Covalent functionalization of graphene

Abstract

The present invention relates to a method for obtaining covalently modified graphene. The present invention also describes a covalently modified graphene obtained by this method and its use as an anchoring surface for bioreceptors in a biosensor device. Finally, the present invention describes a biosensor device comprising a covalently modified graphene obtainable according to the method of the present invention.

Description

Technical Field

[0001] The present invention relates to a method for obtaining covalently modified graphene. Likewise, the present invention relates to a covalently modified graphene obtainable by this method and its use as an anchoring surface for bioreceptors in a biosensor device. Finally, the present invention relates to a biosensor device comprising a covalently modified graphene obtainable by the method according to the present invention. Background Art

[0002] At present, various molecules have been used to covalently and non-covalently functionalize graphene (Plutnar et al., J. Mater. Chem. C., 2018, 6, 6082-6101), and even the anchoring of antibodies has been achieved, but in most cases, it is a reaction in the liquid phase (dissolution), wherein the graphene used is in the form of a thin sheet in a suspension or in the form of a part of an immersed electrode. In the past 5 years, this has produced a large number of publications (Georgakilas et al., Chem. Rev. 2016, 116, 5464-5519). In some other publications, the graphene functionalization is carried out in a solid phase outside the dissolving tank on a layer of previously deposited graphene or graphene oxide. However, these methods do not allow for positional control of the modification performed on the surface of graphene or graphene oxide, without differentiated or distinct regions within the layer (Morales-Narvaez et al., Adv. Mater., 2018, 1805043; Sandoval et al., Chem. Commun., 2019, 55, 12196-12199). An oxidative etching technique known as local anodic oxidation (LAO) or oxidative scanning probe etching (o-SPL) does allow for positional control. However, this technique has so far (1) been applied only at the nanoscale, by adapting microscopes to work at this nanoscale, which is limited to very small and impractical areas of the order of nm (oxidation lithography, R. García et al., Carbon 2018, 129, 281-285; Alvarez et al., 2017, Spanish Conference on Electron Devices (CDE), p. 1-4, doi: 10.1109 / CDE.2017.7905231); and (2) only for oxidation purposes and has not been extended to other types of functionalization or chemical reactions.

[0003] Recently, a method for functionalizing graphene in different regions by utilizing the process of illumination has been published (Valenta et al., Agew.Chem.Int.Ed, 2019, 58, 1324-1328). In this method, the reaction is only advantageous in those regions where a mask is used, and the modified region can be positioned in various positions on the sample surface. However, this process is very limited to certain specific reactions that are easily performed by illumination in the blue light or ultraviolet region, such as those reactions known as Mitsunobu reactions.

[0004] On the other hand, WO 2018098286 A1 describes a biomolecule sensor that utilizes graphene surfaces (and defective graphene). However, the bonding of the sensor molecule (in this case a polymerase) to the graphene as described in said document requires positional control using a mask in the sensor, and only describes the modification of the graphene before its use in the sensor, without any positional control (dispersion or chemical etching of nanoparticles) or the use of a mask to enable such control.

[0005] WO 2018013586 A1 does not describe covalent bonding of sensor molecules to graphene, but uses a functionalization layer deposited on the graphene layer, which covalently bonds to molecules or functional groups on the biomolecules of interest in the sensor. However, the graphene disclosed in the document is not covalently functionalized.

[0006] US2019 / 0079068 A1 and WO2016054550 describe sensor devices that can use carbon conductive materials such as carbon nanotubes, fullerenes or graphene as sensor elements. In addition, the documents describe the use of protein- or antibody-functionalized graphene, wherein the graphene is directly bound to the protein or antibody, or is bound through a linker, and it is specified that the bonding can be covalent. However, the documents do not disclose how the functionalization is performed under any circumstances, nor whether it exists or how its position is controlled.

[0007] Thus, a method for functionalizing graphene characterized by positional control and versatility with respect to the nature of the reagents used has not yet been disclosed, wherein such a method would enable the provision of a physical substrate on which different molecules or functional groups, which could for example be biomarkers or bioreceptors such as antibodies, are anchored on a single miniaturized platform, thereby making the assay much more compact in size, and such a method would enable the provision of a system enabling the detection of analytes in a small space, which is compatible with the development of lab-on-a-chip systems, i.e. systems integrating and automating a variety of laboratory techniques in a chip-sized device with a maximum dimension of a few square centimeters. Summary of the invention

[0008] The present invention relates to a method for obtaining covalently modified graphene, the method comprising:

[0009] i. applying a voltage of at least 10 volts to generate an electric field in the region of the graphene surface in the presence of water and a substance that generates one or more ions and / or one or more free radicals in water or in water and in the presence of an electric field generated by the voltage;

[0010] ii. optionally washing the graphene, and

[0011] Repeating steps (i) and (ii) successively in other regions of the graphene surface to be modified, using the same substance or other different substances that generate one or more ions and / or one or more free radicals in water or in water and in the presence of an electric field generated by the voltage, the other regions being different from the one or more regions in which steps (ii) and (ii) have been performed;

[0012] and wherein the substance that generates one or more ions and / or one or more free radicals in water or in water and in the presence of an electric field is independently selected from the group consisting of:

[0013] - a compound that generates free radicals in water or in water and in the presence of an electric field, the free radicals being selected from the group consisting of superoxide radicals, alkoxy radicals, aryloxy radicals, peroxy radicals, aminooxy radicals, nitric oxide radicals, nitrogen dioxide radicals, nitrosonium cations, nitrene radicals, halogen radicals, alkyl radicals, aryl radicals, carbene radicals and ketyl radicals; or a compound comprising one or more functional groups that generate said free radicals in water or in water and in the presence of an electric field;

[0014] - a compound that generates a cation in water or in water and in the presence of an electric field, wherein the cation is independently selected from the group consisting of a metal cation, an ammonium cation, a nitronium cation, a carbonium ion, a diazonium cation and a guanidinium cation; or a compound comprising one or more functional groups that generate the cation in water or in water and in the presence of an electric field; and

[0015] - a compound that generates anions in water or in water and in the presence of an electric field, wherein the anions are independently selected from the group consisting of peroxide, alkoxide, phenoxide, carboxylate, azide, carbonate, bicarbonate, borate, bromate, iodate, cyanate, isocyanate, cyanide, nitrate, nitrite, halide, sulfate, hydrogensulfate, thiosulfate, sulfite, hydrogensulfite, sulfide, bisulphide, disulphide, phosphate, phosphite, acid phosphate, diacidphosphate and silicate; or a compound comprising one or more functional groups that generate the anions in water or in water and in the presence of an electric field.

[0016] The present invention also relates to a method for obtaining covalently modified graphene, the method comprising:

[0017] i. applying a voltage of at least 10 volts to generate an electric field in the region of the graphene surface in the presence of water and a substance that generates one or more ions and / or one or more free radicals in water or in water and in the presence of an electric field generated by the voltage;

[0018] ii. optionally washing the graphene, and

[0019] Repeating steps (i) and (ii) successively in other regions of the graphene surface to be modified, using the same substance or other different substances that generate one or more ions and / or one or more free radicals in water or in water and in the presence of an electric field generated by the voltage, the other regions being different from the one or more regions in which steps (ii) and (ii) have been performed;

[0020] and wherein the substance that generates one or more ions and / or one or more free radicals in water or in water and in the presence of an electric field is independently selected from the group consisting of:

[0021] - a compound that generates free radicals in water or in water and in the presence of an electric field, the free radicals being selected from the group consisting of alkoxy radicals, aryloxy radicals, nitrosonium cations, nitrogen radicals, halogen radicals, aryl radicals and carbon radicals; or a compound comprising one or more functional groups that generate said free radicals in water or in water and in the presence of an electric field;

[0022] - a compound that generates one or more cations in water or in water and in the presence of an electric field, the cations being independently selected from the group consisting of: Li + 、Na + , K +Mg 2+ , Ca 2+ , Cu 2+ , ammonium, diazonium and carbocations; or compounds comprising one or more functional groups that produce said cations in water or in water and in the presence of an electric field; and

[0023] - A compound that generates one or more anions in water or in water and in the presence of an electric field, the anions being independently selected from the group consisting of chloride, iodide, bicarbonate, alkoxide, phenoxide and carboxylate; a compound comprising one or more functional groups that generate said anions in water or in water and in the presence of an electric field.

[0024] Likewise, the present invention relates to a covalently modified graphene obtainable by the method of the present invention.

[0025] Another aspect of the present invention relates to the use of the covalently modified graphene obtainable according to the method of the present invention in an analyte detection device.

[0026] Finally, the present invention relates to an analyte detection device comprising a covalently modified graphene obtainable according to the method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Relative humidity versus normalized radius of surface area for graphene covalently modified with NaCl, NaHCO3, NaI, and 2-chlorophenol (all at 40 mM) compared to a control using distilled water. DETAILED DESCRIPTION

[0028] The present invention relates to a method for obtaining covalently modified graphene, the method comprising: (i) applying a voltage of at least 10 volts to generate an electric field in the region of the graphene surface in the presence of a solvent and a substance, the substance generating one or more ions and / or one or more free radicals in the solvent or in the solvent and in the presence of the electric field generated by the voltage.

[0029] The term "solvent" refers to a compound that, when mixed with another compound or substance, forms a homogeneous mixture, constituting a single phase, i.e. it forms a mixture in which the physical properties remain homogeneous, "homogeneous" being understood as meaning that all intensive properties (those that do not depend on the amount of the substance) remain unchanged. Usually, the amount of solvent in the mixture is greater than the amount of other substances, which are usually called solutes, and are therefore substances other than the solvent. Preferably, the solvent is water.

[0030] The term "substance that generates one or more ions and / or one or more free radicals in a solvent or in a solvent and in the presence of an electric field" refers to a substance other than a solvent that generates at least one ion and / or at least one free radical when in contact with the solvent and optionally in the presence of an electric field (generated by applying a voltage). Thus, the term includes ionic substances (i.e. substances containing ionic bonds) that release cations and anions that form the ionic substance when dissolved in the solvent, and also includes substances with covalent bonds that are suspended or vaporized in the solvent and that generate the formation of at least one ion and / or the formation of at least one free radical when they are in contact with the solvent or when they are in contact with the solvent and in the presence of an electric field. In other words, in some cases, the substance used generates ions or free radicals only by dissolving in the solvent used, while in other cases, the presence of an electric field is necessary for the substance dissolved in the solvent to generate ions or free radicals.

[0031] In a preferred embodiment of the present invention, once step (i) has been carried out, step (i) is repeated successively in other areas of the graphene surface to be modified, in the presence of the same solvent or other different solvents, and using the same substance or other different substances that produce one or more ions and / or one or more free radicals in the solvent used or in the solvent used and in the presence of the electric field generated by the voltage, wherein the other areas are different from the one or more areas in which this step has been carried out.

[0032] In this way, the present invention enables covalent modification of graphene with one or more types of functional groups at one or more sites, because the method enables covalent modification of graphene with various substances dissolved or suspended in the solvent used and in particular in water (i.e. when the solvent is water), and also enables modification of graphene in one or more regions, the one or more regions comprising the same or different functional groups. Unlike the modification of graphene disclosed in the prior art, the present method enables the use of templates that require the use of additional steps to be avoided. In addition, the present method does not require the use of solutions and substances that may damage the graphene layer because the modification is spatially localized. In this way, the method of the present invention enables covalently modified graphene with different functional groups in different specific regions on its surface to be obtained, wherein both the introduced groups and the modified regions can be selected by the user of the method.

[0033] In one embodiment, the method comprises:

[0034] i. applying a voltage of at least 10 volts to generate an electric field in a region of the graphene surface in the presence of a solvent and a substance that generates one or more ions and / or one or more free radicals in the solvent or in the solvent and in the presence of an electric field generated by the voltage; and

[0035] ii. washing the graphene.

[0036] In one embodiment of the invention, once step (ii) has been carried out, step (i) and optional step (ii) are repeated successively in other areas of the graphene surface to be modified, in the presence of the same solvent or other different solvents, using the same substance or other different substances that produce one or more ions and / or one or more free radicals in the solvent used or in the solvent used and in the presence of the electric field generated by the voltage, wherein the other areas are different from the one or more areas in which this step has been carried out.

[0037] Therefore, the method of the present invention comprises:

[0038] i. applying a voltage of at least 10 volts to generate an electric field in the region of the graphene surface in the presence of a solvent and a substance that generates one or more ions and / or one or more free radicals in the solvent or in the solvent and in the presence of an electric field generated by the voltage;

[0039] ii. washing the graphene, and / or optionally, repeating the previous step successively in other regions of the graphene surface to be modified, different from one or more regions in which the previous step (i) has been performed, in the presence of the same solvent or other different solvents and using the same substance or other different substances that also produce one or more ions and / or one or more free radicals in the solvent used or in the solvent used and in the presence of the electric field generated by the voltage.

[0040] Thus, the method of the present invention comprises covalent modification in regions of graphene according to step (i), and wherein step (i) may be repeated, including between each step (i), with or without a step (ii) in which the graphene is washed before step (i) is performed again.

[0041] In other words, the method of the present invention comprises: step (i), and optionally step (ii), wherein step (i) and optionally step (ii) are repeated successively in multiple regions of the graphene surface in the presence of the same solvent or different solvents and using the same substance or different substances, and step (i) is performed each time.

[0042] In a preferred embodiment of the present invention, the solvent is water, and the present invention relates to a method for obtaining covalently modified graphene, the method comprising:

[0043] i. applying a voltage of at least 10 volts to generate an electric field in the presence of water containing a substance in a region of the graphene surface, the substance generating one or more ions and / or one or more free radicals in the water or in the water and in the presence of an electric field generated by the voltage;

[0044] ii. optionally washing the graphene.

[0045] In one embodiment of the invention, step (i) and optional step (ii) are repeated successively in one or more other areas of the graphene surface to be modified, using the same substance or other different substances that also produce one or more ions and / or one or more free radicals in water or in water and in the presence of the electric field generated by the voltage, wherein the other areas are different from the one or more areas in which the steps have been performed.

[0046] In other words, in a more preferred embodiment of the present invention, the method further comprises: repeating steps (i) and (ii) successively in other areas of the graphene surface to be modified, using the same substance or other different substances that produce one or more ions and / or one or more free radicals in water or in water and in the presence of an electric field generated by the voltage, wherein the other areas are different from one or more areas in which steps (i) and (ii) have been performed.

[0047] Therefore, in a preferred embodiment of the present invention, the covalent modification can be carried out preferably in the presence of water vapor or fine liquid water atomization containing a substance that generates at least one ion and / or at least one free radical in water or in water and in the presence of an electric field. Thus, the graphene surface is contacted with a water-rich atmosphere, referred to as working atmosphere, which contains said substance.

[0048] For the purpose of the present invention, the term "comprising" means that it includes a group of features, but does not exclude the existence of other features, as long as the existence of other features does not make the present invention unworkable. In addition, the terms "consisting of", "containing", "including", "having", "covering" and synonyms of such terms should be interpreted in the same way as the term "comprising".

[0049] In addition, for the purpose of the present invention, the term "comprising" may be replaced by any of the terms "consisting of" or "consisting essentially of". Therefore, in the case where the term "comprising" refers to a group of technical features A, B and C, it should be interpreted as also including other technical features in addition to the technical features A, B and C, as long as the presence of other features does not make the present invention unworkable, but it can also be interpreted as only including such features A, B and C, or substantially including such features A, B and C. Therefore, the term "comprising" referring to the group including features A, B and C should be interpreted as including the group consisting of features A, B and C or consisting essentially of features A, B and C.

[0050] Thus, the solvent optionally generates ions under the action of an electric field generated by applying a voltage (for example, when NaCl salt is present in water as solvent, Cl - Anions and Na + Cations, or for example, R-NH2 amines in the presence of water via H + The proton bonds to the amine to form R-NH3 + ammonium cations), or the formation of free radicals when bond breakage occurs, which produces species with unpaired electrons.

[0051] For the purposes of the present invention, the term "free radical" refers to a chemical species having one or more unpaired electrons. For the purposes of the present invention, examples of free radicals include, but are not limited to: superoxide radicals (O2 ·- ), oxygen diradicals (O2 ·· ), hydroxyl radical (HO·), alkoxy radical (RO·), aryloxy radical (ArO·), peroxy radical (ROO·), aminooxy radical (RNO·), among which TEMPO reagent (tetramethylpyridyl-N-oxyl) and its derivatives, nitric oxide radical (NO·), nitrogen dioxide radical (NO2·), nitrosonium cation (NO2 ·+ ), nitrogen radical (RN·), halogen radical (X·), alkyl radical (R·), aryl radical (Ar·), carbene radical (R··), etc.; wherein R is an alkyl group or H, X is a halogen atom, and Ar is an aryl group.

[0052] On the other hand, for the purpose of the present invention, the term "ion" refers to an atom or group of atoms that carries an electrical charge.

[0053] The method of the present invention is carried out in the presence of an electric field generated by applying a voltage. In some cases, the electric field is also necessary for the formation of ions and / or radicals of the substance used (e.g., for the formation of carbon cations or other types of ions or radicals that require greater energy in the presence of the solvent).

[0054] Thanks to the method of the invention, covalent modification of graphene occurs by forming new bonds between one or more carbon atoms of graphene and one of the atoms of an ion or radical generated by the substance, thereby forming functional groups covalently bonded to the carbon atoms of graphene, wherein a covalent bond is generated between one of the carbon atoms of graphene and an atom of an ion or radical of the substance used.

[0055] In a more preferred embodiment, the voltage of step (ii) is applied by a pointed electrode connected to one pole of a voltage generator. For the purposes of the present invention, the term "voltage generator" is equivalent to a voltage source. In other words, the voltage is externally "applied" between two poles or electrodes by a voltage generator (or voltage source), generating an electric field with field lines from one pole to the other. When one of the electrodes ends in a pointed tip, the generated electric field is concentrated on the tip.

[0056] For the purposes of the present invention, a pointed electrode is an electrode having a tip diameter of less than 30 microns.

[0057] In an even more preferred embodiment, the electric field is applied by a device comprising: a pointed electrode connected to a first pole of a voltage generator, while a second pole of the generator is connected to the graphene; configured to hold a head of the pointed electrode; a dielectric separator accommodating the head and configured to electrically isolate the electrode from the rest of the device; a positioner fastened to the electrical separator and configured to move the head in three dimensions of space; and a damping system configured to allow vertical movement of the head under a predetermined pressure and to attenuate displacements caused by irregularities of the surface over which the head moves; wherein in step (ii), the electric field is generated by the pointed electrode of the device; and a container with a solvent, the container being configured to produce a dispersion of the solvent. In particular, the device is referred to as a functionalized etching device.

[0058] In one embodiment of the invention, the electric field is generated by electrodes, in particular pointed electrodes, having a negative potential relative to the graphene.

[0059] In another embodiment of the invention, the electric field is generated by electrodes, in particular pointed electrodes, having a positive potential relative to the graphene.

[0060] In another embodiment of the invention, the electric field is generated by electrodes, in particular pointed electrodes, having a negative or positive potential relative to the graphene.

[0061] For example, it can be theoretically explained that when an electric field is generated by an electrode (cathode) having a negative potential relative to graphene, graphene acts as a positive electrode (anode) and attracts the negative charge of the species (ions and / or free radicals) generated by the electric field, and combines with the atoms of the species rich in negative charge through an electrophilic addition reaction. On the other hand, also for example, when an electric field is generated by an electrode having a positive potential relative to graphene, the electron cloud π of graphene attracts the positive charge of the species (ions and / or free radicals) generated, and combines with the atoms of the species rich in positive charge through an electrophilic addition reaction.

[0062] Graphene is a single two-dimensional layer of bonded carbon atoms that exhibit sp 2 Hybridized carbon-carbon bonds form hexagons and form a π electron cloud. Due to its structure, graphene behaves as a semi-metal with very high load mobility and has unique properties, among which hardness, elasticity, high conductivity and very low resistance are prominent, but the chemical structure is basically very stable (or not very reactive) to gas. This makes graphene a very interesting material in a variety of technical applications. Preferably, graphene can be a single-layer graphene or a few-layer graphene with 2 to 10 graphene monolayers. For the purposes of the present invention, in the case where the graphene to be covalently modified in the method of the present invention is composed of more than one graphene monolayer, the term "graphene surface" refers to any one of the graphene monolayers. Although graphene is a two-dimensional material, in some cases, the material used includes more than one layer of graphene. Therefore, for the purposes of the present invention, when graphene is formed by more than one layer, covalent modification can occur in any layer of the graphene layer. The formation of covalent bonds between carbon atoms and functional groups involves the conversion of sp to reactive carbon atoms. 3 Hybridization. This produces a material with different properties and reactivity than the initial graphene.

[0063] In one embodiment, the covalent modification is a monotopic modification (i.e., occurs on only one of the two faces of the graphene surface). In another embodiment, the covalent modification is a ditopic modification (i.e., occurs on both sides of the graphene surface).

[0064] For the purposes of the present invention, the solvent contained in the solvent or the substance contained in the solvent and generating one or more ions and / or one or more free radicals in the presence of an electric field is contacted with the graphene surface in an "atomized" or "vaporized" or "dispersed" form without the use of a heat source, so that an atmosphere is generated using the solvent and the substance in contact with the graphene surface. In this manner, for the purposes of the present invention, the terms "atomized", "vaporized" or "dispersed" are used interchangeably and refer to the generation of a homogeneous phase containing the solvent and the substance without the use of a heat source, the homogeneous phase being referred to in terms of its physical properties.

[0065] The method of the invention thus enables positional control of the covalent anchoring of functional groups and, since it is an electrical method based on the application of a local voltage, it enables work with a variety of substances that generate ions or free radicals when in contact with the solvent or when they are otherwise subjected to an electric field.

[0066] For the purpose of the present invention, the term "functional group" refers to an atom or group of atoms that is connected to the molecular structure of a compound having a larger number of atoms and is arranged in a given bond sequence, exhibiting personalized and unique chemical properties. Therefore, in the present invention, the functional group refers to an atom or group of atoms of an ion or free radical of a substance, which generates one or more ions and / or one or more free radicals in a solvent or in a solvent and in the presence of an electric field, and forms covalent bonds with carbon atoms of graphene during the method of the present invention.

[0067] In a preferred embodiment of the method according to the invention, the electric field is applied in a circular region of the graphene surface having a diameter of less than 15 micrometers.

[0068] In a preferred embodiment, the voltage is applied at a distance less than 10 nm from the graphene surface, more preferably at a distance less than 7 nm. In an even more preferred embodiment, the voltage is applied between the pointed electrode and the graphene, keeping the electrode in contact with the surface of the graphene.

[0069] In one embodiment of the method according to the invention, the voltage applied in the region of the graphene surface to be modified is from 10 to 100 Volts; more preferably from 20 to 60 Volts.

[0070] Therefore, a preferred embodiment of the present invention comprises: (i) applying a voltage at a distance of less than 10 nm, preferably at a distance of less than 7 nm and even more preferably between a pointed electrode and graphene with said electrode in contact with a region of the graphene surface, wherein said voltage is from 10 to 100 volts, more preferably from 20 to 60 volts, to generate an electric field in said region of the graphene surface in the presence of a solvent and a substance, said substance generating one or more ions and / or one or more free radicals in said solvent or in said solvent and in the presence of an electric field generated by said voltage.

[0071] In a preferred embodiment, the voltage is applied for a period of at least 1 millisecond (1 ms). In a more preferred embodiment, the voltage is applied for a period of time typically from 1 ms to 30 seconds.

[0072] In a preferred embodiment of the present invention, the solvent is water, and step (i) of the method comprises:

[0073] i. applying a voltage of 10 to 100 volts, more preferably 20 to 60 volts, between a pointed electrode and graphene (as another electrode) at a distance of less than 10 nm, more preferably less than 7 nm and even more preferably in contact with an area of ​​the graphene surface to generate an electric field in said area of ​​the graphene surface, wherein said voltage is applied in the presence of water containing a substance that produces one or more ions and / or one or more free radicals in water or in water and in the presence of an electric field generated by said voltage, and wherein the relative humidity in contact with the graphene surface to be modified is at least 40%, more preferably at least 50%, and even more preferably 60% to 95%.

[0074] For the purposes of the present invention, the term "atmosphere" or "working atmosphere" refers to the solvent phase which is in direct contact with the surface to be modified according to the process of the present invention. According to this text, relative humidity values ​​are expressed in % when water is the solvent used.

[0075] Thus, application of the voltage results in the generation of an electric field in the region of the graphene surface in the presence of a solvent containing a substance (the substance generating one or more ions and / or one or more free radicals in the solvent or in the solvent and in the presence of the electric field), producing a covalent modification of the graphene by forming one or more covalent bonds between carbon atoms of the graphene and one or more atoms of the substance or the solvent in a region having a diameter preferably of about 1 to 12 times the size of the region in which the voltage is generated. Thus, if, for example, an electric field has been generated in a circular region having a diameter of 10 micrometers, covalent modification will occur in a region having a diameter of 10 to 120 micrometers.

[0076] Even more preferably, the solvent is water and step (ii) comprises applying a voltage of 20 to 60 volts in contact with a region of the graphene surface in the presence of water comprising a substance which generates at least one ion and / or at least one free radical in water or in water and in the presence of an electric field, and wherein the relative humidity in contact with the graphene surface to be modified is at least 50%, and more preferably from 60% to 99%.

[0077] In another embodiment of the present invention, the solvent is a polar solvent. Preferably, a protic polar solvent. More preferably, the solvent is water, a carboxylic acid, an alcohol, an amine or a hydrogen halide. Even more preferably, the solvent is water.

[0078] In this way, using the method of the present invention, local electric fields can be generated in different regions separated from each other on the surface of graphene in the presence of the solvent or other different solvents containing the same substance or different substances, thereby using different chemical species that can covalently bond different groups or the same group in the region to treat graphene at mm. 2 Above the functionalization or covalent modification of large areas, the substances generate ions and / or free radicals in the used solvent or in the used solvent and in the presence of an electric field and form bonds with one or more carbon atoms of graphene.

[0079] For example, when the solvent is water, the inorganic salt is ionized in the water, producing a fine liquid atomized phase, in which the salt is transported and responds to the generated electric field. Therefore, when a local electric field is applied in the presence of a fine liquid water atomized phase rich in salt, the electric field mediates the bonding of ionic species or free radical species corresponding to graphene. In order to anchor two substances, one substance in each region, the method is carried out in a sequential manner: first, a first substance is used in a first region, and an electric field is applied, resulting in the modification of graphene with the first substance in the first region. Then, the graphene is washed, and the method is repeated by working with a substance and solvent that is the same or different from the first substance and solvent in another different region.

[0080] In one embodiment of the invention, the graphene is washed with: DMF (dimethylformamide), DMSO (dimethyl sulfoxide), methanol, toluene or ethanol, preferably more than one wash with one or more solvents.

[0081] The process of the invention can be carried out not only with salts, in particular with inorganic salts, but also with any molecules, complexes or substances which generate ions or free radicals in water or other suitable solvents with or without the presence of an electric field generated during the process, and which do not necessarily have to be molecules with bonds of ionic nature.

[0082] Therefore, in a preferred embodiment of the method according to the invention, the substance which generates one or more ions and / or one or more free radicals in a solvent or in a solvent and in the presence of an electric field is independently selected from the group consisting of:

[0083] - a compound that generates free radicals in the solvent or in the solvent in the presence of an electric field, the free radicals being selected from the group consisting of superoxide radicals, oxygen radicals, hydroxyl radicals, alkoxy radicals, aryloxy radicals, peroxy radicals, aminooxy radicals, nitric oxide radicals, nitrogen dioxide radicals, nitrosonium cations, nitrene radicals, halogen radicals, alkyl radicals, aryl radicals, carbene radicals and ketyl radicals; or a compound comprising one or more functional groups that generate said free radicals in the solvent or in the solvent in the presence of an electric field,

[0084] - a compound that generates cations in the solvent or in the solvent and in the presence of an electric field, the cations being independently selected from the group consisting of metal cations, ammonium cations, nitronium cations, iminium cations, iodonium cations, carbonium ions, diazonium cations and guanidinium cations; or a compound comprising one or more functional groups that generate said cations in the solvent or in the solvent and in the presence of an electric field; and

[0085] - a compound that generates anions in the solvent or in the solvent and in the presence of an electric field, the anions being independently selected from the group consisting of peroxide, hydroxide, alkoxide, phenoxide, carboxylate, carbonanion, azide, carbonate, bicarbonate, borate, bromate, iodate, cyanate, isocyanate, cyanide, nitrate, nitrite, halide, sulfate, hydrogensulfate, thiosulfate, sulfite, hydrogensulfite, sulfide, hydrogensulfide, disulfide, phosphate, phosphite, acid phosphate, dihydrogen phosphate and silicate; or a compound comprising one or more functional groups that generate the anions in the solvent or in the solvent and in the presence of an electric field.

[0086] In this way, when an electric field is generated at a distance of the order of nm from the surface area of ​​graphene in the presence of a solvent containing the substance, one or more covalent bonds are generated between one or more carbon atoms of graphene and one of the atoms of the substance, forming a functional group covalently bonded to the carbon atoms of graphene, wherein a covalent bond is generated between one of the carbon atoms of graphene and an atom of the substance. Thus, in a non-limiting example, if the substance is an aryl diazonium salt (aryl-N2 + X -), which can generate N2 and aryl carbocations or aryl radicals in aqueous solution and in the presence of an electric field, which will react with the carbon of graphene to form bonds between the carbon atoms and the carbon atoms of the aryl group, thereby forming aryl functional groups in the functional group.

[0087] In a preferred embodiment of the present invention, the substance that generates one or more ions in a solvent or in a solvent and in the presence of an electric field is independently selected from the group consisting of:

[0088] - a compound that generates free radicals in the solvent or in the solvent and in the presence of an electric field, the free radicals being selected from the group consisting of hydroxyl radicals, alkoxy radicals, aryloxy radicals, nitrosonium cations, nitrene radicals, halogen radicals, aryl radicals and carbene radicals; or a compound comprising one or more functional groups that generate said free radicals in the solvent or in the solvent and in the presence of an electric field;

[0089] - a compound that generates one or more cations in the solvent or in the solvent in the presence of an electric field, the cations being independently selected from the group consisting of: Li + 、Na + , K + Mg 2+ , Ca 2+ , Cu 2+ , ammonium, diazonium and carbocations; or compounds containing one or more functional groups that generate said cations in said solvent or in said solvent and in the presence of an electric field; and

[0090] - a compound that generates one or more anions in the solvent or in the solvent and in the presence of an electric field, the anions being independently selected from the group consisting of chloride, iodide, bicarbonate, hydroxide, alkoxide, phenoxide and carboxylate; a compound comprising one or more functional groups that generate the anions in the solvent or in the solvent and in the presence of an electric field.

[0091] Thus, graphene can be bonded to cations that can be generated by the substance used (e.g., carbonium ions of diazonium salts) or to anions (e.g., I - or HCO3 - ) reaction, depending on whether the nucleophilicity of graphene is higher or lower compared to the generated anion.

[0092] In a more preferred embodiment of the present invention, the method for obtaining covalently modified graphene comprises:

[0093] i. applying a voltage of at least 10 volts to generate an electric field in the region of the graphene surface in the presence of water and a substance that generates one or more ions and / or one or more free radicals in water or in water and in the presence of an electric field generated by the voltage;

[0094] ii. optionally washing the graphene, and

[0095] Repeating steps (i) and (ii) successively in other regions of the graphene surface to be modified, using the same substance or other different substances that generate one or more ions and / or one or more free radicals in water or in water and in the presence of an electric field generated by the voltage, the other regions being different from the one or more regions in which steps (ii) and (ii) have been performed;

[0096] and wherein the substance that generates one or more ions and / or one or more free radicals in water or in water and in the presence of an electric field is independently selected from the group consisting of:

[0097] - a compound that generates free radicals in water or in water and in the presence of an electric field, the free radicals being selected from the group consisting of superoxide radicals, alkoxy radicals, aryloxy radicals, peroxy radicals, aminooxy radicals, nitric oxide radicals, nitrogen dioxide radicals, nitrosonium cations, nitrene radicals, halogen radicals, alkyl radicals, aryl radicals, carbene radicals and ketyl radicals; or a compound comprising one or more functional groups that generate said free radicals in water or in water and in the presence of an electric field;

[0098] - a compound that generates a cation in water or in water and in the presence of an electric field, wherein the cation is independently selected from the group consisting of a metal cation, an ammonium cation, a nitronium cation, a carbonium ion, a diazonium cation and a guanidinium cation; or a compound comprising one or more functional groups that generate the cation in water or in water and in the presence of an electric field; and

[0099] - A compound that generates anions in water or in water and in the presence of an electric field, wherein the anions are independently selected from the group consisting of peroxide, alkoxide, phenoxide, carboxylate, azide, carbonate, bicarbonate, borate, bromate, iodate, cyanate, isocyanate, cyanide, nitrate, nitrite, halide, sulfate, hydrogensulfate, thiosulfate, sulfite, hydrogensulfite, sulfide, hydrogensulfide, disulfide, phosphate, phosphite, acid phosphate, dihydrogen phosphate and silicate; or a compound comprising one or more functional groups that generate the anions in water or in water and in the presence of an electric field.

[0100] In another additional preferred embodiment of the present invention, the substance that produces one or more ions in water in the presence of an electric field is independently selected from the group consisting of: NaCl, NaI, NaHCO3, NaOH, CuCl2 / NH3, 2-chlorophenol, variamine blue and sodium salt of fluorescein.

[0101] In one embodiment of the invention, the electric field is generated by electrodes, in particular pointed electrodes, having a negative potential relative to the graphene.

[0102] In another embodiment of the invention, the electric field is generated by electrodes, in particular pointed electrodes, having a positive potential relative to the graphene.

[0103] Preferably, in the method according to the present invention, the graphene surface to be covalently modified is a monolayer.

[0104] More preferably, in the method according to the present invention, the graphene is obtained by chemical vapor deposition (CVD).

[0105] The highest quality graphene is obtained by chemical vapor deposition (CVD) in the form of a single layer, which can be transferred to any substrate using procedures known in the art.

[0106] Although graphene is a two-dimensional material, in some cases, the material used includes more than one layer of graphene. Therefore, for the purposes of the present invention, when graphene is formed from more than one layer, covalent modification can occur in any of the graphene layers.

[0107] In a specific embodiment of the present invention, the method is carried out using a substance soluble in the solvent used, and before step (i), comprises the following steps:

[0108] a. dissolving the substance in a solvent; and

[0109] b. spreading the obtained solution on the graphene surface;

[0110] c. drying the graphene surface to form a film of the substance on the graphene surface,

[0111] so that the substance deposited on the surface of the graphene is dissolved by placing the surface of the graphene in contact with a solvent that is the same as or different from the solvent in step (a), and by applying a voltage of at least 10 volts in the region of the graphene surface in the presence of the solvent according to step (i).

[0112] In one embodiment of the present invention, the solvent used in step (a) and step (i) is the same. In another embodiment of the present invention, the solvent used in step (a) and step (i) is different.

[0113] In one embodiment, the drying step (c) is performed using a heat source.

[0114] In a preferred embodiment of the present invention, the first solvent and the second solvent are both water, and before step (i), the method comprises the following steps:

[0115] a. dissolving a substance that produces one or more ions in water or in water in the presence of an electric field; and

[0116] b. spreading the obtained solution on the graphene surface;

[0117] c. drying the graphene surface so as to form a film of the substance on the graphene surface,

[0118] so that the substance deposited on the graphene surface is dissolved in water by placing the graphene surface in contact with water, and an electric field is generated in the region of the graphene surface in the presence of water by applying a voltage of at least 10 volts in the region of the graphene surface according to step (i).

[0119] For the purpose of the present invention, the graphene covalent modification method according to the present invention is also referred to as a graphene covalent "functionalization etching process". Therefore, for the purpose of the present invention, the device that generates voltage in the graphene covalent modification method of the present invention can be referred to as a graphene functionalization etching device.

[0120] Preferably, before step (i), the pointed electrode is at a distance equal to or less than 10 nm from the region of the graphene surface to be modified. More preferably, the pointed electrode is in contact with the region of the graphene surface to be modified.

[0121] In one embodiment of the present invention, an electric field is generated before the tip of the electrode is at a distance equal to or less than 10 nm, and the electric field is maintained while it is in contact with the region of the graphene surface to be modified.

[0122] Once step (i) is completed, the procedure is optionally repeated in one or more other regions of the graphene surface. In a preferred embodiment, step (i) is repeated in regions adjacent to the first region to be modified and using the same substance, so that the covalent modifications obtained overlap.

[0123] In another preferred embodiment, step (i) is repeated in regions remote from the first region to be modified and with different substances, so that covalent modifications of different chemical nature are obtained in different regions of the graphene surface.

[0124] In an even more preferred embodiment, the pointed electrode is in contact with the graphene surface during the generation of the electric field in step (i). In a more preferred embodiment of this most preferred embodiment, the pointed electrode is in contact with the graphene surface during the generation of the electric field in step (i) by applying additional pressure on the graphene surface to be covalently modified.

[0125] The electrodes are brought close to the sample until they are within a sufficiently close distance of less than 10 nanometers, or in contact with the surface, such that the electric field reduces the energy barrier (activation energy) for forming covalent bonds between the graphene and the ions or radicals generated by the substance.

[0126] The invention also relates to a covalently modified graphene obtainable by the method according to the invention.

[0127] In a preferred embodiment, the graphene to be modified and the covalently modified graphene obtainable according to the method of the present invention is deposited on a copper layer, or a silicon carbide layer, silicon oxide, quartz, silicon, Teflon, polyethylene terephthalate, or a nickel layer, or a copper / nickel alloy, or a platinum / copper alloy, or a copper / nickel / zinc alloy, or a mineral surface, or a polymer surface.

[0128] Preferably, the covalently modified graphene obtainable according to the invention is in the form of a graphene monolayer. More preferably, the graphene monolayer is in the form of a covalently modified graphene obtainable according to the method of the invention obtained by chemical vapor deposition.

[0129] Therefore, the method of the present invention realizes the covalent modification of graphene with different types of functional groups, controlling the position of the groups. In this regard, the covalently modified graphene that can be obtained according to the method of the present invention makes it possible to provide selective active sites for bonding with different molecules. This makes the graphene useful as an anchoring surface of a bioreceptor in a detection device for one or more analytes, such as an ELISA immunoassay device, wherein the covalently modified graphene that can be obtained according to the method of the present invention is used as an anchoring surface for a receptor substance (e.g., an antigen) that can bond with an analyte to be determined. In addition, the control of the position and type of the covalent modification (different functional groups) makes it possible to anchor or fix different receptor substances, thereby enabling the detection of several analytes on a very small surface. Therefore, the miniaturization of multiple analytes or multi-analyte detection platforms can be carried out more economically in a solid phase.

[0130] Therefore, the present invention also relates to the use of the covalently modified graphene obtainable according to the method described herein in an analyte or biosensor detection device.

[0131] Furthermore, the present invention relates to an in vitro method for detecting an analyte in a human or animal sample, wherein the method comprises anchoring the analyte to a bioreceptor anchored to a covalently modified graphene obtainable by the method described herein.

[0132] For the purposes of the present invention, the device or biosensor is an analytical device that can detect a signal corresponding to a molecule present in an in vitro sample of a human or animal, by generating a measurable signal from the sample using a transducer. The device or biosensor comprises a platform, in particular a surface on which are anchored bioreceptor molecules or bioreceptors capable of binding to or recognizing an analyte whose presence is to be measured, and to which the bioreceptor or analyte is preferably attached, or comprises a physically, chemically, electrically, magnetically, optically (e.g., fluorescently or colorimetrically) or thermally detectable molecule. Thus, the bioreceptor must be able to recognize the analyte to be measured, such as an enzyme, an antibody, a DNA, an RNA or a whole cell.

[0133] The conductivity of graphene enables its use in sensors based on fluorescence transfer, electrochemistry, electron transfer, cyclic voltammetry, differential pulse voltammetry, surface plasmon resonance, field effect transistors (FETs), surface enhanced Raman scattering (SERS), and impedance spectroscopy. Thus, one embodiment relates to the use of covalently modified graphene obtainable by the methods described herein in analyte detection devices based on fluorescence transfer or field effect transistors.

[0134] In one embodiment, the device is a biosensor based on fluorescence transfer, electrochemistry, electron transfer, cyclic voltammetry, differential pulse voltammetry, surface plasmon resonance, field effect transistor (FET), and electrochemical impedance spectroscopy.

[0135] In another embodiment, the device is a graphene field effect transistor (FET) biosensor, or a graphene Förster A resonance energy transfer (FRET) biosensor, an electrochemical impedance spectroscopy device, an LDI-MS device, or a cyclic voltammetry device, or a differential pulse voltammetry device, or a surface plasmon resonance device, or an ELISA type device.

[0136] Preferably, the covalently modified graphene forms part of the anchoring surface of the analyte bioreceptor to be detected in the above device.

[0137] In a preferred embodiment, the bioreceptor anchoring surface is a covalently modified graphene surface, which can be obtained according to the method of the present invention, deposited on a copper layer, or a silicon carbide layer, or a nickel layer, or a copper / nickel alloy, or a platinum / copper alloy, or a copper / nickel / zinc alloy, or a mineral surface, or a polymer surface. Preferably, the polymer surface is polymethyl methacrylate. More preferably, the mineral surface is quartz.

[0138] Such a bioreceptor or bioreceptor molecule may be, without limitation, any protein, peptide, nucleic acid, polysaccharide, nucleotide, nucleoside, sugar, antigen, aptamer, hormone or antibody that can be used in diagnostic methods for any type of analyte present in humans or animals.

[0139] In one embodiment, the bioreceptor or bioreceptor molecule is a peptide, protein, nucleic acid, nucleotide, nucleoside, oligonucleotide, sugar or polysaccharide. In another embodiment, the bioreceptor or bioreceptor molecule is a glycoprotein, more particularly an immunoglobulin. In another further embodiment, the bioreceptor or bioreceptor molecule is a nucleic acid, oligonucleotide, polynucleotide, single-stranded DNA, double-stranded DNA, aptamer or RNA.

[0140] More preferably, the covalently modified graphene obtainable according to the method of the present invention comprises functional groups, whereby molecules with bioreceptor functionality can be attached to analyte detection devices.

[0141] In addition, another aspect of the present invention therefore relates to an analyte detection device comprising a covalently modified graphene obtainable according to the method of the present invention. Preferably, the covalently modified graphene forms part of the anchoring surface of a bioreceptor of the analyte to be detected. More preferably, the covalently modified graphene comprises one or more functional groups for anchoring to a bioreceptor of the analyte to be detected.

[0142] Another embodiment of the present invention relates to the use of graphene obtainable according to the method of the present invention in field effect transistors, graphene field effect transistor (FET) biosensors, or surface enhanced Raman scattering (SERS) biosensors, or graphene Forster resonance energy transfer (FRET) biosensors, or electrochemical detection biosensors.

[0143] Example

[0144] Example 1: Functionalization of graphene using various substances

[0145] The device for covalent modification of graphene is equipped with three electromechanical positioners that enable position control in three directions in space and a pointed electrode with an elastic constant of 300N / m. For each measured value of relative humidity and voltage, covalent modification of graphene is carried out in contact mode (electrode in contact with the graphene surface), wherein an oscilloscope is used to detect the moment of electrical contact. After the contact, the tip is compressed to <3μm to avoid damage to the graphene and then separated. It is then moved to an adjacent area and the method is repeated so that the position and size of the modification can be controlled by the overlap of each oxidation area. The voltage remains unchanged throughout the method and is maintained at -25V. The sample used is CVD graphene deposited on a quartz surface. The chemical reagents used in the manufacture are listed in Table 1 (A, B and C):

[0146] Table 1A:

[0147]

[0148] Table 1B:

[0149]

[0150] Table 1C:

[0151]

[0152] Table 1 (A, B and C): Radius values ​​of covalently modified graphene using different inorganic sodium salts (for each series, normalized with the tip electrode radius measured at the lowest relative humidity).

[0153] Prepare aqueous solutions of each reagent at corresponding concentrations (expressed as mM concentration for salts and expressed as % v / v for NH3). Stir the resulting solution or mixture at room temperature for 10 minutes and place in an atomizing or dispersing device. The atomizing device containing the solution is then placed in a device comprising electrodes, which is enclosed in an insulating chamber. Connect the atomizing device until the corresponding relative humidity value is reached. Monitor the relative humidity value with a humidity-temperature dual-purpose meter. When the desired relative humidity (RH) is reached, start the method of the present invention and keep the RH unchanged. Once the method is completed, the insulating chamber is opened and the sample is washed by continuous immersion in ethanol, methanol and water for one minute and dried by heating it on a hot plate at a temperature of 40-70°C for 3h.

[0154] The expansion speed of the covalent modification of graphene is measured as the radius of the spot or region produced using the reagent at the corresponding concentration and humidity. The radius is normalized using the radius of the pointed electrode used (in Table 1, "Normalized r" column), and the radius of the pointed electrode is estimated as the spot produced under ambient RH in the absence of covalently modified expansion (the first row of each group in Table 1). Each measurement is repeated with 10 spots, and the results are averaged. The radius is measured using a differential contrast microscope (DIC).

[0155] Figure 1 A comparison of normalized radii using the sodium salts listed in Table 1 is shown. Three effects are observed:

[0156] (1) For the same reagent and concentration, the spot radius increases with RH. There is a first stage of slow growth (40 < RH < 80%) and a second stage of exponential growth (RH > 85%). This is because the water meniscus between the tip and graphene is larger at higher RH.

[0157] (2) For the same reagents and RH, the covalent modification of graphene enlarges in the case of using higher concentrations of compounds (Table 1), and in any case is larger than the radius in the case of using distilled water (Table 2).

[0158] (3) For the same RH value and reagent concentration, the greater the nucleophilicity of the chemical reagent used, the larger the radius of the spot. In the case of the compounds in Table 1, the nucleophilicity (polarizability) increases in the order of HCO3<Cl<I.

[0159] This nucleophilicity is related to the reactivity of the anionic additive towards its covalent bonding, following the same mechanism as that of the oxidation with distilled water. Results (2) and (3) allow us to conclude that there is a phenomenon of covalent bonding of graphene with anions.

[0160] To confirm these results, experiments were performed with new chemical reagents such as ammonia or 2-chlorophenol, the nucleophiles should again expand the covalent modification relative to distilled water, and other reagents with lower nucleophilicity than water such as ethanol should slowly increase. The results confirm our conclusions. As an illustration, some selected data were collected in Table 2.

[0161]

[0162] Table 2. Radius values ​​normalized to the tip radius for different humidity values ​​using distilled water (blank for estimating velocity increase) and other additives used to confirm the results obtained in Table 1. In the case of ammonia, the radius appears slightly lower than expected given the high concentration used. This is probably due to its volatility, which causes a portion of the volume to be lost during the atomization process.

Claims

1. A method for obtaining covalently modified graphene, the method comprising: i. applying a voltage of at least 10 volts to generate an electric field in the region of the graphene surface in the presence of an atmosphere of water in vapor form or in the form of a fine liquid water atomization, the atmosphere comprising substances in water and optionally generating one or more ions and / or one or more free radicals in the presence of the electric field generated by the voltage, and contacting the graphene surface with the atmosphere comprising the above substances; i. applying a voltage of at least 10 volts to generate an electric field in the region of the graphene surface in the presence of an atmosphere of water in vapor form or in the form of a fine liquid water atomization, the atmosphere comprising the above substances in water; ii. optionally washing the graphene, and Repeating step (i) and optionally step (ii) successively in other regions of the graphene surface to be modified, different from the one or more regions in which steps (i) and (ii) have been performed, using the same substance or other different substances that generate one or more ions and / or one or more free radicals in water and optionally in the presence of an electric field generated by the voltage; and wherein the substances that generate one or more ions and / or one or more free radicals in water and optionally in the presence of an electric field are independently selected from the group consisting of: - a compound that generates free radicals in water and optionally in the presence of an electric field, the free radicals being selected from the group consisting of superoxide radicals, alkoxy radicals, aryloxy radicals, peroxy radicals, aminooxy radicals, nitric oxide radicals, nitrogen dioxide radicals, nitrosonium cations, nitrene radicals, halogen radicals, alkyl radicals, aryl radicals, carbene radicals and ketyl radicals; or a compound comprising one or more functional groups that generate said free radicals in water and optionally in the presence of an electric field; - a compound that generates a cation in water and optionally in the presence of an electric field, wherein the cation is independently selected from the group consisting of a metal cation, an ammonium cation, a nitronium cation, a carbonium ion, a diazonium cation and a guanidinium cation; or a compound comprising one or more functional groups that generate said cation in water and optionally in the presence of an electric field; as well as - A compound that generates anions in water and optionally in the presence of an electric field, wherein the anions are independently selected from the group consisting of peroxide, alkoxide, phenoxide, carboxylate, azide, carbonate, bicarbonate, borate, bromate, iodate, cyanate, isocyanate, cyanide, nitrate, nitrite, halide, sulfate, hydrogensulfate, thiosulfate, sulfite, hydrogensulfite, sulfide, hydrogensulfide, disulfide, phosphate, phosphite, acid phosphate, dihydrogen phosphate and silicate; or a compound comprising one or more functional groups that generate said anions in water and optionally in the presence of an electric field.

2. The method according to claim 1, wherein the voltage applied in the region of the graphene surface to be covalently modified is 20 to 60 volts.

3. A method according to any one of claims 1 or 2, wherein step (i) comprises applying a voltage of 20 to 60 volts between a pointed electrode and the graphene at a distance of less than 7 nm from a region of the graphene surface to generate an electric field in the region of the graphene surface, wherein the voltage is applied in the presence of water containing the substance, and wherein the relative humidity in contact with the graphene surface to be modified is at least 50%. 4 . The method according to claim 3 , wherein the electric field is generated by a pointed electrode having a negative potential relative to the graphene or having a positive potential relative to the graphene.

5. The method according to any one of claims 1 to 2, wherein the graphene surface to be covalently modified has a graphene monolayer.

6. The method according to any one of claims 1 to 2, wherein the graphene surface to be modified is obtained by chemical vapor deposition.

7. The method according to any one of claims 1 to 2, wherein the electric field is generated in a region having a diameter of less than 30 micrometers in the graphene surface to be modified.

8. The method according to any one of claims 1 to 2, wherein the voltage is applied prior to contact with the graphene surface region to be modified and maintained during the contact.

9. The method of any one of claims 1 to 2, wherein the voltage is applied for a period of at least 1 millisecond.

10. The method according to any one of claims 1 to 2, comprising the following steps before step (i): a. dissolving a substance that produces one or more ions in water and optionally in the presence of an electric field; as well as b. spreading the resulting solution on the graphene surface; c. drying the graphene surface so as to form a film of the substance on the graphene surface, Thereby, by placing the graphene surface in contact with water, the substance deposited on the graphene surface is dissolved in the water, and by applying a voltage of at least 10 volts in the area of ​​the graphene surface according to step (i), an electric field is generated in the area of ​​the graphene surface in the presence of water.

11. The method according to any one of claims 1 to 2, wherein the substance that generates one or more ions and / or one or more free radicals in water and optionally in the presence of an electric field is independently selected from the group consisting of: - a compound that generates free radicals in water and optionally in the presence of an electric field, the free radicals being selected from the group consisting of nitrosonium cations, nitrene radicals, halogen radicals, aryl radicals and carbene radicals; or a compound comprising one or more functional groups that generate said free radicals in a solvent and optionally in the presence of an electric field; - a compound that generates cations in water and optionally in the presence of an electric field, wherein the cations are independently selected from the group consisting of: Li + 、Na + , K + Mg 2+ , Ca 2+ and Cu 2+ ; or a compound comprising one or more functional groups that generate said cations in water, optionally in the presence of an electric field; and - a compound that generates anions in a solvent and optionally in the presence of an electric field, wherein the anions are independently selected from the group consisting of chloride, iodide, bicarbonate, alkoxide, phenoxide and carboxylate; or a compound comprising one or more functional groups that generate said anions in water and optionally in the presence of an electric field.

12. The method according to any one of claims 1 to 2, wherein the relative humidity of the dispersion in contact with the graphene surface region to be modified is from 60% to 99%.

13. The method of any one of claims 1 to 2, wherein the electric field is applied by means of a device comprising: a pointed electrode connected to a first pole of a voltage generator, a second pole of the generator being connected to the graphene; a head configured to hold the pointed electrode; a dielectric separator accommodating the head and configured to electrically isolate the electrode from the rest of the device; a positioner secured to the electrical separator and configured to move the head in three dimensions in space; and a damping system configured to allow vertical movement of the head at a predetermined pressure and to dampen displacements caused by irregularities of a surface over which the head moves; wherein in step (ii), the electric field is generated using a pointed electrode of the device; and a container having a solvent, the container being configured to generate a dispersion of the solvent.

14. A covalently modified graphene obtained by the method according to any one of claims 1 to 13.

15. Use of the covalently modified graphene according to claim 14 in an analyte detection device.

16. Use according to claim 15, wherein the covalently modified graphene forms part of an anchoring surface for a bioreceptor molecule of an analyte to be detected.

17. The use according to any one of claims 15 or 16, wherein the covalently modified graphene comprises functional groups for bonding to bioreceptor molecules in the analyte detection device.

18. An analyte detection device, comprising the covalently modified graphene according to claim 14.

19. The analyte detection device of claim 18, wherein the covalently modified graphene forms part of an anchoring surface for a bioreceptor molecule of an analyte to be detected.

20. The analyte detection device according to any one of claims 18 or 19, wherein the covalently modified graphene comprises one or more functional groups for anchoring to a bioreceptor molecule of an analyte to be detected.

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