ENZYMATIC BIOCATHODE, METHOD FOR PRODUCING IT AND FUEL BIOCELL AND BIOSENSOR COMPRISING THIS ENZYMATIC BIOCATHODE

Abstract

A biomass-based enzymatic biocathode based on glucose, monosaccharide, ketone or aldehyde includes a collector conductor support, conductive particles disposed on and bound to said collector conductor support, and an aldose reductase disposed on said conductive particles, being bound thereto by adsorption and accessible at the surface of the monosaccharide, ketone or aldehyde reagent that is to be reduced when the biocathode is operational.

Claims

1. A biomass-based enzymatic biocathode based on one of monosaccharide, ketone and aldehyde, comprising: a collector conductor support; conductive particles disposed on and bound to the collector conductor support; an aldose reductase disposed on the conductive particles, being bound thereto by adsorption and accessible at the surface for the monosaccharide, ketone or aldehyde reagent that is to be reduced when the biocathode is operational.

2. The enzymatic biocathode according to claim 1, wherein the collector conductor support is selected from: continuous sheets of one of carbon, graphene and graphite; continuous sheets of a metal; continuous indium tin oxide (ITO) sheets; and carbon fibre non-woven fabrics.

3. The enzymatic biocathode according to claim 1, wherein the conductive particles are selected from particles of carbon, graphene, graphite, carbon black or mesoporous carbon nanotubes, and particles of multiwalled carbon nanotubes (MWCNT).

4. The enzymatic biocathode according to claim 1, to the aldose reductase is associated its nicotinamide adenine dinucleotide phosphate (NADPH) cofactor.

5. The enzymatic biocathode according to claim 24, wherein the regeneration agent is an agent for the electroregeneration of the NADPH cofactor at the surface of the biocathode, the electroregeneration agent being at least one redox polymer chosen from benzylpropylviologen, a viologen polysiloxane polymer, polyaniline or polypyrrole.

6. The enzymatic biocathode according to claim 24, wherein the regeneration agent is a photosensitive agent for the regeneration of the NADPH cofactor at the surface of the biocathode, the photosensitive agent being at least one redox photosensitive polymer chosen from methylene green, methylene blue, neutral red, and polyaniline and polypyrrole.

7. The enzymatic biocathode according to claim 24, wherein the regeneration agent is a photosensitive agent for the regeneration of the NADPH cofactor at the surface of the biocathode, the photosensitive agent being at least one non-polymeric photosensitive compound selected from among chlorophyll, acridine, (pentamethylcyclopentadienyl-2,2V-bipyridine aqua) rhodium (III) and proflavine.

8. The enzymatic biocathode according to claim 7, wherein to the non-polymeric photosensitive compound is associated at least one electron donor selected from vitamin C, ferrocene, 8-hydroxyquinoline-5-sulphonic acid hydrate and a quinone, the electron donor being capable, once oxidized by said photosensitive compound, of being reduced at the surface of the biocathode.

9. The enzymatic biocathode according to claim 24, wherein the regeneration agent is a photosensitive agent for the regeneration of the NADPH cofactor at the surface of the biocathode, the photosensitive agent being at least one photosynthesis protein selected from ferrodoxin and ferrodoxin-NADP reductase.

10. The enzymatic biocathode according to claim 1, wherein: (a) aldose reductase; or (b) aldose reductase and its cofactor NADPH; or (c) aldose reductase and its cofactor NADPH and at least one regeneration agent for said cofactor, and optionally at least one electron donor in case the regeneration agent is a photosensitive regeneration agent and is a non-polymeric photosensitive compound is/are encapsulated in a protective shell capable of letting the reagents and reaction products pass through, but not letting (a), (b) or (c) pass through.

11. The enzymatic biocathode according to claim 10, wherein the regeneration agent(s) is (are) at least one redox polymer, the aldose reductase and its cofactor being enclosed in said redox polymer(s), which act(s) as a protective shell, and can be arranged in the form of a layer deposited on the conductive particles.

12. The enzymatic biocathode according to claim 11, wherein the protective shell is made of chitosan, Nafion, polypyrrole, polyacrylic acid.

13. A method of manufacturing a biocathode wherein: (A) on a collector conductor support, conductive particles are fixed by spraying or printing an ink or paste based on these particles dispersed in water and a surfactant or a polymer or a gel, and then drying said ink or paste; and then (B) said conductive particles are deposited on: (a) an aldose reductase, or (b) an aldose reductase and its cofactor NADPH, or (c) an aldose reductase, its cofactor NADPH and a regeneration agent for the cofactor, at least one of (a), (b) and (c) being capable of being deposited in an encapsulated state in a shell capable of letting the reagents and reaction products pass through but not letting (a), (b) or (c) pass through respectively, or it being possible that an encapsulation step be then performed to encapsulate (a), (b) or (c).

14. The method according to claim 13, wherein in step (B), when the regeneration agent for the cofactor is a redox polymer, the latter is deposited on the conductive particles by electropolymerization or electrodeposition or another electrochemical method such as cyclic voltammetry or chronoamperometry or chronopotentiometry, when to aldose reductase is associated its cofactor NADPH, possibly with a protein or proteins, it being possible that the redox polymer be also deposited by chemical polymerisation processes in the presence of an oxidising element, such as iron chloride.

15. A fuel biocell comprising an anode or bioanode and a biocathode as defined in claim 1.

16. The fuel biocell according to claim 15, wherein the fuel is selected from hydrogen and a biomass compound selected from glucose, ethanol, glycerol, cholesterol, an aldehyde.

17. The fuel biocell according to claim 15, wherein the anode is a bioanode, using, as a catalyst for the oxidation reaction, at least one of enzymes, abiotic compounds, microbes and molecular catalysts.

18. The fuel biocell according to claim 17, wherein it is implantable in a human or animal body, for example subcutaneously or in tissue to power an electrically implantable medical device, and optionally externally rechargeable with glucose, monosaccharide, ketone or aldehyde by means of an external injection of a glucose, monosaccharide, ketone or aldehyde solution.

19. The fuel biocell of claim 18, wherein it is implantable in the intestine so as to be used to consume or quantify glucose, ethanol, glycerol, cholesterol, a monosaccharide, a ketone, an aldehyde, or to generate electrical power.

20. The fuel biocell according to claim 15, wherein it comprises a cathode using glucose as oxidant and an anode using glucose as reductant, without the use of dioxygen.

21. The fuel biocell according to claim 18, wherein it comprises an anode based on a conductive material such as platinum, gold, graphite, for producing dioxygen in vivo, by connecting the biocathode and the anode to an electrical generator.

22. The fuel biocell according to claim 15, wherein it is suitable for operation in anaerobic conditions, mines, sea, space.

23. A biosensor for glucose, monosaccharide, ketone or aldehyde comprising an anode consisting of a platinum wire and a biocathode as defined in claim 1, for in vivo and in vitro applications, means for measuring the value of the reduction current of the glucose, monosaccharide, ketone or aldehyde being provided for estimating the level of glucose, monosaccharide, ketone or aldehyde.

24. The enzymatic biocathode according to claim 4, wherein it comprises at least one agent for the regeneration of the NADPH cofactor by catalyzing its reduction at the surface of the biocathode, the regeneration agent allowing an electro- or a photo-regeneration, being in this case photosensitive.

Description

[0056] On this drawing:

[0057] FIG. 1 is a schematic representation of the operation of an enzymatic biofuel cell;

[0058] FIG. 2 is a schematic representation of the operation of a biocell with a biocathode according to the invention;

[0059] FIG. 3 is a schematic representation of the oxidation of interfering molecules for existing glucose biosensors;

[0060] FIG. 4 is a schematic representation of the reduction of a glucose biosensor with a biocathode according to the invention without reduction of interfering molecules;

[0061] FIG. 5 is a schematic representation of a biocathode according to a first embodiment with electro-regeneration of the enzymatic cofactor;

[0062] FIG. 6 is a schematic representation of a biocathode according to another embodiment with electro-regeneration of the enzymatic cofactor;

[0063] FIG. 7 is a schematic representation of a biocathode according to another embodiment with electro-regeneration of the enzymatic cofactor;

[0064] FIG. 8 is a schematic representation of a biocathode in another embodiment with photo-regeneration of the enzyme cofactor;

[0065] FIG. 9 is a schematic representation of a biocathode in another embodiment with photo-regeneration of the enzyme cofactor;

[0066] FIG. 10 is a schematic representation of a glucose biocell with a biocathode in a first variant;

[0067] FIG. 11 is a schematic representation of a glucose biocell with a biocathode in a second variant;

[0068] FIG. 12 is a schematic representation of the reactions taking place at the anode during the operation of the biocell shown in FIG. 11;

[0069] FIG. 13 is a schematic representation of the reactions taking place at the biocathode during the operation of the biocell shown in FIG. 11;

[0070] FIG. 14 is a power versus voltage curve for the biocell shown in FIG. 11 at a glucose concentration of 20 mM in phosphate buffer at pH 7;

[0071] FIG. 15 is a schematic representation of a glucose biosensor with a biocathode according to the present invention; and

[0072] FIG. 16 is a curve of current intensity measured with the biosensor of FIG. 15 as a function of glucose concentration.

[0073] In the figures, the following legend is used: [0074] Cofactor NADPH [0075] Enzyme: aldose reductase [0076] Polymer for encaspulation of enzyme and mediator (nafion, chitosan) [0077] Layer of conductive particles (carbons, metals, . . . ) [0078] Conductor support [0079] Redox mediator for the regeneration of NADPH [0080] ((ex: pentamethylcyclopentadienyl-2,2V-bipyridine aqua)rhodium (III) [0081] Redox polymer [0082] Light [0083] Photosensitive molecule (ex. chlorophyll) [0084] .circle-solid. Electron acceptor (ex. Vitamin C) [0085] Photosensitive polymer [0086] Enzyme 2 for oxidation of glucose (ex: Glucose oxidase) [0087] Redox mediator for enzyme 2 (ex: Naphthoquinone, ferrocene, osmium complex) [0088] Counter electrode (gold, platinum)

[0089] The following examples illustrate the present invention without limiting its scope.

Example 1: Production of a Biocathode with Electroregeneration of the Cofactor at the Surface of the Electrode

[0090] A carbon particle ink is prepared in an aqueous solution containing 0.5% by weight of Tween80 and 5 to 10 mg/mL of carbon particles.

[0091] This ink is deposited on a carbon sheet.

[0092] After drying under vacuum for two hours, a layer of poly(methylene blue) is deposited on the carbon layer by electropolymerisation.

[0093] After rinsing with water, a 1 wt. % Nafion solution containing aldose reductase (100 μM) and its cofactor NADPH (1 mM) is applied and left to dry at room temperature for one hour.

[0094] The obtained biocathode is shown schematically in FIG. 5.

Example 2: Production of a Biocathode with Electroregeneration of the Cofactor Using a Redox Mediator

[0095] A carbon particle ink (5-10 mg/mL) is prepared in an aqueous solution containing 0.5 wt % Tween80.

[0096] This ink is deposited on a carbon sheet.

[0097] A 2 wt. % solution of chitosan containing aldose reductase (100 μM), its cofactor NADPH (1 mM) and a redox mediator pentamethylcyclopentadienyl-2,2V-bipyridine aqua) rhodium (III) (25 μM) is deposited on the carbon sheet, and then left to dry for 6 hours.

[0098] The obtained biocathode is shown schematically in FIG. 6.

Example 3: Production of a Biocathode with Electroregeneration of the Cofactor Using a Redox Polymer

[0099] A carbon particle ink (5-10 mg/mL) is prepared in an aqueous solution containing 0.5 wt % Tween80.

[0100] This ink is deposited on a carbon sheet.

[0101] After drying under vacuum for two hours, a layer of methylene green is electrodeposited on the carbon layer by cyclic voltametry.

[0102] A 2 wt. % solution of chitosan containing aldose reductase (100 μM), its cofactor NADPH (1 mM) is deposited on the methylene green layer, and then allowed to dry for 6 hours.

[0103] The obtained biocathode is shown schematically in FIG. 7.

Example 4: Production of a Biocathode with Photoregeneration of the Cofactor Using a Photosensitive Molecule

[0104] A carbon particle ink is prepared in an aqueous solution containing 0.5% by weight of Tween80.

[0105] This ink is deposited on a carbon sheet.

[0106] After drying under vacuum for two hours, a 1% by volume Nafion solution containing aldose reductase (100 μM), its cofactor NADPH (1 mM), ferrodoxin-NADP protein (100 μM), chlorophyll (100 μM) and vitamin C is applied.

[0107] The obtained biocathode is shown schematically in FIG. 8.

Example 5: Production of a Biocathode with Photoregeneration of the Cofactor Using a Photosensitive Polymer

[0108] A carbon particle ink is prepared in an aqueous solution containing 0.5% by weight of Tween80.

[0109] This ink is deposited on a carbon sheet.

[0110] After drying under vacuum for two hours, a 1% by volume solution of Nafion containing aldose reductase (100 μM) and its cofactor NADPH (1 mM) is applied to this carbon sheet and then left to dry for one hour.

[0111] The obtained biocathode is shown schematically in FIG. 9.

Example 6: Production of 100% Glucose Biocell

Production of a Bioanode

[0112] A carbon particle ink is prepared in an aqueous solution containing 0.5% by volume of Tween80.

[0113] This ink is deposited on a carbon sheet.

[0114] After drying under vacuum for two hours, a 2 wt. % solution of chitosan containing glucose oxidase (100 μM), its mediator naphthoquinone, is applied and everything is left to dry on air at room temperature for six hours.

Production of the Biocell

[0115] A 100% glucose biopile is then produced using the bioanode made above and a biocathode according to Example 3. This biocell oxidises glucose to glucolactone at the bioanode using the enzyme glucose oxidase and its mediator naphthoquinone and reduces glucose to sorbitol at the biocathode.

[0116] The obtained biocell is shown schematically in FIG. 10. In the scheme, the current produced by the biocell flows through a resistor R.

Example 7: Production of 100% Glucose Biocell

Production of the Biocell

[0117] A 100% glucose biocell is made using the bioanode made in Example 6 and a biocathode according to Example 2. This biopile oxidises glucose to gluconic acid at the bioanode using the enzyme glucose oxidase and its mediator naphthoquinone and reduces glucose to sorbitol at the biocathode.

[0118] The resulting biopile is shown schematically in FIG. 11. In the scheme, the current produced by the biocell flows through a voltmeter V.

[0119] FIGS. 12 and 13 show schematically the reactions occurring at the anode and biocathode, respectively.

[0120] At the anode, glucose is oxidised to gluconic acid by the action of glucose oxidase (GOx).

[0121] The mediator of glucose oxidase, in the represented example naphthoquinone (Naphto), is oxidised at the anode surface from its reduced form Naphto.sub.re to its oxidised form Naphto.sub.ox.

[0122] By doing this, electron transfer from the glucose to the bioanode can take place.

[0123] At the biocathode, glucose is reduced to sorbitol by the action of aldose reductase and its cofactor NADPH.

[0124] The NADPH cofactor is regenerated from its NADP form to its NADPH form using the redox mediator pentamethylcyclopentadienyl-2,2V-bipyridine aqua) rhodium (III), denoted RhMed, in FIG. 13, with RhMedred representing the reductant and RhMedox the oxidant of the redox couple.

[0125] The redox mediator is reduced at the cathode surface from its RhMedox form to its RhMedred form.

[0126] In this way, electrons are transferred from the biocathode to the glucose so that it can be reduced to sorbitol.

[0127] The curve of FIG. 14 shows the characteristics of the resulting biocell. The voltage of the open circuit biocell is 120 mV and it is capable of producing a power density of 3 μW/cm.sup.2 at a glucose concentration of 20 mM.

Example 8: Production of a Glucose Biosensor

[0128] A glucose biosensor is made using a biocathode according to Example 3 and a conventional counter electrode such as a gold or platinum counter electrode.

[0129] The resulting biosensor is shown schematically in FIG. 15. In the scheme, the current produced by the biocell flows through a voltmeter V.

[0130] From this biosensor, a calibration curve at zero voltage of the intensity measured using the biosensor versus glucose concentration can be obtained. This curve is shown in FIG. 16.