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    • 专利摘要:
    Covalent functionalization of graphene. A process for obtaining covalently modified graphene is described. Likewise, the covalently modified graphene obtainable through said production process is described, as well as its use as an anchoring surface for bioreceptors in biosensor devices. Finally, a biosensor device comprising the covalently modified graphene obtainable according to the process of the invention is described. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2848798A1
    申请号:ES202030111
    申请日:2020-02-11
    公开日:2021-08-11
    发明作者:Castillo Angel Luis Alvarez；Sánchez Sergio Javier Quesada；Rodrigo Fernando Borrás；Párraga Carmen Coya
    申请人:Universidad Rey Juan Carlos；
    IPC主号:
    专利说明:

    [0004] TECHNICAL FIELD OF THE INVENTION
    [0006] The present invention describes a process for obtaining covalently modified graphene. Likewise, the present invention relates to the covalently modified graphene obtainable through said production process, as well as its use as an anchoring surface for bio-receptors in biosensor devices. Finally, the present invention relates to a biosensor device comprising the covalently modified graphene obtainable according to the process of the invention.
    [0008] STATE OF THE ART
    [0010] Currently, graphene has been functionalized, both covalently and non-covalently, (Plutnar et al, J. Mater. Chem. C., 2018, 6, 6082-6101) with a variety of molecules, even reaching the anchor of antibodies, but in most cases they have been reactions in the liquid phase (in solution), where the graphene used is in the form of flakes in suspension or as part of some submerged electrode. This has led, in the last 5 years, to a large number of publications (Georgakilas et al., Chem. Rev. 2016, 116, 5464-5519). There are also publications in which said functionalization of graphene is carried out outside a dissolution tank, in solid phase on previously deposited layers of graphene, or graphene oxide. However, these methods do not allow a positional control of the modifications made to the surface of graphene, or graphene oxide, without differentiating separate regions or zones within said layers (Morales-Narvaez et al., Adv. Mater., 2018 , 1805043; Sandoval et al., Chem. Commun., 2019, 55, 12196-12199). The oxidation lithography technique, known as local anodic oxidation (LAO), or scanning probe oxidation lithography (o-SPL), does allow positional control. However, this technique has so far been applied (1) only at the nanometric scale by adapting microscopes that work at this scale, being limited to very small and impractical areas, on the order of nm (oxidation lithography, R. García et al., Carbon 2018, 129, 281-285); and (2) only for oxidation purposes, not extended to other types of functionalizations or chemical reactions.
    [0012] Very recently, a way to functionalize graphene in differentiated areas has been published, through a process with irradiation with light (Valenta et al., Agew. Chem. Int. Ed, 2019, 58, 1324-1328). In this method, the reaction is favored only in those areas where a mask is used, and the modified region can be located at various positions on the surface of the sample. However, this process is very limited to certain specific reactions capable of being carried out by irradiation with light in the blue or UV region, such as those known as Mitsunobu reactions.
    [0014] The functionalization that meets the characteristics of positional control and versatility in terms of the nature of the reagent used in a material such as graphene would allow to provide a physical substrate in which to anchor different molecules or functional groups that, for example, can be biomarkers or bio-receptors. (such as antibodies), in a single miniaturized platform, making said assays much more compact in size, and providing a system that allows the detection of analytes in a reduced space, compatible to develop lab-on-chip systems, that is, systems that integrate and automate various laboratory techniques in a chip-sized device with a maximum size of a few square centimeters.
    [0016] BRIEF DESCRIPTION OF THE INVENTION
    [0018] The present invention refers to a process for obtaining covalently modified graphene, comprising:
    [0020] i. applying a voltage of at least 10 volts generating an electric field in an area of the graphene surface in the presence of a solvent and a substance that, in said solvent, or in said solvent and in the presence of the electric field generated by said voltage, gives rise to one or more ions and / or to one or more radicals;
    [0022] ii. wash the graphene and / or, optionally, repeat the previous step successively in another area of the graphene surface to be modified, different from the area, or areas where the previous step has already been carried out, in the presence of the same solvent or a different solvent and, with the same substance, or a different substance than, in the solvent used, or in the solvent used and in the presence of the electric field generated by said voltage, it also gives rise to one or more ions and / or to one or more radicals.
    [0024] The present invention also relates to a process for obtaining covalently modified graphene, comprising:
    [0026] i. apply an electric voltage of at least 10 volts generating an electric field in an area of the graphene surface in the presence of water and a substance that, in water, or in water and in the presence of the electric field generated by said voltage, gives rise to one or more ions and / or one or more radicals;
    [0027] ii. wash the graphene and / or, optionally, repeat the previous step successively in another area of the graphene surface to be modified, different from the area, or areas in which the previous step has already been carried out, with the same u a different substance that, in water, or in water and in the presence of the electric field generated by said voltage, gives rise to one or more ions and / or one or more radicals.
    [0029] Likewise, the present invention relates to covalently modified graphene, obtainable according to the process of the present invention.
    [0031] Another aspect of the present invention relates to the use of covalently modified graphene, obtainable according to the process of the present invention, in an analyte detector device.
    [0033] Finally, the present invention relates to an analyte detector device comprising covalently modified graphene, obtainable according to the process of the present invention.
    [0035] BRIEF DESCRIPTION OF THE FIGURES
    [0036] FIG. 1. Comparison of relative humidity versus normalized radius of the graphene surface area covalently modified with NaCl, NaHCO3, NaI and 2-chlorophenol (all at 40 mM) versus a control with distilled water.
    [0038] DETAILED DESCRIPTION
    [0040] The present invention describes a process for obtaining covalently modified graphene, which comprises: (i) applying an electrical voltage of at least 10 volts to generate an electric field in an area of the graphene surface in the presence of a solvent and a substance that , in said solvent, or in said solvent and in the presence of the electric field generated by said voltage, it gives rise to one or more ions and / or one or more radicals.
    [0042] In a preferred embodiment of the invention, once step (i) has been carried out, said step (i) is repeated, successively, in another area of the graphene surface to be modified, different from the area or areas in the (s) that has already been carried out, in the presence of the same solvent or a different solvent and, with the same substance, or a different substance than, in the solvent used, or in the solvent used and in the presence of the electric field generated by said voltage, gives rise to one or more ions and / or one or more radicals.
    [0043] In one embodiment, the method of obtaining comprises:
    [0045] i. applying an electric voltage of at least 10 volts to generate an electric field in an area of the graphene surface in the presence of a solvent and a substance that, in said solvent, or in said solvent and in the presence of the electric field generated by said voltage , gives rise to one or more ions and / or one or more radicals; and
    [0047] ii. wash the graphene.
    [0049] In one embodiment of the invention, once step (ii) has been carried out, step (i) and, optionally, step (ii) are repeated, successively, in another area of the graphene surface to be modified, different from the area , or areas in which it has already been carried out, in the presence of the same solvent or a different solvent and, with the same substance or a different substance than, in the solvent used, or in the solvent used and in the presence of the electric field generated by said voltage, gives rise to one or more ions and / or one or more radicals.
    [0051] Thus, the method of the present invention comprises:
    [0053] i. applying a voltage of at least 10 volts generating an electric field in an area of the graphene surface in the presence of a solvent and a substance that, in said solvent, or in said solvent and in the presence of the electric field generated by said voltage, gives rise to one or more ions and / or to one or more radicals;
    [0055] ii. wash the graphene and / or, optionally, repeat the previous step successively in another area of the graphene surface to be modified, different from the area, or areas where the previous step has already been carried out, in the presence of the same solvent or a different solvent and, with the same substance, or a different substance than, in the solvent used, or in the solvent used and in the presence of the electric field generated by said voltage, it also gives rise to one or more ions and / or to one or more radicals.
    [0057] Therefore, the process of the invention comprises the covalent modification in a zone of the graphene, according to step (i), and in which said step (i) can be repeated, comprising, between each step (i), a step (ii) in which the graphene is washed, prior to carrying out step (i) again, or not.
    [0059] That is, the process of the invention comprises a step (i), optionally a step (ii), where step (i), and optionally step (ii), is repeated successively in several areas of the graphene surface, in the presence of the same solvent or different solvents, and of the same substance or of a different substance, each time step (i) is carried out.
    [0060] For the purposes of the present invention, the term "comprises" indicates that it includes a group of characteristics, but does not exclude the presence of other characteristics, as long as the presence of the other characteristics does not make the invention impractical. Furthermore, the terms "consists de ”," contains "," includes "," has "," encompasses "and synonyms of these terms must be interpreted in the same way as the term" comprises ".
    [0062] Additionally, for the purposes of the present invention, the term "comprises" may be replaced by any of the terms "consists of", "consists of", "consists substantially of" or "consists substantially of". Thus, when the term "consists of" comprises ”refers to a group of technical characteristics A, B and C, it should be interpreted that it may additionally include other technical characteristics in addition to technical characteristics A, B and C, as long as the presence of the other characteristics does not make the invention impractical. , but it can also be interpreted as comprising only said characteristics A, B and C or, substantially said characteristics A, B and C and, therefore, the term "comprises" referring to a group that comprises characteristics A, B and C must be interpreted to include a group consisting of features A, B, and C, or consisting substantially of features A, B, and C.
    [0064] The term "solvent" refers to a chemical compound that, when mixed with another compound or substance, forms a homogeneous mixture, constituting a single phase, that is, it forms a mixture in which the physical properties remain homogeneous, understood as "homogeneous" part that through which all the intensive properties (those that do not depend on the amount of substance) are constant. In general, the amount of solvent in the mixture is greater than that of the other substance, which is generally called solute.
    [0066] The term "substance that, in a solvent, or in a solvent and in the presence of an electric field, gives rise to one or more ions and / or one or more radicals" refers to a substance that, in contact with said solvent and , optionally in the presence of an electric field (generated by the application of a voltage), generates at least one ion and / or at least one radical. Said term thus includes both ionic substances (that is, substances that comprise ionic bonds) and , when dissolved in said solvent, they release the cations and anions that make it up and, also comprise substances with covalent bonds that, when in contact with said solvent, or when in contact with said solvent and in the presence of an electric field, generate the formation of at least one ion and / or the formation of at least one radical. That is, in some cases, the substance used gives rise to ions or radicals simply by being dissolved in the solvent used, while in other cases it will be the presence of an electric field is necessary for said substance dissolved in said solvent to give rise to ions or radicals.
    [0068] Thus, the solvent, optionally with the effect of the electric field generated by applying a voltage, produces the formation of ions (for example, the generation of a Cl- anion and the Na + cation, when the NaCl salt is in the presence of water as solvent or, for example, the formation of the ammonium cation R-NH3 + of an amine R-NH2, through the union of a proton H + to said amine in the presence of water), or radicals, when a bond is broken that generates species with unpaired electrons.
    [0070] For the purposes of the present invention, the term "radical" refers to that chemical species that possesses one or more unpaired electrons. Examples of radicals, for the purposes of the present invention, include, but are not limited to: superoxide radical (O2- ), diradical oxygen
    [0071] (O2 "), hydroxyl radical (HO-), alkoxyl radical (RO-), aroxyl radical (ArO-), peroxyl radical (ROO-), aminoxyl radical (RNO-) of which the reagent TEMPO ( tetramethylpyridyl-N-oxyl) and its derivatives, nitrogen monoxide radical (NO-), nitrogen dioxide radical (NO2-), nitrosonium cation (NO2 +), nitrene radical (RN--), halogen radical (X-), radical alkyl (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.
    [0073] On the other hand, for the purposes of the present invention, the term "ion" refers to an electrically charged atom or group of atoms.
    [0075] The process of the invention is carried out in the presence of an electric field generated by applying a voltage. In some cases, said electric field is also necessary for the formation of ions and / or radicals of the substance used (for example, for the formation of carbocations, or other types of ions or radicals that require greater energy for their formation. in the presence of said solvent).
    [0077] The covalent modification of graphene occurs, as a result of the process of the invention, through the formation of new bonds between one or more carbon atoms of graphene and one of the atoms of the ion, or of the radical, generated by said substance, forming functional groups covalently linked to carbon atoms of graphene, where the covalent bond occurs between one of the carbon atoms of graphene and an atom of the ion, or of the radical of the substance used.
    [0078] In a more preferred embodiment, the electrical voltage from step (ii) is applied by a pointed electrode connected to one of the poles of an electrical voltage generator. For the purposes of the present invention the term "electrical voltage generator" is equivalent to a voltage source. That is, the voltage is "applied" externally by a voltage generator (or voltage source, or voltage source), between two poles or electrodes, generating an electric field whose lines of force go from one pole to another. When one of the electrodes ends in a point, the generated electric field is concentrated at that point.
    [0080] For the purposes of the present invention, a "tip" electrode is an electrode whose tip has a diameter of less than 30 microns.
    [0082] In an even more preferred embodiment, the electric field is applied by means of a device comprising: a pointed electrode connected to a first pole of an electric voltage generator, while a second pole of said generator is connected to graphene; a head configured to hold the pointed electrode; a dielectric separator that houses said head and that is configured to electrically isolate the electrode from the rest of the device; a positioner that is attached to the electrical separator and that is configured to move the head in three dimensions of space; and a damping system configured to allow vertical movement of the head with a predetermined pressure and to damp the displacements resulting from the irregularities of the surface through which the head moves; wherein in step (ii) the electric field is generated with the pointed electrode of said device; and a container with solvent configured to generate a dispersion of said solvent. In particular such a device is called a functionalization lithography device.
    [0084] In one embodiment of the invention the electric field is generated by an electrode, in particular a pointed electrode, with negative electric potential with respect to graphene.
    [0086] In another embodiment of the invention, the electric field is generated by an electrode, in particular a pointed electrode, with positive electric potential with respect to graphene.
    [0088] For example, it can be explained theoretically that, when the electric field is generated by an electrode with a negative potential with respect to graphene (cathode), the graphene acts as a positive electrode (anode) and attracts the negative charges of the species generated by the field. electrical and binds to an atom of said species rich in negative charge, by means of a nucleophilic addition reaction. On the other hand, also by way of example, when the electric field is generated by an electrode with positive potential with respect to graphene, the cloud of n electrons from graphene attract the positive charges of the generated species and bind to an atom of that species rich in positive charge by an electrophilic addition reaction.
    [0090] Graphene is a single, two-dimensional layer of carbon atoms joined to form hexagons by carbon-carbon bonds that present sp2 hybridization and forming an n cloud of electrons. Due to its structure, graphene behaves like a semi-metal with a very high charge mobility and has unique properties, among which are hardness, elasticity, great conductivity with a very low resistance but a chemical structure, in principle, very stable (or not very reactive) against gases. This makes graphene a very interesting material in many technical applications. For the purposes of the present invention, in the event that the graphene to be covalently modified in the process of the invention consists of more than one graphene monolayer, the term "a graphene surface" refers to any of the graphene monolayers. Although graphene is a two-dimensional material, in some cases, the material used comprises more than one layer of graphene. Thus, for the purposes of the present invention, when graphene is made up of more than one layer, covalent modification can occur in any of the graphene layers The formation of a covalent bond between a carbon atom and a functional group implies the transition to an sp3 hybridization of the reacting carbon atom, which gives rise to materials with properties and reactivity different from that of the initial graphene.
    [0092] In one embodiment the covalent modification is a monotopic modification (that is, it occurs only on one of the two faces of the graphene surface. In another embodiment, the covalent modification is a ditopic modification (that is, it occurs on either of the two graphene surface faces).
    [0094] For the purposes of the present invention, the solvent comprising the substance which, in said solvent, or in said solvent and in the presence of an electric field, gives rise to one or more ions and / or one or more radicals, is in contact with the graphene surface in the form of "atomization" or "vaporization" or "dispersion", without requiring the use of a heat source, in such a way that an atmosphere is generated with said solvent and substance in contact with the surface graphene. In this way, the terms "atomization", "vaporization" or "dispersion" are used interchangeably, for the purposes of the present invention, and indicate the generation of a homogeneous phase, in terms of its physical properties, containing said solvent. and said substance, without the use of a heat source being necessary.
    [0095] Thus, the process of the invention allows a positional control of the covalent anchoring of functional groups and, since it is an electrical procedure, based on the application of a localized electrical voltage, it allows working with a great variety of substances that generate ions or radicals. when in contact with said solvent, or that they generate said ions or radicals when, in addition, they are subjected to an electric field.
    [0097] For the purposes of the present invention, the term "functional group" refers to an atom or set of atoms that, attached to the molecular structure of a compound with a greater number of atoms, and arranged according to a determined sequence of bonds, have chemical properties Therefore, in the present invention said functional group refers to an atom or set of atoms of an ion or radical of a substance that, in a solvent, or in a solvent and in the presence of an electric field, gives gives rise to one or more ions and / or one or more radicals, and which, during the process of the present invention, forms a covalent bond with a carbon atom of graphene.
    [0099] In a preferred embodiment of the process according to the present invention, the electric field is applied in a circular area of the graphene surface with a diameter less than 15 microns.
    [0101] In a preferred embodiment the electrical voltage is applied at a distance from the graphene surface less than 10 nm, more preferably at a distance less than 7 nm. In an even more preferred embodiment the electrical voltage is applied between a pointed electrode and the graphene, keeping said electrode in contact with the surface of the graphene.
    [0103] In one embodiment of the method according to the present invention, the electrical voltage applied in the area of the graphene surface to be modified is between 10 and 100 volts; more preferably between 20 and 60 volts.
    [0105] Therefore, a preferred embodiment of the invention comprises (i) applying an electrical voltage at a distance less than 10 nm, more preferably less than 7 nm, and even more preferably between a pointed electrode and the graphene, keeping said electrode in contact with an area of the graphene surface, in which said voltage is between 10 and 100 volts, more preferably between 20 and 60 volts, to generate an electric field in said area of the graphene surface in the presence of a solvent and a substance that, in said solvent, or in said solvent and in the presence of the electric field generated by said voltage, gives rise to one or more ions and / or one or more radicals;
    [0106] In a preferred embodiment the electrical voltage is applied for a period of at least 1 millisecond (1 ms). In a more preferred embodiment the electrical voltage is generally applied for a period of between 1 ms and 30 seconds.
    [0108] In a preferred embodiment of the invention, the solvent is water and the invention relates to a process for obtaining covalently modified graphene, comprising:
    [0109] i. apply an electric voltage of at least 10 volts to generate an electric field in an area of the graphene surface, in the presence of water comprising a substance that, in water, or in water and in the presence of the electric field generated by said voltage, gives rise to one or more ions and / or one or more radicals;
    [0111] ii optionally, wash the graphene.
    [0113] In one embodiment of the invention, step (i), and optionally step (ii), are repeated successively in other area (s) of the graphene surface to be modified, other than the area, 0 areas in the one (s) that has already been made, with the same substance or a different substance than, in water, or in water and in the presence of the electric field generated by said voltage, also gives rise to one or more ions and / or one or more more radical.
    [0115] Thus, in a preferred embodiment of the invention, the covalent modification can be carried out, preferably, in the presence of water vapor, or of a fine atomization of liquid water comprising a substance that, in water, or in water and in presence of an electric field, gives rise to at least one ion and / or at least one radical. Thus, the graphene surface is in contact with an atmosphere, called a working atmosphere, rich in water, which contains said substance.
    [0117] In a preferred embodiment of the invention, the solvent is water and step (i) of the procedure comprises:
    [0119] 1 apply an electrical voltage of between 10 and 100 volts, more preferably between 20 and 60 volts, between a pointed electrode and the graphene (which acts as the other electrode), at a distance 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 comprising a substance that, in water, or in water and in the presence of the electric field generated by said voltage, it gives rise to one or more ions and / or one or more radicals, and in which the relative humidity in contact with the graphene surface to be modified is of at least 40%, more preferably of at least 50% and even more preferably of between 60% and 95%.
    [0121] For the purposes of the present invention, the term "atmosphere" or working atmosphere "refers to the phase of solvent in direct contact with the surface to be modified according to the process of the present invention. Relative humidity values when water is the solvent used, according to the present document, they are expressed in%.
    [0123] Thus, the application of said electric voltage generates an electric field in an area of the graphene surface, in the presence of a solvent comprising a substance which, in said solvent, or in said solvent and in the presence of an electric field, gives rise to one or more ions and / or one or more radicals, generates the covalent modification of graphene by forming one or more covalent bonds between carbon atoms of graphene and one or more atoms of said substance or said solvent, in an area that has, preferably, a diameter of about 1 to 12 times the size of the area in which the electrical voltage has been generated. Thus, if, for example, an electric field has been generated in a circular area of 10 microns in diameter, the covalent modification will occur in an area with a diameter of 10 to 120 microns.
    [0125] Even more preferably, the solvent is water and step (ii) comprises applying an electrical voltage of between 20 and 60 volts, in contact with an area of the graphene surface, in the presence of water comprising a substance that, in water, or in water and in the presence of an electric field gives rise to at least one ion and / or at least one radical, and in which the relative humidity in contact with the graphene surface to be modified is at least 50% and more preferably between 60% and 99%.
    [0127] In another embodiment of the invention the solvent is a polar solvent. Preferably a polar protic solvent. Most preferably the solvent is water, a carboxylic acid, an alcohol, an amine, or a hydrogen halide. Even more preferably, the solvent is water.
    [0128] In this way, with the process of the invention, graphene can be functionalized, or covalently modified, in extensive regions, of mm2 or greater, with different chemical species, being able to covalently join different groups, or the same, in different zones separated from each other of the surface of the graphene by generating a localized electric field in said areas in the presence of said solvent, or of another different solvent that contains the same substance, or with or different substances than in the solvent used, or in the solvent used and in the presence of an electric field, they generate ions and / or radicals, and that form bonds with one or more carbon atoms of graphene.
    [0129] For example, when the solvent is water, the inorganic salts are ionized in water, a phase of fine liquid atomization is generated in which these salts are transported and respond to the electric fields generated. Thus, when a localized electric field is applied in the presence of said phase of fine liquid atomization of water, enriched with a salt, the average electric field at the junction of the ionic or radical species corresponding to graphene. To anchor two substances, one in each zone, the procedure is carried out sequentially: first a first substance is used in a first zone and the electric field is applied, producing the modification of graphene with said first substance in said first zone. Next, the graphene is washed and the procedure is repeated working on a different area with a substance and solvent, the same or different from the first.
    [0131] In one embodiment of the invention, the graphene is washed with DMF (dimethylformamide), DMSO (dimethylsulfoxide), methanol, toluene, or ethanol, preferably by performing more than one wash with one or more solvents.
    [0133] The process of the invention is not only possible with salts, specifically with inorganic salts, but it can be carried out with any molecule, complex or substance that, in water, or another appropriate solvent, generates ions or radicals, requiring, or no, the presence of the electric field generated during the procedure, and without necessarily being a molecule with ionic bonds.
    [0135] Thus, in a preferred embodiment of the process according to the present invention the substance which, in a solvent, or in a solvent and in the presence of an electric field, gives rise to one or more ions and / or one or more radicals, is selects independently from the group consisting of:
    [0137] - a compound that, in said solvent, or in said solvent and in the presence of an electric field gives rise to a radical selected from the group consisting of: superoxide radical, oxygen diradical, hydroxyl radical, alkoxyl radical, aroxyl radical, peroxyl radical, aminoxyl radical, nitrogen monoxide radical, nitrogen dioxide radical, nitrosonium cation, nitrene radical, halogen radical, alkyl radical, aryl radical, carbene radical and cetyl radical; or a compound comprising one or more functional groups that in said solvent, or in said solvent and in the presence of an electric field, give rise to said radical;
    [0139] - a compound that in said solvent, or in said solvent and in the presence of an electric field gives rise to a cation independently selected from the group consisting in: a metal cation, an ammonium cation, a nitronium cation, an iminium cation, an iodonium cation, a carbocation, a diazonium cation and a guanidino cation; or a compound comprising one or more functional groups that in said solvent, or in said solvent and in the presence of an electric field, give rise to said cation; and
    [0141] - a compound that in said solvent, or in said solvent and in the presence of an electric field, gives rise to an anion independently selected from the group consisting of: peroxide, hydroxide, alkoxide, phenolate, carboxylate, carbanion, azide, carbonate, bicarbonate , borate, bromate, iodate, cyanate, isocyanate, cyanide, nitrate, nitrite, halide, sulfate, bisulfate, thiosulfate, sulfite, bisulfite, sulfide, bisulfide, disulfide, phosphate, phosphite, acid phosphate, diacid phosphate and silicate; or a compound comprising one or more functional groups that in said solvent, or in said solvent and in the presence of an electric field, give rise to said anion.
    [0143] In this way, when an electric field is generated, at a distance of the order of nm from the area of the graphene surface, in the presence of a solvent comprising said substance, one or more covalent bonds are produced between one or more carbon atoms. of graphene and one of the atoms of said substance, forming functional groups covalently bonded to carbon atoms of graphene, where the covalent bond occurs 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 aryldiazonium salt (aryl-N2 + X-), it could be generated, in aqueous solution and in the presence of an electric field, N2 and an aryl carbocation, or an aryl radical, that would react with a carbon of the graphene forming a bond of said carbon atom and a carbon atom of the aryl group, forming, then, an aryl functional group in the graphene.
    [0145] In a preferred embodiment of the invention, the substance that gives rise in the solvent, or in the solvent and in the presence of an electric field, to one or more ions is independently selected from the group consisting of:
    [0147] - a compound that, in said solvent, or in said solvent and in the presence of an electric field gives rise to a radical selected from the group consisting of: hydroxyl radical, alkoxyl radical, aroxyl radical, nitrosonium cation, nitrene radical, halogen radical, aryl radical and carbene radical; or a compound comprising one or more functional groups that in said solvent, or in said solvent and in the presence of an electric field, give rise to said radical;
    [0148] - a compound that, in said solvent, or in said solvent and in the presence of an electric field, gives rise to one or more cations independently selected from the group consisting of: Li +, Na +, K +, Mg2 +, Ca2 +, Cu2 + ammonium, diazonium and carbocation; or a compound comprising one or more functional groups that in said solvent, or in said solvent and in the presence of an electric field, give rise to said cation; and
    [0149] - a compound that, in said solvent, or in said solvent and in the presence of an electric field, gives rise to one or more anions independently selected from the group consisting of: chloride, iodide, bicarbonate, hydroxide, alkoxide, phenolate and carboxylate; a compound comprising one or more functional groups that in said solvent, or in said solvent and in the presence of an electric field, give rise to said anion.
    [0151] Thus, graphene could react with a cation (for example, the carbocation of diazonium salts) or with an anion (for example, I- or HCO3-) that could be generated by the substance used, depending on whether the graphene is more or less nucleophilic than the anion generated.
    [0152] More preferably said solvent is water.
    [0154] In yet another preferred embodiment of the invention, the substance that gives rise to 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 , variamin blue and the sodium salt of fluorescein.
    [0156] In one embodiment of the invention the electric field is generated by an electrode, in particular a pointed electrode, with negative electric potential with respect to graphene.
    [0158] In another embodiment of the invention, the electric field is generated by an electrode, in particular a pointed electrode, with positive electric potential with respect to graphene.
    [0160] Preferably, in the process according to the present invention, the graphene surface to be covalently modified is a monolayer.
    [0162] More preferably, in the process according to the present invention, graphene is obtained by chemical vapor deposition (CVD).
    [0164] The highest quality graphene is obtained in the form of monolayers by means of chemical vapor deposition (CVD), which can be transferred, with procedures known in the state of the art, onto any substrate.
    [0165] Although graphene is a two-dimensional material, in some cases, the material used comprises more than one layer of graphene. Thus, for the purposes of the present invention, when graphene is made up of more than one layer, covalent modification can occur in any of the graphene layers.
    [0167] In a particular embodiment of the invention, the process is carried out with substances soluble in the solvent used and comprises, prior to step (i), the steps of: a. dissolving a substance in a solvent; and
    [0168] b. spreading the solution obtained on the graphene surface;
    [0169] c. drying the graphene surface by forming a film of said substance on the graphene surface,
    [0171] so that, by contacting the graphene surface with a solvent equal to or different from the solvent of step (a), the substance deposited on the graphene surface dissolves and, by applying an electrical voltage of at least 10 volts to said graphene surface zone according to step (i) in the presence of said solvent.
    [0173] In one embodiment of the invention, the solvent used in step (a) and step (i) are the same. In another embodiment of the invention, the solvent used in step (a) and step (i) are different.
    [0175] In one embodiment, drying step (c) is carried out by using a heat source.
    [0177] In a preferred embodiment of the invention, both the first solvent and the second solvent are water and the process comprises, prior to step (i), the steps of: a. dissolving a substance that, in water, or in water and in the presence of an electric field, gives rise to one or more ions; and
    [0178] b. spreading the solution obtained on the graphene surface;
    [0179] c. drying the graphene surface so that a film of graphene is formed on the graphene surface,
    [0181] so that when the graphene surface comes into contact with the water, the substance deposited on the graphene surface dissolves in the water, and when an electrical voltage of at least 10 volts is applied in said area of the graphene surface, According to step (i), an electric field is generated in said area of the graphene surface in the presence of water.
    [0182] For the purposes of the present invention, the graphene covalent modification process according to the present invention is also called graphene covalent "functionalization lithography process". Thus, for the purposes of the present invention, the device that generates the electrical voltage in the graphene covalent modification process of the present invention may be called a graphene functionalization lithography device.
    [0184] Preferably, prior to step (i), the pointed electrode is approached at a distance of equal to or less than 10 nm from the area of the graphene surface to be modified. More preferably, the pointed electrode is in contact with the area of the graphene surface to be modified.
    [0186] In one embodiment of the invention, the electric field is generated before bringing the electrode tip closer to a distance equal to or less than 10 nm, and is maintained when it comes into contact with the area of the graphene surface to be modified.
    [0188] Once step (i) is completed, said procedure is repeated, optionally in one or more other areas of the graphene surface. In a preferred embodiment, step (i) is repeated in a zone adjacent to the first zone to be modified and with the same substance, so that the covalent modifications obtained overlap.
    [0190] In another preferred embodiment, step (i) is repeated in an area remote from the first area to be modified and with a different substance, so that covalent modifications of different chemical nature are obtained in separate areas of the graphene surface.
    [0192] 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 more preferred embodiment, the tip electrode is in contact with the graphene surface during the generation of the electric field of step (i) by exerting additional pressure on the graphene surface to be covalently modified.
    [0194] The electrode approaches the sample to a sufficiently close distance, less than 10 nanometers, or making contact with the surface, so that the electric field reduces the energy barrier (activation energy) of formation of the covalent bond between graphene and the ion or radical generated by the substance.
    [0196] The present invention also relates to covalently modified graphene, obtainable according to the process described in the present invention.
    [0197] In a preferred embodiment, the graphene to be modified, and the covalently modified graphene, obtainable according to the process described in the present invention, are deposited on a copper layer, or a layer of silicon carbide, silicon oxide, quartz. , silicon, teflon, polyethylene terephthalate or on a nickel layer, or on a copper / nickel alloy, or on a platinum / copper alloy, or on a copper / nickel / zinc alloy, or on a mineral surface or on a surface polymeric.
    [0199] Preferably, the covalently modified graphene obtainable according to the present invention is in the form of a graphene monolayer. More preferably in the form of a graphene monolayer obtained by chemical vapor deposition which is covalently modified, obtainable according to the process of the invention.
    [0200] By means of the process of the invention, therefore, the modification of graphene in a covalent manner with different types of functional groups is achieved, controlling the position of said groups. In this sense, the covalently modified graphene, obtainable according to the process described in the present invention, makes it possible to provide selective active sites for binding to different molecules. This makes said graphene useful as an anchoring surface for bio-receptors in detecting devices for one or more analytes, such as ELISA immunoassay devices, in which the covalently modified graphene, obtainable according to the method of the invention, it serves as an anchoring surface for receptor substances (eg antigens) that are capable of binding to analytes to be determined. Furthermore, the control of the position and type of covalent modifications (different functional groups) allows the anchoring or immobilization of different receptor substances, being able to carry out the detection of various analytes on a very small surface area. Thus, it is possible to miniaturize multi-analyte detection platforms, or multi-analyte detection, more economically and in solid phase.
    [0202] Thus, the present invention also relates to the use of covalently modified graphene, obtainable according to the method described herein, in an analyte detector or biosensor device.
    [0204] Furthermore, the present invention relates to an in vitro method for detecting analytes in a sample from a human or animal, in which said method comprises the anchoring of said analytes to bioreceptors anchored to covalently modified graphene, obtainable according to the procedure described in this document.
    [0206] For the purposes of the present invention, said device or biosensor is an analysis device that can detect a signal corresponding to a molecule present in an injected sample. vitro of a human or animal, generating a measurable signal from said sample with the use of a transducer. Said device or biosensor comprises a platform, in particular, a surface on which a bio-receptor molecule, or bio-receptor, is anchored, which is capable of binding or recognizing the analyte whose presence is to be measured, and on which, Preferably, either the bioreceptor or the analyte are bound or comprise a physically, chemical, electrical, magnetic, optical (fluorescence or colorimetric for example) or thermally detectable molecule. Thus, the bio-receptor has to be able to recognize the analyte to be measured, such as an enzyme, an antibody, DNA, RNA or whole cells.
    [0208] The conductive properties of graphene allow its use in sensors based on fluorescence transfer, electrochemistry, electron transfer, cyclic voltammetry, differential pulse voltammetry, surface plasmon resonance, field effect transistors (FET), Raman scattering. surface (SERS) and impedance spectrometry. Thus, one embodiment relates to the use of covalently modified graphene obtainable according to the method described herein in an analyte detector device based on fluorescence transfer or field effect transistors.
    [0210] 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 transistors (FET) and electrochemical impedance spectrometry.
    [0212] In another embodiment the device is a graphene field effect transistor (FET biosensor) biosensor, or a graphene Forster resonance energy transfer biosensor (FRET biosensor), or an electrochemical impedance spectrometry device a LDI-MS, or a cyclic voltammetry device, or a differential pulse voltammetry device, or a surface plasmon resonance device, or an ELISA-type device.
    [0214] Preferably, the covalently modified graphene forms part of the anchoring surface of analyte bioreceptors to be detected in the devices described above.
    [0215] In a preferred embodiment, the bioreceptors anchoring surface is a covalently modified graphene surface, obtainable according to the process described in the present invention, deposited on a copper layer, or a silicon carbide layer, or on a nickel layer, or in a copper / nickel alloy, or in a platinum / copper alloy, or in a copper / nickel / zinc alloy, or on a mineral surface or on a polymeric surface. Preferably said polymeric surface is polymethylmethacrylate. More preferably said mineral surface is quartz.
    [0217] Said bio-receptors, or bio-receptor molecules, can be, without limitation, any protein, peptide, nucleic acid, polysaccharide, nucleotide, nucleoside, sugar, antigen, aptamer, hormone or antibody that can be used for diagnostic methods of any type of analyte present in humans or animals.
    [0219] In one embodiment, the bioreceptor or bioreceptor molecule is a peptide, a protein, a nucleic acid, a nucleotide, a nucleoside, an oligonucleotide, a sugar, or a polysaccharide. In another embodiment, the bioreceptor or bioreceptor molecule is a glycoprotein, more in particular an immunoglobulin. In yet another embodiment the bioreceptor or bioreceptor molecule is a nucleic acid, an oligonucleotide, a polynucleotide, a single stranded DNA, a double stranded DNA, an aptamer or an RNA.
    [0221] More preferably, the covalently modified graphene, obtainable according to the method described in the present invention, comprises functional groups by which molecules with a bio-receptor function can be attached to an analyte detector device.
    [0223] Furthermore, another aspect of the invention therefore relates to an analyte detector device comprising covalently modified graphene, obtainable according to the process described in the present invention.
    [0225] Preferably, the covalently modified graphene forms part of an anchoring surface for the bioreceptors of the analytes to be detected.
    [0227] More preferably, the covalently modified graphene comprises one or more functional groups for its anchorage to bioreceptors of the analytes to be detected.
    [0229] Another embodiment of the invention refers to the use of the graphene obtainable according to the process of the invention in a field effect transistor, in a graphene field effect transistor biosensor (FET biosensor), or in a Raman scattering biosensor. surface augmented (SERS), or in a graphene Foster resonance energy transfer biosensor (FRET biosensor), or in an electrochemical sensing biosensor.
    [0230] EXAMPLES
    [0232] Example 1: Functionalization of graphene with various substances
    [0234] The device used for the covalent modification of graphene is equipped with three electromechanical positioners that allow positional control in the three directions of space, and a pointed electrode with a spring constant of 300 N / m. For each given value of relative humidity and voltage, the covalent modification of graphene is carried out in contact mode (electrode in contact with the graphene surface), using an oscilloscope to detect the moment of electrical contact. The tip is compressed <3 pm after said contact, to avoid damage to the graphene, and is subsequently separated. It is then moved to an adjacent area, and the process is repeated, so as to control the position and size of the overlap modification of individual oxidized areas. The voltage remains constant throughout the process, at -25 V. The samples used are CVD graphene deposited on a quartz surface. The chemical reagents used in manufacturing are listed in Table 1 :
    [0236]
    [0239]
    [0240]
    [0242] Table 1 : Covalently modified graphene radius values (normalized with the radius of the pointed electrode measured at the lowest relative humidity for each series) using different inorganic sodium salts.
    [0244] An aqueous solution of each reagent was prepared at the corresponding concentration (expressed as mM concentrations in the case of salts and except for NH3 which is expressed as% v / v). The resulting solution or mixture was stirred for 10 min. at room temperature and introduced into the atomization or dispersion device. The atomization device containing the solution was then introduced into the device comprising the electrode, which is closed in an insulating chamber. The atomization equipment was connected, until the corresponding relative humidity value was reached. The value of relative humidity was monitored with a hygrometer-thermometer. When the desired relative humidity (RH) was reached, the process of the invention was started, keeping the RH constant. Once the process was finished, the insulating chamber was opened and the sample was washed by consecutive immersion in ethanol, methanol and water for one minute, and allowed to dry by heating on a hot plate for 3h at a temperature between 40-70 ° C.
    [0246] The expansion rate of the covalent modification of graphene was measured as the radius of the spots or zones generated using said reagent at the corresponding concentration and humidity. The radius was normalized with the radius of the tip electrode used (in Table 1 , column " r norm "), which was estimated as the spot generated at ambient RH, where there is no expansion of the covalent modification (first row of each group in Table 1 ) Each measurement was repeated with 10 spots and the results were averaged Radii were measured using a differential contrast microscope (DIC).
    [0247] Figure 1 shows the comparison of the normalized radii using the sodium salts listed in Table 1 . Three effects are seen:
    [0249] (1) For the same reagent and concentration, the radius of the spot increases with RH. There is a first phase of slow growth (40 <RH <80%), and a second phase of exponential growth (RH> 85%). This is because the water meniscus generated between the tip and the graphene is larger at higher RH.
    [0251] (2) For the same reagent and RH, the covalent modification of graphene expands at a higher compound concentration ( Table 1 ), being in any case greater than the radii with distilled water ( Table 2 ).
    [0253] (3) For the same RH value and reagent concentration, the spot radius is greater the greater the nucleophilicity of the chemical reagent used. In the case of the compounds in Table 1 , nucleophilia (polarizability) grows in the order HCO3 <Cl <I.
    [0255] This nucleophilicity is associated with the reactivity of the anion additive with regard to its covalent bond, in the event that a mechanism equivalent to that of oxidation with distilled water is followed. Results (2) and (3) lead us to conclude that there is a phenomenon of covalent bonding to graphene of the anions.
    [0257] To confirm these results, experiments were carried out with new chemical reagents, such as ammonia or 2-chlorophenol, nucleophilic reagents that again had to expand the covalent modification with respect to distilled water, and others such as ethanol, less nucleophiles than water and that therefore had to retard growth. The results confirm our conclusion. By way of example, some selected data is collected in Table 2 .
    [0259]
    [0260] Table 2 . Radius values normalized with the radius of the tip for different humidity values using distilled water (blank to estimate the speed increase), as well as other additives to confirm the results obtained in Table 1 . In the case of ammonia, the radii appear to be somewhat lower than expected in view of the high concentration that was used. This may be due to its volatility, which causes some of the volume to be lost in the atomization process.
    权利要求:
    Claims (20)
    [1]
    1. Procedure for obtaining covalently modified graphene, comprising: i. applying a voltage of at least 10 volts generating an electric field in an area of the graphene surface in the presence of a solvent and a substance that, in said solvent, or in said solvent and in the presence of the electric field generated by said voltage, gives rise to one or more ions and / or to one or more radicals;
    [2]
    2. Process according to claim 1, wherein, it additionally comprises, following step (i):
    ii. wash the graphene.
    [3]
    3. Process according to any of claims 1 or 2, in which step (i) is repeated successively in another area of the graphene surface to be modified, different from the area or areas in which the previous step has been carried out, with the same solvent or another different solvent, and the same substance or another different substance that, in the solvent used, or in the solvent used and in the presence of the electric field generated by said voltage, also gives rise to one or more ions and / or to one or more radicals.
    [4]
    4. Process according to any one of claims 1 to 3, in which the electrical voltage applied in the area of the graphene surface to be covalently modified is between 20 and 60 volts.
    [5]
    5. Process according to any one of claims 1 to 4, in which the graphene surface to be covalently modified is a graphene monolayer.
    [6]
    6. Process according to any of claims 1 to 5, in which the graphene surface to be modified is obtained by chemical vapor deposition.
    [7]
    7. Process according to any of claims 1 to 6, in which the electric field is generated in an area of the graphene surface to be modified with a diameter of less than 30 microns.
    [8]
    Method according to any one of claims 1 to 7, in which the electrical voltage is applied prior to contact with the area of the graphene surface to be modified, and is maintained during said contact.
    [9]
    9. Process according to any of claims 1 to 8, comprising, prior to step (i), the steps of:
    to. dissolving a substance in a solvent; and
    b. spreading the solution obtained on the graphene surface;
    c. drying the graphene surface by forming a film of said substance on the graphene surface,
    so that, by contacting the graphene surface with a solvent equal to or different from the solvent of step (a), the substance deposited on the graphene surface dissolves and, by applying an electrical voltage of at least 10 volts to said zone of the graphene surface according to step (i) in the presence of said solvent, an electric field is generated in said zone of the graphene surface.
    [10]
    10. Process according to any of claims 1 to 9, in which the substance that, in the solvent, or in said solvent and in the presence of an electric field, gives rise to one or more ions and / or to one or more radicals, is independently selected from the group consisting of:
    - a compound that, in said solvent, or in said solvent and in the presence of an electric field gives rise to a radical selected from the group consisting of: superoxide radical, oxygen diradical, hydroxyl radical, alkoxyl radical, aroxyl radical, peroxyl radical, aminoxyl radical, nitrogen monoxide radical, nitrogen dioxide radical, nitrosonium cation, nitrene radical, halogen radical, alkyl radical, aryl radical, carbene radical and cetyl radical; or a compound comprising one or more functional groups that in said solvent, or in said solvent and in the presence of an electric field, give rise to said radical;
    - a compound that, in said solvent, or in said solvent and in the presence of an electric field, gives rise to a cation, wherein said cation is independently selected from the group consisting of: a metal cation, an ammonium cation, a nitronium cation , a carbocation, a diazonium cation and a guanidino cation; or a compound comprising one or more functional groups which, in said solvent, or in said solvent and in the presence of an electric field, give rise to said cation; and
    - a compound that, in said solvent, or in said solvent and in the presence of an electric field, gives rise to an anion, where said anion is independently selected from the group consisting of: peroxide, hydroxide, alkoxide, phenolate, carboxylate, azide , carbonate, bicarbonate, borate, bromate, iodate, cyanate, isocyanate, cyanide, nitrate, nitrite, halide, sulfate, bisulfate, thiosulfate, sulfite, bisulfite, sulfide, bisulfide, disulfide, phosphate, phosphite, acid phosphate, diacid phosphate, and silicate; or a compound comprising one or more functional groups which, in said solvent, or in said solvent and in the presence of an electric field, give rise to said anion.
    [11]
    11. Process according to any one of claims 1 to 10, in which the solvent of step (i) is water.
    [12]
    12. Process according to claim 11, in which the relative humidity of the dispersion in contact with the area of the graphene surface to be modified is between 60 and 99%.
    [13]
    13. Method according to any of claims 1 to 12, in which the electric field is applied by means of a device comprising: a pointed electrode connected to a first pole of an electric voltage generator, a second pole of said generator connected to graphene; a head configured to hold the pointed electrode; a dielectric separator, which houses said head, and which is configured to electrically isolate the electrode from the rest of the device; a positioner that is attached to the electrical separator and that is configured to move the head in three dimensions of space; and a damping system configured to allow vertical movement of the head with a predetermined pressure and to damp the displacements resulting from the irregularities of the surface through which the head moves; wherein in step (ii) the electric field is generated with the pointed electrode of said device; and a container with solvent configured to generate a dispersion of said solvent.
    [14]
    14. Covalently modified graphene obtainable according to the method of any of claims 1 to 13.
    [15]
    15. Use of the covalently modified graphene of claim 14 in an analyte detector device.
    [16]
    16. Use according to claim 15, in which the covalently modified graphene forms part of the anchoring surface of bio-receptor molecules of analytes to be detected.
    [17]
    17. Use according to any of claims 15 or 16, wherein the covalently modified graphene comprises functional groups for binding to bioreceptor molecules in said analyte detector device.
    [18]
    18. Analyte detector device comprising covalently modified graphene, according to claim 14.
    [19]
    19. Analyte detector device according to claim 18, in which the covalently modified graphene forms part of an anchoring surface of bioreceptor molecules of the analytes to be detected.
    [20]
    20. Analyte detector device according to any of claims 17 or 18, in which the covalently modified graphene comprises one or more functional groups for anchoring to bio-receptor molecules of the analytes to be detected.
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    公开号 | 公开日
    WO2021160911A1|2021-08-19|
    ES2848798B2|2021-12-29|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2016054550A1|2014-10-03|2016-04-07|Rite Taste, LLC|Device and method for chemical analysis|
    WO2018013586A1|2016-07-12|2018-01-18|Nanotech Biomachines, Inc.|Graphene bio-electronic sensing technology|
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