NANOSTRUCTURED BIOELECTRODE FOR GLUCOSE OXIDATION, FROM ELECTROGENERATED AROMATIC COMPOUNDS

Abstract

The invention relates to a bioelectrode comprising a conductive material, on the surface of which are deposited carbon nanotubes, a redox mediator based on pyrene or a derivative thereof, oxidized in-situ, and an enzyme capable of catalyzing the glucose oxidation. The invention also relates to a process for producing such a bioelectrode, and to the uses thereof.

Claims

1-10. (canceled)

11. A bioelectrode comprising a conductive material and having a surface on which are deposited carbon nanotubes, a redox mediator based on pyrene or a derivative thereof, oxidized in-situ, this oxidation forming ketone bonds on the aromatic ring of pyrene, and an enzyme capable of catalyzing the glucose oxidation.

12. The bioelectrode according to claim 1, wherein the enzyme is a flavin adenine dinucleotide-dependent glucose dehydrogenase or a flavin adenine dinucleotide-dependent glucose oxidase.

13. The bioelectrode according to claim 1, wherein the mediator is obtained by chronoamperometry and comprises the application, for a given time, of a potential of 1 V to the bioelectrode and to pyrene, or a derivative thereof, deposited in-situ on the surface of the bioelectrode.

14. The bioelectrode according to claim 3, wherein the given time preferably ranges from 30 seconds to 3 minutes.

15. The bioelectrode according to claim 1, wherein the mediator oxidized in-situ is obtained by cyclic voltammetry and comprises the application to pyrene, or a derivative thereof, deposited in-situ on the surface of the electrode, of a potential varying cyclically from 0.4 V to 1 V.

16. The bioelectrode according to claim 5, wherein a number of cycles varying from 3 to 20 is applied.

17. A process for producing a bioelectrode capable of glucose oxidation, the method comprising: a) a step involving the oxidation of pyrene, or a derivative thereof, wherein the pyrene or the derivative is pre-deposited on the surface of a conductive material, a conductive material on the surface of which carbon nanotubes are also deposited, and b) a step subsequent to step a), involving depositing an enzyme capable of catalyzing the glucose oxidation on the surface of the electrode.

18. The process according to claim 7, wherein the pyrene oxidation step is carried out by chronoamperometry and comprises applying a potential of 1 V at the surface for a given time.

19. The process according to claim 8, wherein the given time ranges from 30 seconds to 3 minutes.

20. The process according to claim 7, wherein the oxidation step is carried out by cyclic voltammetry and comprises applying a voltage varying cyclically from 0.4 V to 1 V at the surface.

21. The process according to claim 10, wherein a number of cycles is applied, the number varying from 3 to 20.

22. The process according claim 7, wherein the enzyme is a flavin adenine dinucleotide-dependent glucose dehydrogenase or a flavin adenine dinucleotide-dependent glucose oxidase.

23. A bioelectrode produced by the process described in claim 5.

Description

[0038] An embodiment of the invention is given by way of non-limiting example and which includes appended drawings which show the following:

[0039] FIG. 1: (A) Voltammograms of a glassy carbon/MWCNT/pyrene electrode before (black) and after chronoamperometry of 1 V vs, Ag/AgCl for 30 seconds (phosphate buffer 0.2 M pH=7)

[0040] (B) Electrochemical response of the electrosynthesis of a glassy carbon/MWCNT/pyrene electrode (black=1st cycle/gray=cycles 2 to 6)

[0041] FIG. 2: (A) Voltammograms of a glassy carbon/MWCNT/pyrene redox electrode at different scan rates (phosphate buffer 0.2 M pH=7).

[0042] (B) Represents the intensities of the anode and cathode peaks of a glassy carbon/MWCNT/pyrene redox electrode as a function of the scan rate.

[0043] FIG. 3: (A) Voltammograms of a glassy carbon/MWCNT/pyrene redox electrode at different pH values (2, 3, 4, 5, 6, 7, 8)

[0044] (B) Representation of the evolution of the standard potential as a function of the pH of a glassy carbon/MWCNT/pyrene redox electrode

[0045] FIG. 4: (A) Electrochemical response of the modified MWCNT/pyrene redox/FAD-GDH electrode in the absence (black curve) and in the presence of 200 mM glucose (gray curve)

[0046] (B) Chronoamperometry at 0.2 V vs. Ag/AgCl of the modified MWCNT/pyrene redox/FAD-GDH electrode during glucose injection (1, 2, 5, 10, 20, 50, 100, 200 mM glucose) (cf. insert) Representation of the evolution of the catalytic current as a function of the glucose concentration obtained during chronoamperometry at 0.2 V vs. Ag/AgCl

[0047] FIG. 5: (A) Electrochemical response of the electrosynthesis of a glassy carbon/MWCNT/anthracene electrode

[0048] (B) Electrochemical response of the electrosynthesis of a glassy carbon/MWCNT/perylene electrode

[0049] FIG. 6: Electrochemical response of the modified MWCNT/phenanthene redox/FAD-GDH electrode in the absence (black curve) and in the presence of 200 mM glucose (gray).

[0050] FIG. 7: Comparison of pyrenedione with 1,4-naphthoquinone by CV in terms of efficiency and stability of electron transfer (catalytic streams, A and C) and in terms of stability of redox activity after 100 cycles (non-catalytic stream, B and D).

[0051] Production of a Bioelectrode According to the Invention

[0052] A commercial 0.071 cm.sup.2 glassy carbon electrode (sold by Bio-Logic, France) is modified by the addition of carbon nanotubes (suspension of 5 mg.Math.mL.sup.1 in carbon nanotubes).

[0053] This suspension is made by adding 10 mg of unfunctionalized multi-wall carbon nanotubes (MWCNT Nanocyl, 97%) in 2 mL of NMP (N-methyl-2-pyrrolidone). The dispersion is placed under ultrasonic stirring for 2 hours. 20 L of this previously stirred MWCNT suspension is then deposited on the surface of the glassy carbon electrode.

[0054] The electrode is then placed under vacuum in a desiccator. The electrode is then removed from the desiccator when the solvent has evaporated and the carbon nanotubes are dry (on average a few hours, generally from 3 to 5 hours).

[0055] Functionalization of Electrodes Via Dropcasting with Pyrene

[0056] After functionalization of the electrode with carbon nanotubes, it is modified by adding 20 L of a 10 mM-concentrated solution of pyrene dissolved in dichloromethane (conc. 5 mg/mL). The solvent is then evaporated at atmospheric pressure (approx. 100 kPa) and ambient temperature (approx. 25 C.).

[0057] Electrosynthesis of the Electrode by Chronoamperometry and Cyclic Voltammetry

[0058] The electrode modified with pyrene is placed in an electrolytic solution (phosphate buffer 0.2 M Na.sub.2HPO.sub.4 and 0.2 M NaH.sub.2PO.sub.4 of pH 7) degassed beforehand under argon. The electrode is then subjected by chronoamperometry to a current of 1 V using as a counter electrode a platinum electrode and a reference electrode of the Ag/AgCl type for 30 seconds. The electrode is then rinsed with distilled water to remove all traces of electrolyte carrier or organic molecules.

[0059] It should be noted that the activation of pyrene was also carried out by successive cyclic voltammetry scans ranging from 0.4 V to 1 V vs. Ag/AgCl. The number of cycles varies from 3 to 20 and the electrode is then rinsed with distilled water to remove all traces of electrolyte carrier or organic molecules. The results presented below were generally carried out using the electrode obtained by chronoamperometry but similar results were obtained by cyclic voltammetry (for example FIG. 1 (right)), and these two electrodes are considered to be of almost identical structure and performance.

[0060] Functionalization of the Electrode Via Dropcasting of the Biocompound

[0061] The FAD-GDH used in this example is a FAD-GDH from Aspergillus sp. (SEKISUI DIAGNOSTICS, Lexington, Mass., Catalog No. GLDE-70-1192) which has the following characteristics:

[0062] Appearance: lyophilized yellow powder.

[0063] Activity: >900 U/mg powder 37 C.

[0064] Solubility: readily dissolves in water at a concentration of: 10 mg/mL.

[0065] One activity unit: the amount of enzyme that will convert one micromole of glucose per minute at 37 C.

[0066] Molecular weight (gel filtration) 130 kD.

[0067] Molecular weight (SDS-PAGE): diffuse band at 97 kD indicative of a glycosylated protein.

[0068] Isoelectric point: 4.4.

[0069] K.sub.m value: 5.Math.10.sup.2 M (D-glucose).

[0070] This enzyme is specific. Sugars other than D-glucose have been tested at a concentration of 30 mM. 2-deoxy-D-glucose exhibits only 25% activity compared to that of D-glucose.

[0071] D-xylose exhibits 11%, D-galactose 0.7%, D-mannose 0.4%, D-trehalose 0.2% and D-fructose 0.1%, activity compared to that of D-glucose. L-glucose, D-mannitol, D-lactose, D-sorbitol, D-ribose, D-maltose and D-sucrose each exhibit less than 0.1% activity compared to that of D-glucose.

[0072] Beforehand, a 5 mg.Math.mL.sup.1 solution of FAD-GDH is prepared in a buffer solution (phosphate buffer 0.2 M Na.sub.2HPO.sub.4 and 0.2 NaH.sub.2PO.sub.4 pH 7) and stored at 20 C. Before each deposit, the solution is removed from the freezer and defrosted. 20 L of this solution is deposited by dropcasting on the modified electrode. The solvent is then evaporated at atmospheric pressure (approx. 100 kPa) and ambient temperature (approx. 25 C.).

[0073] Characterization of the Bioelectrode

[0074] The bioanode obtained is used in a standard electrolytic cell (with a platinum counter electrode and a reference electrode of the Ag/AgCl type) to constitute a cell when positioned in a glucose-concentrated medium. This cell is studied below and has the following characteristics:

[0075] Electrochemical Characterization

[0076] 1. Electrosynthesis

[0077] FIG. 1 (left) shows the electrochemical response of a glassy carbon electrode coated with carbon nanotubes and pyrene. The black curve represents the electrochemical response of the electrode, only a capacitive current is observed corresponding to the contribution of the carbon nanotubes. The gray curve was recorded after having imposed a potential of 1 V for 30 seconds. A faradic signal is observed at a potential of 0.036 V vs. Ag/AgCl. The application of a potential of 1 V therefore induces the synthesis of a new species exhibiting redox properties.

[0078] The previous experiment was carried out by imposing a potential for a given time. It is also possible to electrogenerate the redox probe by successive scans. The different electrochemical cycles are shown in FIG. 1 (right), The black curve represents the first scan cycle and the gray curves represent subsequent cycles. During the first cycle, the absence of a redox signal at 0.05 V in the outward cycle is noticed. The redox peak appears on the return cycle. This behavior is similar to electropolymerization reactions.

[0079] Here it is not the formation of a redox polymer but the electrosynthesis of an electroactive system. At potentials close to 1 V, oxidation of the compound occurs, forming ketone bonds on the aromatic compounds which then become electroactive (Diagram 1). The molecules formed contain quinone functions giving them redox properties.

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[0080] 2. Characterization of the Redox Signal

[0081] The electrochemical response of the electrogenerated redox electrode is characteristic of a species immobilized at an electrode i.e. E close to 0 mV (10 mV to 2 mV.Math.s.sup.1) and the intensity of the oxidation and reduction peak is proportional to the scan rate (FIG. 2).

[0082] The nature of this product was also studied by varying the pH of the electrolytic solution (FIG. 3). The variation of the pH generates a modulation of the redox potential. The slope is 0.056, which is close to the theoretical value 0.059, indicating that this is a redox system involving the exchange of the same number of protons as electrons. It is very likely that this is an exchange of 2 electrons and 2 protons like many aromatic redox probes (naphthoquinone, anthraquinone, etc.). We can therefore assume that electrosynthesis generates the formation of ketone functions on the aromatic rings. In the case of this electrode which is functionalized with a pyrene unit, the assumed product formed is 1,6-pyrenedione or 1,4-pyrenedione. The electrogenerated redox couple is therefore pyrenedione/dihydroxypyrene with an exchange of 2 electrons and 2 protons. However, the literature (e.g. P. Barathi, A. Senthil Kumar, Langmuir, 29 (2013) 10617-10623, cited) differs on the exact nature of the compound formed and it is not necessarily possible to conclude on the number of ketone functions formed.

[0083] Study of the Catalytic Properties of Bioelectrodes

[0084] FIG. 4 shows the electrochemical response of the bioanode described above (MWCNT/pyrene redox/FAD-GDH) in the absence and presence of glucose. The black curve in the absence of glucose shows only the reversible electrochemical response of the immobilized redox probe. Conversely, in the presence of an aqueous solution of 200 mM glucose, a wave of oxidation is observed and characteristic of catalytic activity. Catalytic current occurs at the potential of the redox probe. This shows that the electrogenerated redox probe allows mediated electron transfer between the electrode and the FAD-GDH enzyme. The figure on the right shows the flow of current as increasing amounts of glucose are added. The maximum catalysis current of the range of 1.3 mA (6.5 mA.Math.cm.sup.2) is reached for glucose concentrations of 200 mM. This is also observed by the insert in the right drawing (4B) showing the evolution of the current as a function of the concentration which reaches a plateau for concentrations of 200 mM. The apparent Michaelis-Menten constant of the system is 39.8 mM.

[0085] Comparative Studies of Other Activated Polyaromatic Compounds

[0086] In order to establish the surprising properties of the enzyme electrode according to the invention, several comparative studies have been carried out using base materials other than pyrene. The bioanodes were produced in the same way and according to the same steps as for the activated pyrene electrode described above. The chosen activation method was cyclic voltammetry (FIGS. 5A and 5B) and chronoamperometry (FIG. 6) which generates the same behaviors. The only modification was the nature of the polycyclic compound.

[0087] Thus, FIG. 5 (left) shows the electrochemical response of the oxygenated derivative of anthracene after activation by cyclic voltammetry. The signature matches exactly that of anthraquinone, a commercial product. The drawing on the right shows the electrosynthesis of a perylene derivative under the same conditions. It is most likely a perylenequinone but such derivatives are not marketed which does not make it possible to determine the exact structure. The potential of these two components (0.5 V for anthraquinone and 0.2 for perylenequinone) does not allow electron transfer with FAD-GDH.

[0088] FIG. 6 shows the electrochemical response of the oxygenated derivative of phenanthrene after activation by cyclic voltammetry. This shows a transfer of electrons after electro-oxidation. The signature exactly matches that of phenanthraquinone, a commercial product. The catalytic current nevertheless remains low (a few tens of A) compared to electro-oxidized pyrene (several hundred A)

[0089] FIG. 7 shows the comparison of a pyrenedione electrode (according to the invention) with a 1,4-naphthoquinone electrode by cyclic voltammetry in terms of the efficiency and stability of electron transfer. In the context of the production of biofuel cells, it is necessary to avoid the release of the redox mediator in solution, which induces a decrease in performance over time as well as possible pollution in the case of the implantation of biofuel cells in living organisms. After 100 cycles of cyclic voltammetry of the electrode in the presence of 200 mM glucose, the catalytic current decreases by 60% for the pyrenedione derivative while it decreases by more than 93% in the case of the electrode functionalized by the 1,4-naphthoquinone unit (FIGS. 2A and C). A decrease of 47% and 77% of the non-catalytic faradic signal is observed respectively for pyrenedione and 1,4-napthoquinone (FIGS. 2B and D).

[0090] In the case of the pyrene unit, this has certain advantages for use in bioanodes as a redox mediator for FAD-GDH. The product is easily electrosynthesized and exhibits rapid electron transfer. The redox potential of the pyrene-quinone' pyrene-dihydroquinone couple has a potential close to the redox potential of the active site of the enzyme. In the presence of the FAD-GDH enzyme and glucose, a catalysis current is observed (FIG. 7A). In our example, the maximum catalytic current obtained for an MWCNT/pyrene-quinone IFAD-GDH electrode is 1.4 m. This catalytic wave appears at potentials close to the redox potential of FAD-GDH and therefore makes it possible to obtain high open-circuit voltages (OCV) in the case of the integration of this bioanode in a biofuel cell device. The OCV is a crucial parameter to obtain devices delivering high power.