ES2525365B1 - MATERIALS OPERATED WITH NAD * OR NADP * AND ITS APPLICATION IN BIOSENSORS AND OTHER DEVICES - Google Patents

Abstract

Materials functionalized with NAD {sup, +} or NADP {sup, +} and its application in biosensors and other electrochemical devices. # The present invention relates to a functionalized material comprising a support on which a NAD cofactor is fixed {sup , +}, NADP {sup, +}, NADH or NADPH through the amino group of adenine by a bridge group from epoxides. Said material is intended to interact with the dependent cofactor enzymes in the presence of the enzyme substrate. Due to its properties, this material is useful as a bio-electrode for the elaboration of biosensors based on dehydrogenase enzymes as well as bio-anode in biopiles.

Description

image 1

Materials functionalized with NAD + or NADP + and their application in biosensors and

other devices.

DESCRIPTION

The present invention relates to a material with transducer capacity comprising a support on which a NAD +, NADP +, NADH or NADPH cofactor is fixed through the amino group of the adenine by a bridge group derived from epoxides. The key advantage of this invention is that the cofactor in this case is not denatured, but is covalently immobilized and reacts with the NAD (P +) -dependent enzymes in the presence of an enzymatic substrate. Said material is suitable for obtaining biosensors and other electrochemical devices such as biopiles or biofuel cells.

STATE OF THE PREVIOUS TECHNIQUE

The development of immobilization and regeneration methods of nicotinamide adenine dinucleotide (NAD +) or nicotinamide adenine dinucleotide phosphate (NADP +) [also called diphosphopyridine nucleotide or nucleotide phosphate o / and Coenzyme I] cofactors has been the subject of considerable scientific and technological interest in The last three decades. This interest has had a considerable interdisciplinary nature due to the important role that the two cofactors of this type play in the transfer of electrons between redox metabolic reactions and / or in the functioning of several enzymes called oxido-reductases, dehydrogenases or NAD (P) enzymes. + -dependents. Thus, areas such as bioelectrochemistry, including the manufacture of biosensors, the development of biofuel cells and the production of bio-batteries, as well as other less related areas such as enzymatic synthesis and the development of biotechnological processes can be highlighted as examples for the purification and separation of enzymes.

However, the great limitation when considering these developments has been marked by the difficulty of developing functionalized bioelectrodes with the NAD (P) + cofactor and that this is accessible to the enzymes in play, as well as being recyclable on the surface of the electrode relatively long.

image2

Several research groups in the area of bioelectrochemistry have focused their efforts on this objective. Examples of physical immobilization of the cofactor and enzymes in a sol-gel matrix have been described in the literature (Th. Noguer, D.Szydlowska, JL, Marty, M. Trjanowicz. Pol. J. Chem 78 (2004) pp. 1679-1689) and cellulose acetate (SD. Sprules, JP. Hart, SA. Wring, R. Pittson, Anal. Chim. Acta 304 (1995) pp. 74-24) by simple encapsulation, and other chemicals using 3-glycidoxypropyltrimethoxysilane for non-oriented immobilization of the NAD + cofactor in a three-dimensional sol-gel matrix (Z. Wang, M. Etiennne, F. Quilès, GW. Kohring,

A. Walcarius Biosens Bioelectron 32 (2012) pp 111-117) or by performing covalent bonds thanks to functional groups of polymers such as dextran (M. Montagné, JL. Marty. Anal. Chem. Acta. 315 (1995) pp. 297-302 ), polyethylene glycol

(KKW Mak, U. Wollenberger, FW Scheller, R. Rennneberg. Biosens. Bioelectron 18 (2003) pp. 1059-1100), Chitosan (M. Zhang, C. Mullens, W. Gorski. Anal. Chem 79 (2007 ) pp. 2446-2450) or polyethyleneamine (H. Zheng, J. Zhou, J. Zhang, R. Huang, H. Jia, S. Suye. Microchim. Acta 165 (2009) pp. 109-115). However, in most of these designs, several relevant factors have not been taken into account. Among them it is important to highlight the following: conservation of the cofactor's native state, the probability of accessibility of the enzyme to the binding site in the cofactor structure; and the performance of the biocomponents introduced in the immobilization matrix.

To resolve these limitations, several pioneer groups have proposed new methodologies for oriented immobilization of the cofactor and the enzyme in several stages, achieving a biosensor design similar to the so-called layer-by-layer. In this context, bioelectrode designs based on interlinked molecular chains that consist of sequences of: electrode / chemical mediator / cofactor / enzymes have been developed. However, the highlight of these works is the design of an affinity complex between the cofactor and the enzyme. In all these examples this was achieved thanks to the oriented immobilization of the cofactor in its "native" state by means of 3-aminophenylboronic acid that selectively interacts with the ribose unit of the cofactor. The advantage that is used for this design is that the nicotinamide unit is left free and accessible, which allows to justify a supposed bioaffinity (YM. Yan, O. Yehezkeli, I. Willner. Chem. Eur. J. 13 (2007) pp 10168-10175; BL Hassler, RM Worden, Biosens, Bioelectron 21 (2006) pp. 2146-2154). However, given the great abundance of the ribose group in nature and the weakness of the hydrogen bond established between the boron group and the hydroxides of the ribose subunit of NAD (P) +, this system can be attributed a certain instability in the continuous operation, especially in the presence of sugars since these compounds can easily replace the ribose group and establish other hydrogen bonds with the boron atom. For this reason it is easy to imagine in this case an inevitable detachment of the cofactor. To correct these disadvantages, immobilization is usually attempted by cross-linking the enzyme.

image3

DESCRIPTION OF THE INVENTION

This invention describes a new method of immobilization of the cofactor NAD +, NADP +, NADH or NADPH in its active form and in an oriented manner. Through this method of immobilization, it has been possible to establish strong chemical bonds between functional groups of the cofactor and of the transducer surface without damaging the joint center of the cofactor-enzyme.

Unlike other approximations of the state of the art, in the present invention the surface of the substrate has been modified in stages, thus producing ordered layers of the components and taking advantage of the high affinity of the enzymes in question to the cofactor efficiently immobilized and with its enzyme-oriented binding site.

In this invention, the functionalization of transducer surfaces of the physical signal that acts as a probe is obtained to obtain real-time information on the redox state of the cofactor in question and to follow the biocatalytic process, which makes it present suitable characteristics to be applied to the manufacture of biosensors. In the biosensor obtained, the resulting biocomponents are in intimate contact with the physical transducer. Obviously this design ensures a great sensitivity of the measurement system and can be used to use the same surface in applications where it is necessary to recycle the same cofactor between several enzymes.

image4

Unlike other similar biofunctionalized materials described where the oriented immobilization of the NAD (P) + cofactor is described by its ribose subunit and thanks to the 3-aminophenylboronic acid that acts as a bridge between the transducer surface and the cofactor, in this invention the cofactor 5 through the use of epoxy groups that react with the amine group of the adenine unit. In this case, strong chemical bonds are established between the cofactor and the surface, which entails enormous advantages. Among them the durability, flexibility and the possibility of applying the surface thus functionalized in various applications and designs. For example, in this case the immobilization of the enzyme is not essential

10 and the application of the same surface is possible for the control of several processes by several enzymes.

Therefore, in a first aspect the present invention relates to a functionalized material of general formula (I): S-L-C

(I)

where:  S is an electrode support,  L is a bridge group comprising the group of formula (II)

twenty

image5

(II)  C is a cofactor that is selected from NAD +, NADP +, NADH or NADPH,

characterized in that C is linked to L by a covalent bond through the primary amino group at position 6 of the adenine of C.

In the present invention, "electrode" support is understood as a solid material capable of detecting and / or transmitting the electron flow produced by changes from NAD (P +) to NAD (P) H or NAD + to NADPH after its reaction with a enzyme

30 dependent cofactor. This electrode support is used to transmit physical signals of any electrochemical, optical, mass, magnetic, etc. type.

image6

The supports capable of being part of the functionalized material of the present invention can be: conventional electrodes used in chemical elect as electrodes of noble metals (or ro, platinum, silver, palladium ...), including a deposition on non-conductive surfaces of these metals via sputtering, screen printing or the like, such as coal and graphite derivatives. Among these: vitrified carbon electrodes and graphite carbon electrodes, carbon nanotubes, fullerene, graphene, black carbon, carbon fiber or the like, as long as they are solidified with adhe rentes type (sol-gel materials, ionic liquids, Teflon, epoxy resin, etc).

Two electrodes that are conventionally used in spectrum-electrochemistry and solar cells based on transition metal oxides (for example tin, zinc) without doping doped with indium or fluorine (ITO, FTO electrodes, etc.) may also be used. .), as well as optical electrodes like the previous ones but based on optical fiber, mica, glass, and the like. In addition, any substrate that can be used in the transduction of physical signals such as changes in voltage, temperature, weight, resonance, or / and any change in the physical properties of the transducer and / or its bio-physical environment.

In a preferred embodiment, the support is read from gold, silver, platinum or palladium.

In another preferred embodiment, the material is selected from carbon nanotubes, graphite, graphene, fullerene or carbon microfibers.

In another preferred embodiment, the support is a tin oxide or zinc doped with indium

or fluoride

In another preferred embodiment, the possible supports mentioned above can preferably be found in the form of nanoparticles.

In another preferred embodiment, the support comprises an additional coating that joins the bridge group L. In a more preferred embodiment, this coating can be a polymer or polymer mixture or a silane layer which has been synthesized by a sol-gel and deposited procedure on the electrode by means of conventional methods of surface modification (spin-coating, dip-coating or simply casting solution).

image7

In another preferred embodiment, the material of the invention further comprises at least one enzyme linked to cofactor C. In a more preferred embodiment, the enzyme is a dehydrogenase.

In another preferred embodiment, the enzyme is immobilized, and more preferably

10 by a crosslinking reagent such as glutaraldehyde or by a membrane.

Another aspect of the present invention relates to a process for obtaining the functionalized material described above comprising the following steps: a) epoxidation of the substrate or S by means of a compound comprising at least one group of formula (III),

image8

b) and adding a solution of the coffer ctor C to the product obtained in step (a), where S and C are defined as above.

Another aspect of the present invention relates to a process for obtaining the functionalized material described above comprising the following steps: a) epoxidation of cofactor C with a compound comprising at least one group of formula (III):

image9

b) and deposition of the product obtained in step (a) on a support S, where C and S are defined as above.

image10

In a preferred embodiment, the process of the invention further comprises a step c) of immobilization of an enzyme with affinity for cofactor C. In a more preferred embodiment, the enzyme is a dehydrogenase.

The compound used in the epoxidation stage can be an organic compound, an organic or inorganic polymer, a metal oxide, carbon nanomaterials, metal nanoparticles and other materials as long as they contain at least one epoxide group or is designed to convert to this group, to then react with the N6 amino group of the adenine subunit of cofactor C. Preferably, the compound used in the epoxidation step is selected from 3-glycidoxypropyltrimethoxysilane, glycidol, epichlorohydrine, polyethylene glycol diglycidyl ether or 1,4-butanediol diglycidyl ether.

In another preferred embodiment, a coating comprising active functional groups is attached to the support S. In a more preferred embodiment the active functional groups are selected from hydroxyls, amines or carboxyls.

Another aspect of the invention relates to the use of the functionalized material described above for the manufacture of biosensors, bioelectrodes, biopiles, biobatteries or biofuel cells.

Another aspect of the present invention relates to a biosensor comprising the functionalized material described above.

In the present invention a device with the capacity to measure biological or chemical parameters is understood as a biosensor. It consists of at least three basic components that are the functionalized material described above, which acts as a biological sensor, a transducer that couples the other components and translates the signal emitted by the sensor and the detector that can be optical, piezoelectric, thermal, magnetic, etc.

The operation of the biosensor of the present invention is based on the oxidation mechanisms of the enzyme on the substrate, and the reduction of the cofactor from NAD (P) + to NAD (P) H. This movement of electrons and H + is detected by the support and transmitted to the transducer.

image11

The transduction systems used in the present invention may be based on the detection of the changes:

a) in the redox state of cofactor C, directly or indirectly (using a biochemical mediator), as in the amperometric and optical transduction processes.

b) in the physical state of the SLC surface, comparing parameters (such as weight, resonance, conductivity, potential, magnetic moment…) before and after the interaction of the surface with the C-dependent enzyme or / and a complex of them.

Another aspect of the present invention relates to an enzymatic fuel biopile, so named because redox enzymes are involved in the anode and cathode in the electrodes of the cell. The energy in this case is generated thanks to the establishment of a potential difference between the two electrodes, which is due to the oxidation of the enzymatic cofactor or a cofactor-mediator complex in the anodic compartment and the reduction of oxygen in the cathodic behavior

In the biopile of the present invention, the anode is the previously functionalized material which reacts with a mixture of enzymes and at least one biofuel, as long as at least one of the enzymes is C-dependent. In this case, the anode is the S-L-C-E complex-shaped material made from S-L-C, where E is a selective C-dependent enzyme for biofuel.

Another aspect of the invention relates to a method for the qualitative and / or quantitative determination of the substrate of an enzyme in a sample comprising the following steps:

a) contacting said sample with the biosensor described above, b) subjecting the biosensor to conditions that result in an electrochemical signal, c) measuring the electrochemical signal obtained in (b) and d) qualitatively and / or quantitatively determining the substrate from of the measure of

The electrochemical signal.

image12

More specifically, this method comprises the following steps to perform an electrochemical measurement:

a) placing the biosensor described above, in the working electrode position in an electrochemical cell, generally composed in addition to the working electrode of a reference electrode and an auxiliary electrode.

b) Fill the electrochemical cell with a measurement solution, whose

Composition depends on enzymes. c) Polarize if the biosensor is preferred to a specific potential. d) Inject the sample to be measured in the electrochemical cell. e) Measure the electrochemical signal obtained in (d). f) Determine the substrate qualitatively and / or quantitatively from the measure of

The electrochemical signal.

In a more detailed way, this procedure can be carried out as follows: a) Contact the biosensor described above with a measurement solution.

b) Subject the product obtained in step (a) to transduction conditions capable of controlling the redox state of compound C or the bio-affinity of C with C-dependent enzymes.

c) Inject a mixture of enzymes into the measurement solution, where at least one of them is C-dependent. d) Continue monitoring and recording the input signal indicative of the redox state of C

or of the succession of the bio-affinity C C-dependent enzyme. e) Inject a sample containing a substrate to be determined. f) And finally correlate the difference between the input signals obtained in the

stages d and e with the concentration of the substrate. In this case, steps c) and d) may appear inverted.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

image13

DESCRIPTION OF THE FIGURES

Figure 1: represents the reactions involved in the mechanism of transduction of the electrochemical signal. Figure 2: represents the electro-catalytic response recorded in amperometric mode of the glucose biosensor (performed according to example 1) in the presence of meldola blue and several glucose concentrations. Figure 3: represents the glucose calibration line, obtained from the current data recorded at 10 s in the amperogram of Figure 2. It also represents the analytical characteristics of this line and the detection limit (LOD) of the biosensor of glucose Figure 4: represents the electro-catalytic response recorded in chronoamperometric mode of the alcohol biosensor (performed according to example 1) in the presence of meldola blue and various alcohol concentrations. Figure 5: represents the calibration line of the percentage by volume of the alcohol, in a sample obtained from the current data recorded in the chronoamperogram of Figure 4. The analytical characteristics of that line and the detection limit (LOD) are also included. ) of the alcohol biosensor. Figure 6: represents the electro-catalytic response recorded in chronoamperometric mode of the malic acid biosensor (performed according to example 1) in the presence of meldola blue and various concentrations of malic acid. Figure 7: represents the electro-catalytic response recorded in chronoamperometric mode of the glucose biosensor developed in example 3 in the presence of various concentrations of the analyte.

EXAMPLES

Example 1: obtaining the material of the invention with a gold support by prior hydroxylation thereof.

The hydroxylation of gold electrodes was first performed thanks to the deposition

image14

of a silane layer synthesized via sol-gel. The epoxidation of this new surface with epoxy groups was carried out by reaction with 3glycidoxypropyltrimethoxysilane (GPS), while the oriented immobilization of the cofactor has been carried out by the reaction of the epoxide group and the amino (N6) of the adenine unit .

5 mL of Mili-Q water was placed in a reaction vessel, 1.93 mL of tetraethylorthosilicate (TEOS) (CAS: 78-10-4 sigma-aldrich) was added and under magnetic stirring, 6 were added. , 25 mL of a 0.01M hydrochloric acid solution dropwise as an acid catalyst. The whole was left under stirring for 10 hours, sufficient to obtain a completely miscible solution. After dilution by a factor of 3, to obtain a final concentration of Si equal to 0.25M, a precise volume was taken and deposited on a previously washed and dried electrode according to conventional methods. The volume to be deposited has been optimized at 0.7 µL / mm2; where the denominator corresponds to the geometric surface of the electrode. The deposited drop was dried for two hours at room temperature and then at 200 ° C for 10 min to evaporate all the ethanol generated and end the sol-gel condensation processes. After this step, the electrode was cooled in the desiccator and immersed in a toluene solution containing 0.17 mol / L of 3glycidoxypropyltrimethoxysilane (GPS), (CAS: 2530-83-8 Sigma-Aldrich). It was then left under stirring overnight and finally dried at room temperature. With these processes, the electrode functionalization with epoxy groups was achieved, a previous step for the oriented immobilization stage of the NAD (P) +.

For cofactor immobilization, a solution of NAD + 2mg / mL at basic pH was prepared, the electrode was introduced therein and allowed to react for a minimum of 24 hours under stirring and at room temperature. After this time the electrode was taken, washed gently with water, and bioaffinity was performed between the cofactor and the specific enzyme to the analyte of interest. Finally, the enzyme was immobilized by conventional processes: using glutaraldehyde (diluted in water to a percentage of 0.5%) as a crosslinking agent or simply by retention under a dialysis membrane fixed by an O-ring.

Following this procedure, three prototype biosensors were developed: glucose biosensor based on glucose dehydrogenase (EC 1.1.1.47), ethanol biosensor based on alcohol dehydrogenase (EC 1.1.1.1) and malic acid biosensor based on malate dehydrogenase (EC 1.1.1.37). It should be noted that for the good recycling of the cofactor, in all these prototypes the enzyme diaphorase (EC 1.8.1.4) was used, which was immobilized by the same process together with the enzymes in question and after the bioaffinity was carried out with the cofactor immobilized.

image15

The mechanism for generating the electrochemical signal in the presence of the substrate and the graphs of responses recorded by the biosensors developed in this example are illustrated in Figures (1-6). The measurement data were obtained in an electrochemical cell, equipped with three electrodes; reference, auxiliary and working (silver / silver chloride, platinum and the biosensor manufactured according to the description, respectively) and using a potentiostat (PalmSens Electrochemical Sensor Interfaces, Palm Instruments BV Holland) equipped with own software for the establishment of the signal of excitation of the system and for recording the electrochemical response. The electro-analytical methods used in these examples were the amperometric and / or chrono-amperometric measurements, so named because they consist in establishing a potential difference between the reference electrode and the working electrode and recording the evolution of the current with the time between this same electrode and the auxiliary electrode.

In all cases of this example, the cell contains a pH 7 phosphate buffer solution and an amount of meldola blue equal to 0.1 mmolar. Figure 2 shows results of amperograms obtained by the glucose biosensor. To perform these measurements, a calculated volume of a standard glucose solution is added to the measurement solution, a potential difference is established between the working electrode and the reference electrode equal to 50 mV and the current variation between the current is recorded. Work electrode and auxiliary electrode. The system under continuous polarization reaches a limit current independent of the time also called diffusion current or Cottrell current. This current is directly proportional to the concentration of glucose, so the calibration line illustrated in Figure 3 was obtained directly by correlating the current registered at a time of enough to reach the limit current with the concentration of glucose present in the cell. (0, 0.1; 0.4; 0.6; 1 g / L).

image16

In the rest of the cases, alcohol and malic acid biosensors, chronoamperometric measurements have been used. After introducing the three electrodes into the measuring cell containing the buffer and the mediator, the working electrode is polarized to a potential of 50 mV (vs Ag / AgCl) and the evolution of the current produced is recorded. The system under continuous polarization is established reaching the same limit current. When the enzyme substrate is injected into the cell, re-oxidation of the electrochemical mediator occurs on the surface of the electrode, which translates into jumps of the current recorded as shown in Figures 3 and 5. The magnitude of these jumps is directly proportional to the concentration of the enzyme substrate and serves to prepare the calibration lines directly as illustrated in figure 4.

In the case of choosing not to immobilize the enzymes, these substances were retained with an O-ring or added directly to the electrochemical cell. The enzymatic activity was monitored by recording the same kinetic-electrochemical curves.

Example 2: obtaining the material of the invention with a gold support by direct epoxidation thereof.

A gold electrode was applied with a reduction with royal water to generate OH groups on its surface, then dried in a nitrogen environment and immediately introduced into a toluene solution containing 0.17 mol / L of GPS that was left overnight under stirring and under nitrogen atmosphere. With this process the hydrolysis of the organosilane was carried out giving rise to a gold surface equipped with epoxy groups. This same electrode was introduced in a solution of 2 mg / mL of NAD + at basic pH and the oriented immobilization of this cofactor was carried out for a minimum of 24 hours at room temperature. Enzyme immobilization was performed as in the previous example.

: obtaining the material of the invention with a support of silica nanoparticles by functionalizing them in a colloidal suspension.

image17

Following the same base chemistry of Examples 1 and 2, the functionalization of nanoparticles with epoxy group and their reaction with the cofactor can also take place in a colloidal suspension. This has been carried out by the following procedure: 3,025 mmol of TEOS corresponding to 0.674 mL was added to a solution of ethanol 21.37 mL, and then 1.07 mL of the ammonia catalyst was added dropwise under magnetic stirring and The reaction was left in process for 8h. At the end of this time, a heating plate was dried at 80 ° C to evaporate all the ethanol. Once the solid was recovered, 25 mg were taken and dissolved in 10 mL of toluene. To this solution, 10 + was added as in Examples 1 and 2. A dialysis with a 2000 membrane was applied to the result of that reaction. The product was extracted and stored in the refrigerator to be applied directly to the surface of the previously washed electrode. 5 µL of this suspension was applied to a vitrified carbon electrode (Metrohm AG 3mm) and the enzymes (specifically glucose dehydrogenase and diaphorase) were also added. The tetrathiafulvalene chemical mediator (TTF), (CAS No. 31366-25-3) was then added and retention was applied on a dialysis membrane (10000) secured with an O-ring. Example of the chrono-amperometer curves in the presence of the analyte are illustrated in Figure 7. The measurements were made as in Example 1, except that in this case it was not necessary to add the mediator to the solution.

25 buffer and that the applied potential was equal to 100mV vs Ag / AgCl.

Claims (21)

image 1 1. Functionalized material of general formula (I): S-L-C 5 (I) where:  S is an electrode support,  L is a bridge group comprising the group of formula (II) image2 10 image3 (II)  C is a cofactor that is selected from NAD +, NADP +, NADH or NADPH, characterized in that C binds to L through a covalent bond through the primary amino group at position 6 of the adenine of C. fifteen 2. Material according to claim 1 wherein the support S is selected from noble metals, carbon materials, transition metal oxides, doped or undoped, fiber optics, polymers, mica, glass, silicon or silica. 3. Material according to the preceding claim wherein the support is selected from gold, silver, platinum or palladium. 4. Material according to claim 2 wherein the material is selected from Carbon nanotubes, graphite, graphene, fullerene or carbon microfibers. 25 5. Material according to claim 2 wherein the support is a tin or zinc oxide doped with indium or fluorine.

6.

Material according to any one of claims 1 to 5 wherein the support is in the form of nanoparticles.

7. Material according to any of the preceding claims wherein the support comprises an additional coating that joins the bridge group L.

8.

Material according to claim 7 wherein the coating is a polymer or mixture of polymers.

image4 9. Material according to claim 7 wherein the coating is a silane layer synthesized by sol-gel. 10. Material according to any of the preceding claims which further comprises at least one enzyme linked to cofactor C. 11. Material according to claim 10 wherein the enzyme is a dehydrogenase. 12. Material according to any of claims 10 or 11, wherein the enzyme is immobilized. 13. Material according to the preceding claim wherein the enzyme is immobilized by a crosslinking reagent or by a membrane. twenty 14. Method of obtaining the material according to any of claims 1 to 13 comprising the following steps: a) epoxidation of the substrate S by a compound comprising at least one group of formula (III), image5 B) and adding a solution of cofactor C to the product obtained in step (a), wherein S and C are defined as in claim 1. 15. Method for obtaining the material any of claims 1 to 13 comprising the following steps: image6 a) epoxidation of cofactor C with a compound comprising at least one group of formula (III): image7 b) and deposition of the product obtained in step (a) on a support S, 5 where C and S are defined as in claim 1. 16. The method according to any of claims 14 or 15, further comprising a step c) of immobilization of an enzyme with affinity for cofactor C. 10

17.

Process according to the preceding claim wherein the enzyme is a dehydrogenase.

18.

Process according to any of claims 14-17 wherein the compound

15 used in the epoxidation step is selected from 3-glycidoxypropyltrimethoxysilane, glycidol, epichlorohydrino, polyethylene glycol diglycidyl ether or 1,4-butanediol diglycidyl ether. 19. A method according to any of claims 14-18 wherein a coating comprising active functional groups is attached to the support S. 20. The method according to the preceding claim wherein the active functional groups are selected from silanes, hydroxyls, amines or carboxyls. Use of the functionalized material according to any one of claims 1 to 13 for the manufacture of biosensors, bioelectrodes, biopiles, biobatteries or biofuel cells. 22. Biosensor comprising the functionalized material described in any one of claims 1 to 13. image8 23. Biopila comprising the functionalized material described in any one of claims 1 to 13. 24. Method for qualitative and / or quantitative determination of the substrate of an enzyme in a sample comprising the following steps: a) contacting said sample with the biosensor of claim 22, b) subjecting the biosensor to conditions resulting in an electrochemical signal, c) measure the electrochemical signal obtained in (b) and d) determine the substrate qualitatively and / or quantitatively from the measurement of The electrochemical signal. 25. The method of claim 24 comprising the following steps: a) contacting the biosensor of claim 22 with a measurement solution, b) subjecting the product obtained in step (a) to transduction conditions capable of controlling the redox state of compound C or bio-affinity of C with C-dependent enzymes, c) inject a mixture of enzymes into the measurement solution, where at least one of them is C-dependent, d) continue monitoring and recording the input signal indicative of the redox state of C or of the succession of the bio-affinity C C-dependent enzyme, e) inject a sample containing a substrate to be determined and f) correlate the difference between the input signals obtained in stages d and e with the concentration of the substrate. 26. A method according to claim 24 comprising the following steps: a) contacting the biosensor of claim 22 with a measurement solution, b) subjecting the product obtained in step (a) to transduction conditions capable of controlling the redox state of compound C or bio-affinity of C with C-dependent enzymes, c) continue monitoring and recording the input signal indicative of the redox state of C or from the succession of the bio-affinity C C-dependent enzyme, image9 d) inject a mixture of enzymes into the measurement solution, where at least one of them is C-dependent, e) inject a sample containing a substrate to be determined and f) correlate the difference between the input signals obtained in stages d and e with the concentration of the substrate.