamine T-BAB as reducing agent, which acts on the oxidized functions. Calcium propionate exhibits risks, especially associated with a prolonged use by an operator, who may be subject to cough, skin and eye irritation or gastrointestinal disorders, in case of swallowing. A riskiest situation is related to the use of T-BAB, which is harmful by inhalation, ingestion and skin contact as well as irritating to eyes, respiratory system and skin. Disadvantageously, the traditional treatments involving the use of such substances are aggressive towards paper and it is not possible to modulate and monitor the action of the two substances on the paper. Moreover, from an optical point of view, excessive alterations of the colorimetric components may occur, producing an undesired whitening of the restored papers, not always respectful of the historical value of the specimen. The traditional treatment solution involves the use of water or ethyl alcohol or even isopropyl alcohol to disperse the calcium propionate or T-BAB. The application of the solutions thus produced can take place either by immersion or by spraying. The immersion in an aqueous basic solution is more effective to neutralize the acids present in the paper. However, it is often impossible to intervene by subjecting the paper support to immersion in aqueous solutions. In fact, although washing in aqueous solution facilitates the action of the de-acidifying and reducing treatment, it also results in a strong physical stress of the paper, which is subject to a change of its size and to a reduction of its resistance as a result of the fiber spreading (swelling), with consequent damage of the paper support itself.

Spraying, instead, is not sufficiently effective to achieve the restoration of the paper support.

The need for products for the restoration of paper supports and a related production process which allows overcoming the aforesaid drawbacks is therefore felt.

Document A Ezhil Vilian et al., Analytical Methods, vol. 7, no. 13, pages 5627- 5634, DOI: 10.1039/C5AY01005E, describes the production of a drop casting modified sensor, in particular it describes the functionalization of an electrode surface. To this end, graphene oxide in liquid phase is first generated by a purely chemical method, in which graphite powder is attacked chemically. Thereafter, a part of the liquid is withdrawn and fed into a different container, in particular into a three-electrode electrolysis cell. By a Cyclic Voltammetry technique, the surface of a Glassy Carbon working electrode is modified and covered with a layer of graphene oxide. Thereafter, the modified electrode undergoes electrochemical reduction of the layer of graphene oxide, which becomes therefore reduced graphene. The subsequent functionalization with L-GSH (reduced glutathione) always takes place via a Cyclic Voltammetry technique in aqueous solution containing L-GSH. In particular, the working electrode surface is functionalized with L-GSH, since L-GSH binds to the reduced graphene layer.

Also document Qiong Zeng et al., Advanced Functional Materials, Wiley, vol. 20, no. 19, pages 3366-3372, DOI: 10.1002/ADFM.201000540 describes the production of an electrochemical sensor. Also the procedure of this document describes the production of graphene oxide in liquid phase by means of a purely chemical method, in which graphite powder is attacked chemically. Disadvantageously, the use of a chemical template is provided, in particular sodium dodecyl benzene sulfonate (SDBS).

None of the processes described in the above documents enables large-scale productions. In particular, the known processes do not allow the large scale production of graphene functionalized with a biomolecule. Note that Hummer's method, as well as variants thereof, cannot lead to the production of large amounts of graphene which is functionalized with biomolecules.

Document Haiqun Chen et al., Advanced Materials, vol. 20, no.18, pages 3557- 3561 , DOI: 10.1002/ADMA.200800757 describes a graphene bucky paper produced by vacuum filtration, starting from a dispersion of graphene oxide produced with a purely chemical method. Using vacuum filtration, the graphene oxide deposited on the membrane, which is made of alumina, is disadvantageously subject to peel off.

It would be advantageous to have one or more products for efficient restoration of paper supports which is also harmless to the operator who uses them.

Summary of the invention

It is an object of the present invention to provide a better restoration of paper supports.

It is another object of the present invention to provide a product for a more effective restoration of paper supports and which preferably is at least less toxic and less harmful to the operators' health compared to the prior art. In addition, this new product is advantageous from the point of view

Therefore, the present invention achieves at least one of the objects discussed above by providing an electrochemical process for the production of at least one nanometric graphene sheet functionalized with at least one biomolecule, wherein there is provided an electrochemical cell, comprising two electrodes, of which at least one electrode, which is anode, is made of graphite, said two electrodes being at least partially immersed in an electrolysis bath and connected to a power supply unit, wherein each electrode is cylindrical in shape with a diameter φ/height L ratio = 1 /100±10%, wherein the electrolysis bath comprises an electrolytic solution and said at least one biomolecule,

the process comprising a step in which a predetermined electric potential is applied to the electrodes by means of the power supply unit, whereby the at least one biomolecule mainly covalently binds with said electrode made of graphite, which acts as an anode, which exfoliates producing at least one graphene sheet functionalized with said at least one biomolecule. According to one aspect, the invention provides a graphene sheet functionalized with at least one biomolecule obtainable by the above process.

According to a further aspect, the invention provides the use of a graphene sheet functionalized with at least one biomolecule as a de-acidifying and/or reducing agent for the restoration of paper supports, in particular deteriorated manuscripts containing iron-gall inks.

According to yet another aspect, the invention provides a bucky-paper comprising a porous support and at least one graphene sheet functionalized with at least one biomolecule.

Advantageously, the production process of graphene, in particular of graphene sheets of nanometer size in terms of thickness, width and area of the sheet, functionalized with biomolecules, is of the electrochemical type. In particular, the process involves electrolysis with anodic etching.

This allows having a good control on the end product, in particular in terms of shape, size and functionalization degree, and it also ensures the production thereof on a large scale.

Note that the process of the invention, being of electrochemical type, involves the exfoliation of a graphite electrode for producing graphene sheets functionalized with one or more biomolecules. Advantageously, the functionalization takes place in situ, i.e. it takes place substantially simultaneously to the exfoliation. It is therefore a process very different from the purely chemical processes of the prior art. Moreover, according to the process of the invention, by suitably varying the process time it is possible to obtain graphene in aggregation states and in different forms. In particular, graphene can be produced as nano-dispersions (i.e. dispersions containing nanometric particles), bucky-gel (i.e. a gelatinous material comprising multi-layer graphene functionalized with biomolecules and/or ionic liquids) or colloidal systems (i.e. mixtures containing solid or liquid particles, with a diameter from 5 to 200 nm, dispersed in a fluid, liquid or gas). Typically, single-layer graphene sheets are substantially present in nano-dispersions. Typically, substantially multi- layer graphene sheets are present in the bucky-gel and in colloidal systems. Such a multi-layer graphene may spontaneously organize itself in the form of "nano- ribbons" (GNR), namely in the form of strips cut from each two-dimensional graphene sheet - with a precise crystallographic direction, and periodic boundary conditions in only one direction. Nano-dispersions, bucky-gels and colloidal systems may be applied to the paper support by spraying or by means of a brush, for example according to the restoration methods set forth in the Technical Specifications (Capitolato Tipo Tecnico) (for technical-scientific details, reference should be made to the ICRCPAL - Istituto Centrale per il restauro e la Conservazione del Patrimonio Archivistico e Librario - website;

http://www.icpal.beniculturali.it/lab_restauro.html).

The term "biomolecule" means any organic substance or organic molecule having functional groups capable of binding to graphene. Examples of biomolecules are carbohydrates, lipids, proteins, including enzymes, nucleic acids, primary and secondary metabolites, or natural substances.

The term "mainly covalently" means that a covalent bond is formed, not excluding that even weak bonds may form, such weak bonds being less in number than the covalent bonds.

Preferably, but not exclusively, the biomolecules used in the invention are enzymes, such as alkaline phosphatase, or peptides, such as GSH.

Preferably, the graphene sheets are functionalized with at least one biomolecule, i.e. with at least one type of biomolecule. Preferably, the graphene sheets are functionalized with one or two biomolecules. For example, the graphene sheets are functionalized with a peptide and/or a protein.

When the graphene sheets are functionalized with a single biomolecule, the bond between such a biomolecule and the graphene sheets is manly covalent. When the graphene sheets are functionalized with two biomolecules, preferably the graphene sheets are mainly covalently bound to the first biomolecule and bound to the second biomolecule by entrapment or mechanical intercalation or by weak bonds.

The invention provides for at least the electrode acting as the anode being made of graphite, while the electrode acting as the cathode may be made of metal such as platinum or gold or graphite. Preferably, both electrodes are made of graphite. According to the process of invention, when the electric potential is applied to the electrodes, the graphite anode oxidizes and exfoliates due to the action of the oxygen radicals induced by electrolysis of the aqueous solution, where the aqueous part is mainly provided by the buffer. Graphene oxide sheets are thus generated. The graphene oxide thus generated molecularly interacts with the biomolecule present, such as dissolved, in the electrolysis bath by means of the following interactions: covalent, hydrogen bonds and electrostatic interactions, mechanical entrapment between the graphene layers which each sheet comprises.

The fact of providing cylindrical electrodes with a ratio of the base diameter to the height of about 1 :100 ensures a semi-infinite linear diffusion process on the surface of the working electrode. Using different shapes of the electrodes or different diameter to height ratio, the transport processes supplying the surface of the working electrode by providing the enzyme which binds to such an electrode significantly change and this results in a different final product. Although the 1 :100 ratio is a particular preferred, it is apparent that a minimal tolerance is allowed, such as 10%, preferably 5%, falling within the scope of the present invention.

Advantageously, the manufacturing process is environmentally friendly, and the resulting products are non-toxic for the operator. Moreover, through the process of the invention a product is obtained, which can be used effectively in the field of restoration, and in particular in the restoration of archival and book heritage such as books and manuscripts.

One embodiment of the invention provides a process for producing graphene sheets functionalized with the alkaline phosphatase enzyme PA. Advantageously, the graphene sheets functionalized with alkaline phosphatase have a strong de- acidifying effect, particularly useful for the restoration of substrates deteriorated due to acidity, like ancient manuscripts. The de-acidifying properties of the sheets of the invention are clear to a greater extent when the substrate to be restored has foxing stains due to biodeterioration, and/or pitting stains due to biological and physical- chemical causes in synergy.

In addition, the alkaline phosphatase strengthens the antimicrobial properties of graphene, yielding a synergistic action directed against biodeterioration.

Another embodiment of the invention provides a process for producing graphene sheets functionalized with reduced glutathione GSH, which is a tripeptide with among the strongest antioxidant properties known and tested (see for example David L Nelson, Michael M Cox, I principi di biochimica di Lehninger, Sixth Edition, Edon Melloni, Franca Salamino, 2014 Zanichelli Editore).

A further embodiment provides for functionalizing graphene sheets with at least two biomolecules, preferably two biomolecules, such as an enzyme and a peptide. In particular, according to an embodiment, the sheets are first functionalized with GSH and then the sheets already functionalized with GSH are functionalized with alkaline phosphatase. A synergistic effect is obtained thereby with strong antioxidant and de-acidifying properties.

An advantageous aspect of the invention lies in the fact that when the sheets are functionalized with a single biomolecule (e.g. GSH or PA), the latter binds to them in a mainly covalent manner. When the sheets are functionalized with two biomolecules with a consecutive two-step process, the first biomolecule (e.g. GSH) binds covalently to the graphene sheets, and the second biomolecule (e.g. PA) is entrapped or mechanically intercalated between the layers of graphene which make up each sheet, or by means of "weak" interactions. Thereby, a final product can be obtained which has different release kinetics, depending on the bond between biomolecule and graphene sheets. Therefore, a specific product can be produced for the degree of deterioration of the substrate to be restored, depending on the progress of the damage of the supports themselves. Therefore, it will be preferable to restore a highly deteriorated support, applying graphene functionalized with biomolecules through weak interactions of the matter, so that the restoration agent (e.g. PA, de-acidifying agent) can be available to carry out its task in a very short time, or even instantly (just to quickly counter the damage of the surface to be treated). On the other hand, in case of parchment and/or paper supports characterized by medium-low intensity damage, then it might be advisable to apply graphene sheets functionalized with biomolecules through strong covalent interaction, capable of carrying out over time, with a gradual mechanism, a preservative and stabilization action of the treated surfaces.

An advantage of using graphene for the restoration of paper materials is the similarity between its structure and that of cellulose, both having hexagonal shapes, allowing the intercalation of graphene in the cellulose chains. Moreover, graphene is also a consolidating agent of damaged supports.

As mentioned above, the functionalization of graphene with biomolecules significantly improves the properties thereof useful for the purposes of restoration. In fact, graphene functionalized with alkaline phosphatase PA is particularly suitable as a de-acidifying agent and graphene functionalized with reduced glutathione GSH is particularly suitable as a reducing agent.

The functionalized nano-graphene of the invention also has advantageous antimicrobial and antibacterial properties.

Advantageously, the expedient of using phosphate buffered saline as electrolyte solution does not alter the activity and stability of the biomolecules, and in particular of the alkaline phosphatase PA and of the reduced glutathione GSH. To the same end, a different buffer may also be used, such as carbonate buffer, which is suitable for both the alkaline phosphatase PA and for the reduced glutathione GSH. These buffers ensure maximum activity of the prosthetic group. Preferably, the electrolyte solution has a concentration from 0.01 M to 0.2 M and ionic strength (μ) from 0.05 M to 0.2 M.

Also, in order not to alter the activity of the biomolecules, the temperature at which the process is conducted is preferably monitored, for example with a thermocouple, and possibly it is predetermined based on the biomolecule. Typically, the process takes place at room temperature.

Compared to the ideal substantially pure, or pristine, single-layer graphene, which has a thickness of about 3.354 A, the functionalized graphene of the invention includes both shapes, such as "nano-ribbons" (GNR) and sizes which are different due to the greater degree of aggregation due to the functionalization with the biomolecule.

In general, each electrochemically exfoliated sheet of the invention has both the width and the length from about 20 to 200 nm, typically of about 100 nm. Each sheet may consist of 2 to about 20 superimposed layers, typically between 2 and 10 superimposed layers depending on the duration of the electrochemical synthesis. For each functionalized graphene sheet, the distance between each plane, or layer, of the multi-layer is between about 6 and 14 A, where distance between each plane means the distance π-π, i.e. inter-layer stacking.

The distance between the layers is substantially due to the functionalization of graphene oxide induced by oxygen radicals, which are byproducts of the water electrolysis, considering that the working buffer is in aqueous solution, and to the fact that the biomolecules contribute to expand the graphene layers even more, increasing the overall thickness of each sheet. In fact, the biomolecule can be mechanically entrapped in the electrochemically exfoliated layers or it is bound by covalent or electrostatic interaction to the graphene carbon. Usually, the overall thickness of each sheet is typically from 20 nm to 200 nm, typically about 100 nm, depending on the number of layers.

The specific surface area, or surface area, of each layer, as determined with BET measurements, is typically from 2000 to 3000 m²/g. BET measurements are well known to those skilled in the art, and "surface area" means one of the values which form part of the BET table.

As will be described further, the shape, number of layers and state of aggregation of graphene depend on the kinetics of the process.

The products obtained with the process of the invention are particularly suitable for the restoration of paper supports deteriorated due to the presence of iron-gall and/or printing inks as well as due to the chemical composition of the support combined with natural aging. For such applications, and irrespective of the state of aggregation of the graphene sheets, it was found that the percentage by weight of biomolecule with respect to graphene is preferably but not exclusively 1 % (w/w), which can be obtained by providing a concentration of biomolecule in the electrolysis bath of about 1 mg/ml. Other possible concentrations of biomolecule are between 0.1 and 10 mg/ml and between 0.1 and 5 mg/ml.

Advantageously, the process of the invention, in particular the functionalization step, is carried out without a chemical template, such as an anionic and/or cationic surfactant, or an emulsifying agent.

The dependent claims describe embodiments of the invention.

Brief description of the drawings

Further features and advantages of the invention will become more apparent from the detailed description of non-exclusive embodiments of a process for the production of graphene sheets functionalized with a biomolecule and related uses, described by way of a non-limiting example with the aid of the accompanying drawings, in which: Fig. 1 shows a schematic representation of an electrochemical cell provided for the process of the invention;

Fig. 2 shows a schematic representation of a graphene sheet comprising three layers, functionalized according to the invention;

Figs. 3a and 3b respectively show an image of a sheet of the invention, obtained through electronic scanning (Field Emission-Scanning Electron Microscopy/Energy Dispersive x-ray MicroAnalysis) and a schematic representation of the same sheet; Fig. 4a shows an enlarged detail of Fig. 3a;

Fig. 4b shows an enlargement of Fig. 4a;

Fig. 4c shows a schematic representation of the detail in Fig. 4b;

Fig. 5 shows an enlarged detail of Fig. 4a at a higher magnification than in Fig. 4b. The same reference numerals in the figures identify the same elements or components.

Detailed description of embodiments of the invention

With reference to the figures, the present invention provides a process for producing graphene sheets functionalized with at least one biomolecule. In the non-limiting embodiments hereinafter, the alkaline phosphatase enzyme PA and/or the reduced glutathione peptide GSH are used.

The process involves the use of an electrochemical cell 1 which comprises two electrodes, which in following embodiments are two identical graphite electrodes, and in particular a working electrode 2, which is the anode, and a reference electrode 3, which serves as the cathode. Alternatively, the electrode serving as the cathode may be made of platinum or gold. Electrodes 2, 3 are each connected to a power supply unit 4 so as to be electrically connected to each other. Preferably, a direct current (DC) power supply unit 4 is used to prevent the concentrations at the ends of the two electrodes from varying, as it happens under alternating current regime.

Each electrode 2, 3 is substantially cylindrical in shape, whose diameter is indicated by reference φ and whose height is indicated by reference L. The diameter (cp)/height (L) ratio is φ/L = 1 /100±10%, preferably 1 /100 ±5%. A non-exhaustive example of the dimensions is φ = 3 mm and L = 300 mm. It has been experimentally observed that this aspect ratio is particularly important to obtain the functionalized sheets. In fact, experiments were conducted with aspect ratios other than those mentioned above.

A reference example was conducted with electrodes having an aspect ratio of 1 :50. In this case, the electrode geometry is such that it is quickly consumed, thinning as a truncated cone. GSH and alkaline phosphatase diffusion processes at the electrode surface are inhibited. The massive exfoliation process is blocked and no more graphene sheets or flakes are produced in the electrochemical cell.

Other reference examples were conducted with an aspect ratio of 1 :200, 1 :500 and 1 :1000, respectively. In all cases, the electrode no longer has the ideal flat geometry necessary for the diffusion of GSH and alkaline phosphatase, a necessary condition to initiate the exfoliation. In fact, the electrode substantially becomes, at most, an infinite wire run through by current. The generated radicals prefer to flow through the electrode with their electrons unpaired rather than chemically attacking the electrode (diffusing towards it) and starting the exfoliation and functionalization. Again with respect to the geometry of the measurement and also taking into account the wiring configuration which connects it to the direct current power supply, it is assumed that the working and the reference electrode distance is independent of the operator but meets the geometrical requirements of the cell parameters in the measurement system.

The electrochemical cell 1 also comprises a receptacle 5, preferably of borosilicate glass, containing an electrolysis bath 6, or electrolytic bath, in which electrodes 2, 3 are partially immersed. The electrolysis bath 6 comprises an electrolysis solution, or supporting electrolyte, which is preferably Dulbecco phosphate buffered saline PBS and the biomolecule for functionalization, which may for example be phosphatase PA or reduced glutathione GSH. Alternatively, the buffer may be carbonate buffer. The electrolyte solution has an ionic strength μ from 0.05 to 1 M, preferably of about 0.1 M.

The biomolecule is present in the electrolytic bath at a concentration from 0.1 to 10 mg/ml, preferably from 0.5 to 5 mg/ml, for example it is of about 1 mg/ml. The process of the invention provides for applying a constant and controlled electrochemical potential E to electrodes 2, 3 which can be between 1 and 24 V, or 1 and 20 V, or 1 and 12 V, using a reference graphite or platinum or gold electrode. For example, when the biomolecule is GSH, it is preferable to work at a potential from 1 to 8 V, preferably using a graphite electrode as a reference electrode. By way of another example, when the biomolecule is alkaline phosphatase, it is preferable to work at a potential from 1 to 4 V, preferably using a graphite electrode as a reference electrode.

The electrochemical potential can be selected on the basis of the biomolecule. The potential can also be selected according to the material of which the cathode is made. By applying such a potential, the biomolecule covalently binds with the working electrode 2, which at the same time exfoliates electrolytically, thereby producing graphene sheets 7 functionalized with the biomolecule present in the electrolytic bath 6. The potentials applied for GSH and alkaline phosphatase were properly optimized on empirical basis. Also the choice to conduct a continuous electrolysis exfoliation (electrolysis with anodic etching) allows both the organic biomolecules to perform fully their roles and their functionalities; and at the same time, it allows the micrometric graphite working electrode to undergo an etching and thereby exfoliate. In fact, by way of example, working with the modern controlled and constant potential electrochemical techniques, such as Differential Pulse Voltammetry (DPV, which applies a square wave potential function) or Cyclic Voltammetry (CV, which applies a sawtooth potential function) on conventional graphite, platinum, gold, glassy carbon (GC) electrodes, etc., would lead for: a) GSH to a chemical recombination of radicals (having unpaired electrons) and therefore a termination/ending of the exfoliation reaction; b) the alkaline phosphatase, the formation of dimers resulting in the loss of the whole enzymatic activity.

In order to ensure a homogeneous mixing of the electrolysis solution, preferably during the electrolytic bath it is subjected to stirring, such as by immersing a magnet 8 in the bath and placing receptacle 5 on a magnetic plate, not shown.

According to a first embodiment of the invention, a process is provided for producing graphene sheets functionalized with the alkaline phosphatase enzyme PA. The electrolysis bath comprises an electrolytic solution and the biomolecule present in the electrolytic bath is alkaline phosphatase PA. The electrolyte solution is Dulbecco phosphate buffered saline PBS at the concentration from 0.01 M to 0.2 M, preferably of about 0.1 M, pH from 7.3 to 7.5, preferably of about 7.4 and ionic strength μ of about 0.1 M. The alkaline phosphatase enzyme PA is provided at a concentration from 0.1 to 10 mg/ml, preferably from 0.5 to 5 mg/ml, for example about 1 mg/ml. The process of the invention provides for applying a constant and controlled electrochemical potential E from 1 to 4 V, preferably of about E = + 1 V for a predetermined time and under controlled flow of inert gas, preferably nitrogen. The electrochemical phenomenon which takes place at the ends the working electrode is an anodic etching, whereby graphene sheets 7 are produced from the exfoliation of anode 2. Each graphene sheet 7 produced is functionalized with the alkaline phosphatase enzyme PA. In particular, the alkaline phosphatase enzyme PA is covalently entrapped in the graphene sheets which are oxidized at the carbon chemical shift, since the electrolysis of aqueous medium produces oxygen radicals. Evidence of the fact that the alkaline phosphatase mainly covalently binds to the graphene sheets is given by the fact that formation of foam in the electrolysis bath solution is observed during the process. The graphene sheets thus obtained are particularly suitable when a slow release of the alkaline phosphatase (which therefore functions as an extended release alkaline reserve of the appropriate pH units) on the substrate to be treated is desired, in particular to be preserved and stabilized (preservation and stabilization typically are the other two steps of a delicate operation of restoration specific for manuscripts which are not at a too advanced state of damage).

It is not excluded that part of the oxidized and electrolytically exfoliated graphene/enzyme interaction is weak in nature, for example of the hydrogen bond and/or electrostatic interaction type; or mechanical entrapment.

The anodic etching is assisted by gaseous nitrous flow at about 0.1 cm³/min. This is a relatively low flow to minimize the turbulence phenomena in the electrolysis cell. A second embodiment provides a process for producing graphene sheets functionalized with reduced glutathione GSH. Advantageously, GSH is a powerful radical initiator for the exfoliation of the working electrode, or in other words it is an exfoliation promoter. In addition, GSH functionalizes the graphene sheets by binding to them covalently.

The electrolysis bath comprises an electrolytic solution and the biomolecule is the reduced glutathione peptide GSH. The electrolyte solution is Dulbecco phosphate buffered saline PBS at the concentration from 0.01 M to 0.2 M, preferably of about 0.1 M, pH from 7.3 to 7.5, preferably of about 7.4 and ionic strength μ of about 0.1 M. The reduced glutathione peptide GSH is provided in the electrolysis bath at a concentration from 0.1 to 10 mg/ml, preferably from 0.5 to 5 mg/ml, for example about 1 mg/ml. The process of the invention provides for applying a constant and controlled electrochemical potential E from 1 to 8 V, preferably of about +8 V for a predetermined time and under controlled flow of inert gas, preferably nitrogen. For example, nitrogen is flushed with a flow of about 0.1 cm³/min. Thereby, the reduced glutathione GSH present in the electrolysis bath undergoes the same process as described above in detail for the PA.

A third embodiment provides for functionalizing the graphene sheets with both GSH and with alkaline phosphatase.

In particular, two steps performed in succession are provided.

In the first step, a first functionalization of graphene sheets is carried out with GSH. In the first step, the process is substantially identical to that described for the second embodiment.

In the second step, the sheets functionalized with GSH obtained in the first step are functionalized with the alkaline phosphatase. In particular, using the same cell, the potential is being adapted, going from 1 -8 V to 1 -4 V. The solution containing alkaline phosphatase described for the first embodiment is added to the cell, preferably gradually. In particular, the alkaline phosphatase enzyme PA is present at a concentration from 0.1 to 10 mg/ml, preferably from 0.5 to 5 mg/ml, for example of about 1 mg/ml.

Preferably, the solution containing alkaline phosphatase is added after about 1 -8 hours or 6-8 hours of operation of the cell configured to produce graphene sheets functionalized with GSH alone. In addition, it is preferable that when the solution containing alkaline phosphatase is added, GSH is still present in the electrolysis bath. In particular, in this case, alkaline phosphatase is predominantly mechanically entrapped or mechanically intercalated within the graphene layers to which GSH is already covalently bound. The evidence that, in this case, the alkaline phosphatase mainly binds by mechanical entrapment or mechanical intercalation with the graphene sheets, is given by the fact that when the alkaline phosphatase is added, no foam formation is observed. In this case, the release of alkaline phosphatase on the substrate to be restored takes place very quickly. This effect is particularly advantageous when the substrate to be treated has a very thorough deterioration, so the technician would require a traditional restoration rather than preserving and/or protecting the manuscript. In order to achieve this quick release effect, it is particularly preferred to carry out the process by functionalizing with GSH first and then with PA.

Both for the first and for the second and third embodiment, the final synthesis product contains single-layer or multi-layer (consisting of 2 up to 20 superimposed layers) graphene sheets functionalized with the biomolecule or biomolecules present in the electrolysis bath. For example, Fig. 2 shows a graphene sheet functionalized with GSH, comprising three superposed graphene layers 1 1 . The graphene functionalized with the biomolecule exhibits different forms of aggregation, depending on the electrolysis/synthesis time.

In particular, when the duration of the process, namely the operating time of the power supply unit, is of about 1 hour, a nano-dispersion which is stable over time is obtained which contains substantially single-layer oxidized graphene sheets functionalized with the biomolecule present in the electrolysis bath. This type of product may also be obtained by washing the working electrode with distilled water or buffer after about 1 hour of electrolysis. In fact, the graphene sheets which detach from the working electrode during the washing are substantially single layer.

When the duration of the process is of about 6 hours, a colloid or colloidal system is produced which can be used as such or collected on the bottom of the electrolysis cell, and then dried. The solid bottom body, once dried (for example in an oven at 100 °C) looks like a black graphene powder. Such a powder substantially consists of multi-layer graphene sheets, with a minimum of 3 to a maximum of 10 layers. The powder can then be re-dispersed in the liquid phase in a suitable dispersion medium, depending on the desired final application, typically at a titer of 1 mg/ml. When the process exceeds 6 hours achieving no more than 12 hours, the synthesis product takes the consistency of an actual bucky-gel. The gel comprises multi-layer graphene sheets with a number of layers greater than 10.

According to another aspect thereof, the invention provides for using the products obtained by means of the processes described above for the restoration of parchment and/or paper supports, in particular containing iron-gall inks and/or printing inks which have impaired the nature and stability thereof over time. An example of an application in the restoration of archival and book heritage is described hereinafter: the graphene powder functionalized with peptide can be used to prepare colloidal phases dispersed in a liquid, which is preferably phosphate buffered saline PBS or an alcohol, such as ethyl alcohol and/or isopropyl alcohol. The colloidal phase can then be applied as aerosol to the paper support to be restored by means of a sprayer device.

According to another example the bucky-gel, as is or further diluted with alcohol or with phosphate buffered saline PBS, can be directly applied to the paper support to be restored with the aid of a brush.

According to yet another example, the colloidal dispersion of multi-layer graphene sheets, as is or further diluted with alcohol or phosphate buffered saline PBS, can be sprayed using a pressurized sprayer on the paper support to be restored.

According to a further aspect, the invention also provides a bucky-paper, i.e. a porous support, typically consisting of filter paper (preferably Whatman pure cellulose) or a polycarbonate membrane or a sponge, where the graphene multi- layers functionalized with biomolecules, preferably in the form of black powder, have been deposited.

According to the invention, the bucky-paper thus obtained is typically used for the restoration of paper supports, in particular paper supports deteriorated by iron-gall inks, membranous supports containing inks or supports such as polychrome paintings on canvas. In fact, bucky-paper is used as adhesive substrate to remove dirt deposits which compromise the surfaces involved, such as of historic-artistic type. Alternatively, it is used as black sponge, if the bucky-paper is subjected to treatment with cryogenic liquid, for example with liquid nitrogen for about 2 hours. The sponge bucky-paper can also be prepared as "white sponge" when the bucky- paper is subjected to a chemical treatment in egg whites and subsequent passage into liquid nitrogen. In this case, the sponge removes dirt or undesired material from the surface to be treated, for example by physical adsorption, not by weak interaction of a magnetic nature.

An example of production of the bucky-paper provides for the bottom body obtained after about 6 hours of electrolysis is washed with distilled and vacuum-filtered in flask. During the vacuum filtration, the solvent is completely removed and the multi- layer graphene functionalized with biomolecules is deposited on a Whatman paper filter of pure cellulose with pore size of about 20 nm fitted on a Buchner funnel. Bucky-papers are also suitable for different applications in the field of sensors, electronic circuits and electro-chemical devices.

While the invention has been described with reference to alkaline phosphatase and GSH, in the light of the present description those skilled in the art can select other biomolecules with which the graphene sheets can be functionalized.

Claims

1 . An electrochemical process for the production of at least one nanometric graphene sheet functionalized with at least one biomolecule, wherein there is provided

an electrochemical cell (1 ) comprising two electrodes (2, 3), of which at least one electrode (2), which is anode, is of graphite,

said two electrodes (2, 3) being at least partially immersed in an electrolysis bath (6) and connected to a power supply unit (4)

wherein each electrode (2, 3) is cylindrical in shape with a diameter (cp)/height (L) ratio = 11\ 00±10%,

wherein the electrolysis bath (6) comprises an electrolytic solution and said at least one biomolecule,

the process comprising a step in which a predetermined electric potential (E) is applied to the electrodes (2, 3) by means of the power supply unit (4), whereby the at least one biomolecule mainly covalently binds with said graphite electrode (2), which exfoliates producing at least one graphene sheet (7) functionalized with said at least one biomolecule.

2. The process according to claim 1 , wherein said at least one biomolecule is an enzyme, or wherein said at least one biomolecule is a peptide.

3. The process according to claim 1 or 2, wherein said at least one biomolecule is dissolved in the electrolysis bath (6) at a concentration in the range between 0.1 and 10 mg/ml, preferably between 0.5 and 5 mg/ml.

4. The process according to any one of claims 1 to 3, wherein said at least one biomolecule is alkaline phosphatase (PA) or reduced glutathione (GSH).

5. The process according to any one of claims 1 to 4, wherein said electric potential (E) is in the range between 1 V and 20V, preferably between 1 and 8V.

6. The process according to any one of claims 1 to 5, wherein said electrolytic solution is a buffer, preferably phosphate buffered saline (PBS).

7. The process according to any one of claims 1 to 6, wherein the other electrode (3) of said two electrodes (2, 3) is made of graphite or platinum or gold.

8. The process according to any one of the preceding claims, wherein a further step is provided, wherein in said further step a further electrolytic solution comprising a further biomolecule is added to the electrolysis bath.

9. The process according to claim 8, wherein said further biomolecule is an enzyme, preferably alkaline phosphatase.

10. A graphene sheet functionalized with at least one biomolecule obtainable with the process according to any one of claims 1 to 9.

1 1 . The graphene sheet according to claim 10, wherein said sheet comprises a plurality of graphene layers.

12. The graphene sheet according to claim 10 or 1 1 , having a specific surface area in the range between 2000 and 3000 m²/g determined by BET measurements and consists of 2 to 20 layers, the distance between two graphene layers being in the range from 6 to 14A.

13. Use of a sheet according to any one of claims 10 to 12 as de-acidifying and/or reducing agent for the restoration of deteriorated paper supports.

14. Buckypaper comprising a porous support and at least one graphene sheet according to any one of claims 10 to 12.