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WO2008062216A1 - Électrode - Google Patents

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Publication number
WO2008062216A1
WO2008062216A1 PCT/GB2007/004499 GB2007004499W WO2008062216A1 WO 2008062216 A1 WO2008062216 A1 WO 2008062216A1 GB 2007004499 W GB2007004499 W GB 2007004499W WO 2008062216 A1 WO2008062216 A1 WO 2008062216A1
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Prior art keywords
alkyl
electrode
hydrogen
phenyl
residue
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Inventor
Fraser Andrew Armstrong
Christopher Francis Blanford
Rachel Sarah Heath
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Oxford University Innovation Ltd
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Oxford University Innovation Ltd
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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M8/00—Fuel cells; Manufacture thereof
    • H01M8/16—Biochemical fuel cells, i.e. cells in which microorganisms function as catalysts
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M4/00—Electrodes
    • H01M4/86—Inert electrodes with catalytic activity, e.g. for fuel cells
    • H01M4/88—Processes of manufacture
    • H01M4/8803—Supports for the deposition of the catalytic active composition
    • H01M4/8807—Gas diffusion layers
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M4/00—Electrodes
    • H01M4/86—Inert electrodes with catalytic activity, e.g. for fuel cells
    • H01M4/88—Processes of manufacture
    • H01M4/8817—Treatment of supports before application of the catalytic active composition
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M4/00—Electrodes
    • H01M4/86—Inert electrodes with catalytic activity, e.g. for fuel cells
    • H01M4/90—Selection of catalytic material
    • H01M4/9008—Organic or organo-metallic compounds
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M4/00—Electrodes
    • H01M4/86—Inert electrodes with catalytic activity, e.g. for fuel cells
    • H01M4/90—Selection of catalytic material
    • H01M4/9075—Catalytic material supported on carriers, e.g. powder carriers
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
    • H01M4/00—Electrodes
    • H01M4/86—Inert electrodes with catalytic activity, e.g. for fuel cells
    • H01M2004/8678—Inert electrodes with catalytic activity, e.g. for fuel cells characterised by the polarity
    • H01M2004/8689—Positive electrodes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
    • Y02E60/30—Hydrogen technology
    • Y02E60/50—Fuel cells

Definitions

  • the invention relates to electrodes or electrode precursors for use in fuel cells, and to methods of production of said electrodes or electrode precursors.
  • Fuel cells are electrochemical devices that convert the chemical energy of a fuel directly into electrical and thermal energy.
  • a fuel cell consists of an anode and a cathode, which are electrically connected via an electrolyte.
  • a fuel which is often hydrogen, is fed to the anode where it is oxidised with the help of an electrocatalyst.
  • an oxidant such as oxygen (or air) takes place.
  • the electrochemical reactions which occur at the electrodes produce a current and thereby electrical energy.
  • thermal energy is also produced which may be harnessed to provide additional electricity or for other purposes.
  • Fuel cells may also be adapted to utilise other hydrocarbon fuels such as methanol or natural gas.
  • Fuel cells have many advantages over traditional energy sources. The major attractions of these systems are their energy efficiency and their environmental benefits. Fuel cells can be operated at an efficiency which is higher than almost all other known energy conversion systems and this efficiency can be increased further by harnessing the thermal energy produced by the cell. Further, fuel cells are quiet and produce almost no harmful emissions, even when running on fuels such as natural gas, since the system does not rely on the combustion of the fuel. Particularly advantageous are cells which operate on hydrogen, as these systems produce no emissions other than water vapour and their fuel source is renewable. There is therefore a significant interest in developing commercially viable fuel cells. Aside from the obvious environmental benefits, there is a considerable need for a new and renewable source which will provide the necessary security, in terms of energy provision in the future, to our highly energy dependent society.
  • a number of enzymes have been identified as useful as electrocatalysts in fuel cells. Examples include hydrogenase and laccase.
  • Examples include hydrogenase and laccase.
  • a new fuel cell is therefore required which utilises an enzyme but which overcomes or ameliorates the problems identified above, allowing for the enzyme to remain catalytically active for a longer time and/or to demonstrate an increased activity.
  • the present invention addresses these difficulties by providing a new electrode or electrode precursor and a method for producing the electrode or electrode precursor wherein the electrode or electrode precursor is modified with an organic residue to which an enzyme can bind. It has been surprisingly found that the organic residue improves physical attachment to and electrical contact with the enzyme, resulting in both an increased stability and an increased activity of the enzyme. Furthermore, the straightforward methodology used to prepare the novel electrodes and electrode precursors makes them a commercially- viable prospect.
  • the present invention therefore provides a method of preparing an enzyme-modified electrode or enzyme-modified electrode precursor comprising:
  • a monocyclic aromatic ring selected from an unsubstituted or substituted phenyl or 5- or 6-membered heteroaryl ring, said aromatic ring bearing a -N 2 + substituent and a further conjugated substituent selected from C 2-6 alkenyl and C 2-6 alkynyl; and (b) a counter ion; and (ii) exposing the modified electrode or electrode precursor surface to a blue copper oxidase enzyme such that the enzyme binds to the residue R.
  • the electrode or electrode precursor further comprises a blue copper oxidase enzyme which is bound to residue R.
  • the invention also provides a fuel cell comprising: (a) a fuel source which provides hydrogen to an anode;
  • a cathode at which the oxidant is reduced and which is electrically connected to the anode via an electrical conductor; and (e) an electrolyte which serves as a conductor for ions between the anode and the cathode, wherein the cathode is an enzyme-modified electrode in accordance with the invention.
  • a monocyclic aromatic ring selected from an unsubstituted or substituted phenyl or 5- or 6-membered heteroaryl ring, said aromatic ring bearing a conjugated substituent selected from C 2-6 alkenyl and C 2-6 alkynyl.
  • the enzyme for use in the invention is electrocatalytic, that is, it is useful in catalysing an electrochemical reaction which occurs in the region of the electrode.
  • Figure 1 depicts the relative rate of inactivation of TvL III film on both an unmodified and modified PGE (pyrolytic graphite electrode) electrode.
  • Figure 2 depicts a typical cyclic voltammetry scan of a stationary pyrolytic graphite 'edge' plane electrode in 4 mM anthracene-2-diazonium (A2D) solution (electrode geometric area 0.1 cm 2 , scan rate 50 mV s "1 ).
  • A2D anthracene-2-diazonium
  • Figure 3 demonstrates the change in electrocatalytic activity with time of a film of TvL III on an (a) A2D-modified PGE electrode and an (b) unmodified PGE electrode.
  • the black scan with italic label was taken with enzyme still present in the electrochemical cell solution.
  • Figure 4 depicts epifluorescence micrographs of TvL III tagged with fluorescein-5-EX on (a) a sanded PGE electrode surface and (b) a A2D-modified PGE electrode surface.
  • Figure 5 depicts an exemplary fuel cell according to the invention.
  • Figure 6 depicts the results of lifetime measurements carried out on four carbon cloth electrodes modified in accordance with the invention.
  • a Ci -4 alkyl group or moiety is a linear or branched alkyl group or moiety containing from 1 to 4 carbon atoms, for example a Ci -2 alkyl group or moiety containing from 1 to 2 carbon atoms.
  • Examples of Ci -4 alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl and t-butyl.
  • the alkyl moieties may be the same or different.
  • two alkenyl moieties may be the same or different.
  • a C 2-6 alkynyl group or moiety is a linear or branched alkynyl group or moiety containing from 2 to 6 carbon atoms, more preferably a C 2-4 alkynyl group or moiety containing from 2 to 4 carbon atoms, such as -C ⁇ CH, -C ⁇ C-C ⁇ CH or -C ⁇ C-CH 3 , in particular -C ⁇ CH or -C ⁇ C-C ⁇ CH, more preferably -C ⁇ CH.
  • two alkynyl moieties may be the same or different.
  • a halogen is typically chlorine, fluorine, bromine or iodine, preferably chlorine, bromine or fluorine, more preferably chlorine or fluorine.
  • Ci -4 alkoxy group is typically a said Ci -4 alkyl group which is attached to an oxygen atom.
  • a 5- or 6-membered heteroaryl group or moiety is a monocyclic 5- or 6- membered aromatic ring containing at least one heteroatom, for example 1, 2, 3 or 4 heteroatoms, selected from O, S and N.
  • the heteroaryl group or moiety is a 6- membered ring, the heteroatom or heteroatoms present are nitrogen atoms.
  • R is hydrogen or a Ci -4 alkyl group.
  • Preferred heteroaryl groups or moieties include pyrrolyl, pyrazolyl, imidazolyl, triazolyl, furanyl, oxazolyl, isoxazolyl, oxadiazolyl, thienyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and benzoquinone, more preferably pyrrolyl, pyrazolyl, imidazolyl, furanyl, oxazolyl, isoxazolyl, thienyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl and benzoquinone, still more preferably pyrrolyl, furanyl, thienyl, pyridinyl and benzoquinone, most preferably pyrrolyl
  • a 5- or 6-membered heterocyclyl group or moiety is a monocyclic non- aromatic, saturated or unsaturated C 5 -C 6 carbocyclic ring in which one or more, for example 1, 2 or 3, of the carbon atoms are replaced with a moiety selected from N, O, S, S(O) and S(O) 2 , and wherein one or more of the remaining carbon atoms is optionally replaced by a group -C(O)- or -C(S)-.
  • a group -C(O)- or -C(S)- preferably only one such carbon atom is replaced.
  • Suitable heterocyclyl groups and moieties include azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, tetrahydro furanyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, dithiolanyl, dioxolanyl, pyrazolidinyl, piperidyl, piperazinyl, thiomorpholinyl, S-oxo-thiomorpholinyl, S,S-dioxo- thiomorpholinyl, morpholinyl, 1,3-dioxolanyl, 1,4-dioxolanyl, trioxolanyl, trithianyl, imidazolinyl, pyrazolinyl, thioxolanyl, thi
  • Preferred heterocyclyl groups are piperidinyl, pyrrolidinyl, piperazinyl, imidazolidinyl, pyrazolidinyl, pyrazolinyl, imidazolinyl, thioxothiazolidinyl, oxopyrrolidinyl, oxothiazolidinyl and oxopyrazolidinyl groups. More preferred heterocyclyl groups are piperazinyl, piperidinyl, thioxothiazolidinyl, oxopyrrolidinyl, oxothiazolidinyl and oxopyrazolidinyl groups.
  • heteroaryl and heterocyclyl groups refer to an "N" moiety which can be present in the ring, as will be evident to a skilled chemist the N atom will be protonated (or will carry a substituent) if it is attached to each of the adjacent ring atoms via a single bond.
  • a C 3-7 carbocyclic moiety is a monocyclic non-aromatic saturated or unsaturated hydrocarbon ring having from 3 to 7 carbon atoms.
  • it is a saturated or mono-unsaturated hydrocarbon ring (i.e. a cycloalkyl moiety or a cycloalkenyl moiety) having from 3 to 7 carbon atoms.
  • Examples include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and their mono-unsaturated variants.
  • an electrode or electrode precursor is provided which is modified with an organic residue R.
  • an electrode is modified to provide a modified electrode.
  • an electrode precursor is modified to provide a modified electrode precursor.
  • An electrode precursor as used herein is a material which can undergo one or more subsequent processing steps in order to form an electrode.
  • an electrode precursor is typically a material which can be used to manufacture an electrode (e.g. carbon cloth or carbon powder).
  • the modification with a group R can therefore be carried out at any stage during the production of the electrode, including as an intermediate or final stage in the process. Unless otherwise indicated, any reference to 'an electrode' hereinbelow includes 'an electrode' and 'an electrode precursor'.
  • residue R results from diazonium coupling of a diazonium salt described above.
  • Diazonium coupling involving the electroreduction of diazonium salts, was chosen to create specific functional groups on the electrode surface. This flexible method allows a number of electrode surfaces, such as carbon-containing surfaces, to be covalently bound with organic molecules.
  • the electroreduction process first involves the use of a compound containing a terminal amine which is converted to a nitronium ion by addition of acid and nitrite at ice temperatures. This can be depicted as follows:
  • step (A) the reagents in step (A) being acid and nitrite on ice.
  • the diazonium salt, RN 2 + , and an appropriate counter ion usually stay in solution.
  • the RN 2 + cation is then coupled to the surface via formation of a radical with the release of nitrogen followed by a one-electron electrochemical reduction.
  • the organic residue R binds to an atom at the surface of the electrode.
  • the organic residue can bind to the surface of the electrode via a carbon-carbon bond between a carbon atom on the surface of the electrode and a carbon atom on organic residue R.
  • this is achieved by adding the diazonium salt to a cell containing an acid and the electrode which is to be modified, and allowing modification of the surface of the electrode. This can usefully be achieved by scanning through a range of potentials.
  • the modified electrode can then be removed from the cell and washed, for example with ethanol and water.
  • modification of the electrode surface occurs by heating the electrode in the presence of the diazonium salt (RN 2 + with an appropriate counter ion).
  • the electrode may be immersed in, or coated with, a diazonium salt solution and heated, e.g. at at least 4O 0 C (preferably at least 5O 0 C, for example up to 100 0 C or up to 8O 0 C). Heating may be continued for a suitable period, such as at least 10 minutes or up to 1 hour. About 30 minutes heating at 65 0 C has been found to provide effective results.
  • US5,554,739 discusses alternative means for linking diazonium salts to carbon surfaces.
  • a monocyclic aromatic ring selected from an unsubstituted or substituted phenyl or 5- or 6-membered heteroaryl ring, said aromatic ring bearing a -N 2 + substituent, and also bearing a further conjugated substituent selected from C 2-6 alkenyl and C 2-6 alkynyl.
  • R represents hydrogen or Ci -4 alkyl.
  • the diazonium salts of the invention comprise a cation of formula RN 2 + and a suitable counter ion.
  • the group R is an organic group, and represents the organic residue which modifies the surface of the electrode as a result of the electroreduction of the diazonium salt.
  • the organic residue comprises three or four fused or linked aromatic rings
  • the aromatic rings are fused or linked in a linear or substantially linear fashion.
  • the organic residue moiety comprises three phenyl rings fused together, preferably it represents anthracene, rather than phenanthrene.
  • one or more of the aromatic rings in such a moiety are 5- membered heteroaryl rings, these will not fuse or link in a completely linear way but should be fused or linked in such a way as to provide an aromatic moiety which is as close as is possible to a linear moiety.
  • At least one of the aromatic rings in the organic residue R is a phenyl ring.
  • the residue R comprises three aromatic rings, preferably two or three of the rings are phenyl rings.
  • the residue R comprises four aromatic rings, preferably two, three or four, more preferably three or four, most preferably four of the rings are phenyl rings.
  • the aromatic rings which make up the residue R are selected from phenyl and 5- or 6- membered heteroaryl rings.
  • suitable rings include pyrrolyl, pyrazolyl, imidazolyl, triazolyl, furanyl, oxazolyl, isoxazolyl, oxadiazolyl, thienyl, thiazolyl, isothiazolyl and thiadiazolyl, more preferably pyrrolyl, pyrazolyl, imidazolyl, furanyl, oxazolyl, isoxazolyl, thienyl, thiazolyl and isothiazolyl, still more preferably pyrrolyl, furanyl, and thienyl.
  • Most preferred 5- membered heteroaryl rings are pyrrolyl rings.
  • aromatic rings making up the residue R are 6-membered heteroaryl rings
  • suitable rings include pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl and triazinyl, more preferably pyridinyl, pyrimidinyl and pyrazinyl, still more preferably pyridinyl.
  • aromatic rings making up the residue R are 6-membered heteroaryl rings having at least one carbon atom, more preferably one or two carbon atoms, most preferably two carbon atoms, which are replaced, preferred groups include benzoquinone. Where a benzoquinone group is present, preferably it is present between and fused to two phenyl rings.
  • the residue R is a fused aromatic moiety where all aromatic rings present are fused together.
  • the aromatic rings are selected from phenyl, pyrrolyl, furanyl, thienyl, pyridinyl and benzoquinone.
  • the fused aromatic moiety comprises three aromatic rings, preferably each ring is a phenyl ring, or two rings are phenyl rings and the third ring is a pyridinyl or benzoquinone ring.
  • each ring is a phenyl ring, or one ring is a phenyl ring and the other ring is a pyrrolyl, furanyl or thienyl ring.
  • the residue R is a naphthalenyl, indolyl, benzofuranyl, benzothienyl, anthracenyl, benzoquinolinyl, acridinyl or anthracenyl-9,10-dione moiety, most preferably an anthracenyl moiety.
  • preferred aromatic rings are phenyl and pyridinyl rings, more preferably phenyl rings.
  • preferred residue R groups include biphenyl, triphenyl, 1,2- diphenylethene and 1 ,4-distyrylbenzene moieties.
  • one of the aromatic rings which subsequently forms the residue R bears the -N 2 + substituent. Where 3 or 4 aromatic rings are present, preferably this is a terminal aromatic ring.
  • the aromatic rings may then be further unsubstituted or substituted. It is possible for every carbon atom in the residue R to be substituted (excluding, of course, the bridgehead carbon atoms in the fused variants and the carbon atom bearing the -N 2 + substituent). If every possible carbon atom is substituted, then the preferred substituents are halogen atoms, more preferably fluorine atoms.
  • the aromatic rings which make up the residue R are each unsubstituted or substituted by 1, 2 or 3, more preferably 1 or 2, unsubstituted substituents which are the same or different when two or more are present, and which are selected from halogen atoms and hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C M alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R, -NR R",
  • R is hydrogen or Ci -4 alkyl and R' and R", which are the same or different, represent hydrogen, C M alkyl or phenyl.
  • More preferred substituents include halogen atoms and hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, Ci -4 alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R, -NR'R ", -SO 2 NR'R", phenyl and 5- or 6-membered heteroaryl groups, wherein R is hydrogen or Ci -4 alkyl, R' represents hydrogen or Ci -4 alkyl, and R" represents hydrogen, Ci -4 alkyl or phenyl.
  • Still more preferred substituents include halogen atoms and hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, Ci -4 alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R and -S0 2 NR'R", wherein R is hydrogen or C M alkyl, R' represents hydrogen or Ci -4 alkyl, and R" represents hydrogen, C M alkyl or phenyl.
  • substituents include halogen atoms and hydroxy, nitro, Ci -2 alkyl, -COR -COOR, -SH, -SO 3 R, cyano and -SCN groups wherein R is hydrogen or Ci -2 alkyl, more preferably halogen atoms and methyl, hydroxy, nitro and -COOH groups, e.g. halogen atoms and hydroxy, nitro and -COOH groups.
  • the residue R is unsubstituted or substituted with 1 , 2 or 3 substituents as defined above, more preferably it is unsubstituted or substituted with 1 or 2 substituents as defined above, still more preferably it is unsubstituted or substituted with 1 substituent as defined above.
  • the fused aromatic moiety is unsubstituted.
  • the aromatic cation comprises a monocyclic aromatic ring selected from an unsubstituted or substituted phenyl and 5- or 6-membered heteroaryl ring, preferably the aromatic ring is a phenyl or pyridinyl group, more preferably a phenyl group.
  • the monocyclic aromatic ring bears, in addition to the -N 2 + substituent, a conjugated substituent selected from C 2-6 alkenyl and C 2-6 alkynyl.
  • a conjugated substituent selected from C 2-6 alkenyl and C 2-6 alkynyl.
  • this substituent in addition to containing a conjugated portion, is also in conjugation with the monocyclic aromatic ring.
  • the monocyclic aromatic ring which make up the residue R is further unsubstituted or substituted by 1, 2 or 3, more preferably 1 or 2, unsubstituted substituents which are the same or different when two or more are present, and which are selected from halogen atoms and hydroxy, Ci -4 alkyl, C 2 ⁇ alkenyl, C 2-4 alkynyl, C 1-4 alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R, -NR'R", -S0 2 NR'R", phenyl, C 3-7 carbocyclyl, 5- or 6-membered heteroaryl and 5- or 6- membered heterocyclyl groups, wherein R is hydrogen or Ci -4 alkyl and R' and R", which are the same or different, represent hydrogen, Ci -4 alkyl or phenyl.
  • the monocyclic aromatic is further unsubstituted or substituted by only 1 or 2 substituents selected from halogen atoms and hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, Ci -4 alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R, -NR'R", -S0 2 NR'R", phenyl and 5- or 6-membered heteroaryl groups, wherein R is hydrogen or Ci -4 alkyl, R' represents hydrogen or Ci -4 alkyl, and R" represents hydrogen, Ci -4 alkyl or phenyl.
  • substituents include halogen atoms and hydroxy, C) -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C M alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R and -S0 2 NR'R", wherein R is hydrogen or Ci -4 alkyl, R' represents hydrogen or Ci -4 alkyl, and R" represents hydrogen, Ci -4 alkyl or phenyl, more preferably Ci -4 alkyl, C 2-4 alkenyl and C 2-4 alkynyl. Most preferably the monocyclic aromatic ring is further unsubstituted.
  • most preferable aromatic cations include 4-vinylbenzenediazonium, this being the cation of a diazonium salt derived from 4- aminostyrene (otherwise known as 4-vinylaniline).
  • the diazonium salt preferably comprises (a) an aromatic cation of formula (I) or (II), and (b) a counter ion:
  • rings A and D represent phenyl or a 5- or 6-membered heteroaryl ring
  • rings B and C may each be present or absent and, when present, each represent phenyl or a 5- or 6-membered heteroaryl ring
  • the rings A, B, C and D are fused together via a pair of adjacent carbon atoms.
  • ring B is absent, then ring A is fused to ring C (where present) or ring D (when ring C is also absent).
  • ring C is absent, then ring D is fused to ring B (where present) or ring A (when ring B is also absent).
  • A, B, C and D are phenyl or pyridinyl rings, more preferably phenyl rings.
  • B or C is absent, for example both B and C may be absent.
  • X 2 represents a bond.
  • X 3 represents a bond.
  • rings A, B, C and D are unsubstituted or substituted by 1, 2 or 3 substituents which are the same or different and are selected from halogen atoms and hydroxy, C M alkyl, C 2-4 alkenyl, C 2-4 alkynyl, Ci -4 alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R, -NR'R' ', -S0 2 NR'R' ', phenyl, C 3-7 carbocyclyl, 5- or 6-membered heteroaryl and 5- or 6-membered heterocyclyl groups, wherein R is hydrogen or Ci -4 alkyl and R' and R", which are the same or different, represent hydrogen, C M alkyl or phenyl.
  • substituents include halogen atoms and hydroxy, C M alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C M alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R, -NR'R", -S0 2 NR'R", phenyl and 5- or 6-membered heteroaryl groups, wherein R is hydrogen or Ci -4 alkyl, R' represents hydrogen or Ci -4 alkyl, and R" represents hydrogen, Ci -4 alkyl or phenyl.
  • Still more preferred substituents include halogen atoms and hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C M alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R and -S0 2 NR'R", wherein R is hydrogen or C M alkyl, R' represents hydrogen or C M alkyl, and R" represents hydrogen, C M alkyl or phenyl.
  • substituents include halogen atoms and hydroxy, nitro, Cj -2 alkyl, -COR -COOR, -SH, -SO 3 R, cyano and -SCN groups wherein R is hydrogen or Ci -2 alkyl, more preferably halogen atoms and hydroxy, nitro and -COOH groups.
  • substituents on ring A in formula (II) include C M alkyl and Ci -4 alkoxy.
  • Preferred substituents on rings B, C and D in formula (II) include halogen atoms and hydroxy, nitro, C U2 alkyl, -COR -COOR, -SH, -SO 3 R, cyano and -SCN groups wherein R is hydrogen or Ci -2 alkyl, more preferably halogen atoms and hydroxy, nitro and -COOH groups.
  • Particularly preferred substituents on ring D in formula (II) include nitro groups. More preferably the diazonium salt comprises (a) an aromatic cation of formula (IA), and (b) a counter ion:
  • A represents a bond, or carbon or nitrogen; either:
  • E and F are the same or different and represent a bond or carbon or nitrogen, provided that at least one of E and F represents carbon or nitrogen; - either:
  • clearly no substituent is present at that position.
  • R 1 or R 2 respectively is absent.
  • the -N 2 + substituent present in the cation of formula (IA) is preferably borne at either the B or the C position.
  • the group to which it is bonded i.e. either B or C
  • the carbon atoms present in the aromatic rings containing A, B, C, D, E and F are unsubstituted or substituted by a substituent selected from halogen atoms and hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, Ci -4 alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R, -NR'R", -S0 2 NR'R", phenyl, C 3-7 carbocyclyl, 5- or 6-membered heteroaryl and 5- or 6-membered heterocyclyl groups, wherein R is hydrogen or Ci -4 alkyl and R' and R", which are the same or different, represent hydrogen, C M alkyl or phenyl.
  • any combination of A, B, C, D, E and F may be chosen provided that the rings of which they are members are aromatic. More preferred substituents on the carbon atoms in the rings of formula (IA), including at R 1 and R 2 when these represent a substituent, include halogen atoms and hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, Ci -4 alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R, -NR'R", -SO 2 NR R", phenyl and 5- or 6-membered heteroaryl groups, wherein R is hydrogen or Ci -4 alkyl, R' represents hydrogen or Ci -4 alkyl, and R" represents hydrogen, C M alkyl or phenyl.
  • Still more preferred substituents include halogen atoms and hydroxy, C M alkyl, C 2-4 alkenyl, C 2-4 alkynyl, Ci -4 alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R and -S0 2 NR'R", wherein R is hydrogen or Ci -4 alkyl, R' represents hydrogen or Ci -4 alkyl, and R" represents hydrogen, Ci -4 alkyl or phenyl.
  • substituents include halogen atoms and hydroxy, nitro, Ci -2 alkyl, -COR -COOR, -SH, -SO 3 R, cyano and -SCN groups wherein R is hydrogen or Cj -2 alkyl, more preferably halogen atoms and hydroxy, nitro and -COOH groups.
  • the cation of formula (IA) is substituted in total by only 1, 2 or 3 substituents as defined above, more preferably it is unsubstituted or substituted with 1 or 2 substituents as defined above, still more preferably it is unsubstituted or substituted with 1 substituent as defined above.
  • the cation of formula (IA) is unsubstituted.
  • A represents a bond or carbon or nitrogen, more preferably A represents a carbon.
  • A represents a bond
  • B represents -NR-, -O- or -S- where R represents hydrogen or Cj -4 alkyl, more preferably where R represents hydrogen or Ci -2 alkyl.
  • R represents hydrogen or Cj -4 alkyl, more preferably where R represents hydrogen or Ci -2 alkyl.
  • a represents a bond preferably C represents carbon.
  • a represents a bond preferably D represents carbon.
  • A represents carbon or nitrogen
  • B represents carbon.
  • C represents carbon.
  • D represents carbon.
  • the ring containing A, B, C and D is a phenyl, pyridinyl, pyrrolyl, furanyl or thienyl ring, more preferably a phenyl ring.
  • the ring containing A, B, C and D is unsubstituted or substituted with 1, 2 or 3 substituents selected from halogen atoms and hydroxy, C M alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C M alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R and -SO 2 NR'R", wherein R is hydrogen or Ci -4 alkyl, R' represents hydrogen or Ci -4 alkyl, and R" represents hydrogen, C M alkyl or phenyl.
  • the ring containing A, B, C and D is unsubstituted or substituted with 1 or 2, more preferably 1, substituents selected from halogen atoms and hydroxy, cyano, nitro, -SCN and -COOH, more preferably from halogen atoms (in particular chlorine atoms) or cyano, nitro, -SCN or -COOH groups.
  • substituents selected from halogen atoms and hydroxy, cyano, nitro, -SCN and -COOH, more preferably from halogen atoms (in particular chlorine atoms) or cyano, nitro, -SCN or -COOH groups.
  • the ring containing A, B, C and D is unsubstituted.
  • E and F are the same or different and represent a bond or carbon or nitrogen, provided that at least one of E and F represents carbon or nitrogen. Furthermore, in formula (IA) when both E and F represent carbon or nitrogen then G and H are the same or different and represent carbon or nitrogen. However, when one of E and F represents a bond, then one of G and H represents carbon and the other of G and H represents -NR-, -O- or -S- where R represents hydrogen or Ci -4 alkyl.
  • R 1 and R 2 are either both hydrogen or a substituent (e.g. they are both hydrogen), or they form, together the carbon atoms (i.e.
  • a phenyl ring or a 5- or 6-membered heteroaryl ring said phenyl ring or 5- or 6-membered heteroaryl ring being unfused or fused to a further phenyl rriinnge o orr 55-- o orr 66--mmeemmbbeerreedd hheetteerrooaarrvyll r riinnge..
  • R 1 and R 2 are both hydrogen or a substituent (e.g. they are both hydrogen), preferably the ring containing E, F, G and H is phenyl, pyrrolyl, furanyl or thienyl. In a preferred embodiment, when R 1 and R 2 are both hydrogen or a substituent (e.g.
  • the ring containing E, F, G and H is unsubstituted or substituted with 1 or 2, more preferably 1 , substituents selected from halogen atoms and hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, Ci -4 alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R and -S0 2 NR'R", wherein R is hydrogen or Ci -4 alkyl, R' represents hydrogen or Ci -4 alkyl, and R" represents hydrogen, Ci -4 alkyl or phenyl.
  • R 1 and R 2 together with the carbon atoms to which they are bonded, form an unfused or fused phenyl ring or a 5- or 6-membered heteroaryl ring, preferably the ring containing E, F, G and H is unsubstituted or substituted with 1 or 2, more preferably 1, substituents selected from halogen atoms and hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, Ci -4 alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R and -S0 2 NR'R", wherein R is hydrogen or Cj -4 alkyl, R' represents hydrogen or C M alkyl, and R" represents hydrogen, C M alkyl or phenyl.
  • R 1 and R 2 together with the carbon atoms to which they are bonded, form an unfused or fused phenyl ring or a 5- or 6-membered heteroaryl ring, preferably they form a phenyl ring.
  • R 1 and R 2 together with the carbon atoms to which they are bonded, form a phenyl ring or a 5- or 6-membered heteroaryl ring which is fused to a further phenyl ring or 5- or 6-membered heteroaryl ring, preferably they are fused to a further phenyl ring.
  • R 1 and R 2 together with the carbon atoms to which they are bonded, form an unfused or fused phenyl ring or a 5- or 6-membered heteroaryl ring, preferably they form a phenyl ring or a naphthalene group.
  • R 1 and R 2 form a ring or a fused ring
  • preferred substituents include halogen atoms and hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, Ci -4 alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R, -NR'R", -SO 2 NR'R", phenyl, C 3-7 carbocyclyl, 5- or 6-membered heteroaryl and 5- or 6-membered heterocyclyl groups, wherein R is hydrogen or Ci -4 alkyl and R' and R", which are the same or different, represent hydrogen, C M alkyl or phenyl.
  • R 1 and R 2 together with the carbon atoms to which they are bonded, form a phenyl ring or a 5- or 6-membered heteroaryl ring
  • the carbon atoms in the ring which they form (and which are capable of being substituted) are each unsubstituted or substituted by a substituent selected from halogen atoms and hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C M alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R, -NR'R", -SO 2 NR'R", phenyl and 5- or 6-membered heteroaryl groups, wherein R is hydrogen or Ci -4 alkyl, R' represents hydrogen or Ci -4 alkyl, and R" represents hydrogen, Ci -4 alkyl or phenyl.
  • Still more preferred substituents include halogen atoms and hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, Ci -4 alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R and -SO 2 NR R", wherein R is hydrogen or Ci -4 alkyl, R' represents hydrogen or Ci -4 alkyl, and R" represents hydrogen, Ci -4 alkyl or phenyl.
  • substituents include halogen atoms and hydroxy, nitro, Ci -2 alkyl, -COR -COOR, -SH, -SO 3 R, cyano and -SCN groups wherein R is hydrogen or Ci -2 alkyl, more preferably halogen atoms and hydroxy, nitro and -COOH groups. Where more than one carbon atom in the ring is substituted, the substituents may be the same or different. In a most preferred embodiment, when R 1 and R 2 , together with the carbon atoms to which they are bonded, form a phenyl ring or a 5- or 6-membered heteroaryl ring, preferably the carbon atoms in the ring which they form are each unsubstituted.
  • the diazonium salt comprises (a) an aromatic cation of formula (IB), and (b) a counter ion:
  • A, B, D, E and F are as defined above in relation to formula (IA);
  • I and J are the same or different and represent a bond, or a carbon or nitrogen, provided that at least one of I and J represents carbon or nitrogen; and wherein the carbon atoms in each ring are themselves unsubstituted or substituted by a substituent, which are the same or different when two or more are present, selected from halogen atoms and hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C M alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R, -NR'R", -SO 2 NR'R", phenyl, C 3-7 carbocyclyl, 5- or 6-membered heteroaryl and 5- or 6-membered heterocyclyl groups, wherein R is hydrogen or Ci -4 alkyl and R' and R", which are the same or different, represent hydrogen, Ci
  • R is hydrogen or a C M alkyl group.
  • preferred substituents on the ring containing I and J include halogen atoms and hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, Ci -4 alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R, -NR 'R", -SO 2 NR'R", phenyl and 5- or 6-membered heteroaryl groups, wherein R is hydrogen or Ci -4 alkyl, R' represents hydrogen or Ci -4 alkyl, and R" represents hydrogen, Ci -4 alkyl or phenyl.
  • Still more preferred substituents include halogen atoms and hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, Ci -4 alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R and -S0 2 NR'R", wherein R is hydrogen or C M alkyl, R' represents hydrogen or Ci -4 alkyl, and R" represents hydrogen, Q -4 alkyl or phenyl.
  • substituents include halogen atoms and hydroxy, nitro, Ci -2 alkyl, -COR -COOR, -SH, -SO 3 R, cyano and -SCN groups wherein R is hydrogen or Ci -2 alkyl, more preferably halogen atoms and hydroxy, nitro and -COOH groups. Where more than one carbon atom in the ring is substituted, the substituents may be the same or different. In a most preferred embodiment, the ring containing I and J is unsubstituted.
  • the diazonium salt comprises (a) an aromatic cation of formula (IC), and (b) a counter ion:
  • each R a which is the same or different when m is 2 or 3, represents a halogen atom or a hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, Ci -4 alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R or -SO 2 NR'R" group, wherein R is hydrogen or Cj -4 alkyl, R' represents hydrogen or Ci -4 alkyl, and R" represents hydrogen, Ci -4 alkyl or phenyl; each R b , which is the same or different when n is 2, represents a halogen atom or a hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, Ci -4 alkoxy, cyano, nitro, - -
  • R is hydrogen or Ci -4 alkyl
  • R' represents hydrogen or Ci -4 alkyl
  • R" represents hydrogen, Ci -4 alkyl or phenyl.
  • m is zero, 1 or 2, more preferably zero or 1.
  • preferred R a groups include halogen atoms and hydroxy, cyano, nitro, -SCN and -COOH groups, more preferably halogen atoms (in particular chlorine atoms) or cyano, nitro, -SCN or -COOH groups, still more preferably a halogen atom or a nitro or -SCN group, most preferably a halogen atom.
  • Preferred halogen atoms for R a include chlorine and fluorine, more preferably chlorine. Most preferably m is zero.
  • n is zero or 1.
  • preferred substituents include hydroxy, Ci -4 alkyl, nitro, -COOR and -S0 2 NR'R", wherein R is hydrogen or Cj -4 alkyl, R' represents hydrogen or Ci -4 alkyl, and R" represents hydrogen, Ci -4 alkyl or phenyl. Most preferably it is unsubstituted or substituted with 1 substituent selected from hydroxy, Ci -2 alkyl, nitro, -COOH or -SO 2 NHPh, more preferably selected from hydroxy and Ci -2 alkyl (e.g. methyl). Most preferably n is zero.
  • R c groups include halogen atoms and hydroxy, Ci -4 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, Ci -4 alkoxy, cyano, nitro, -SCN, -COR, -COOR, -SR, -SO 3 R and -SO 2 NR R" groups, wherein R is hydrogen or Ci -4 alkyl, R' represents hydrogen or C ⁇ alkyl, and R" represents hydrogen, Ci -4 alkyl or phenyl.
  • R c groups include halogen atoms and hydroxy, nitro, Ci -2 alkyl, -COR -COOR, -SH, -SO 3 R, cyano and -SCN groups wherein R is hydrogen or Ci -2 alkyl, more preferably halogen atoms and hydroxy, nitro and -COOH groups. Most preferably p is zero.
  • the diazonium salts for use in the invention can readily be prepared from the corresponding amine derivatives (i.e. containing a group -NH 2 in place of the -N 2 + group). Many of these amine derivatives are commercially available and, where they are not commercially available, can be prepared by analogy with synthetic strategies which will be known to the person skilled in the art. As noted above, a suitable method for conversion of an amine derivative into a diazonium salt involves the conversion of a terminal amine to a nitronium ion by the addition of acid and nitrite at ice temperatures.
  • aromatic cation is a fused aromatic moiety
  • preferred cations include: 3-nitroanthracene-2 -diazonium, 1 -thiocyanatoanthracene-2-diazonium, benzo[g]quinoline-3-diazonium, - 3-chloro-9,10-dioxo-9,10-dihydroanthracene-2-diazonium,
  • aromatic cation is a fused aromatic moiety
  • a preferred subset of cations are tricyclic aromatic moieties such as: acridine-2-diazonium, acridine-3-diazonium, anthracene-2-diazonium, anthracene- 1 -diazonium - 3-nitroanthracene-2-diazonium, l-thiocyanatoanthracene-2-diazonium, benzo[g]quinoline-3-diazonium, and
  • preferred cations include: lH-indole-5-diazonium,
  • the diazonium salt of formula (IC) is an anthracene-2- diazonium.
  • the residue R is an anthracene group.
  • the diazonium salt comprises the cation RN 2 + and also a suitable counter ion which balances the charge.
  • suitable counter ions There are many suitable counter ions which will be apparent to the person skilled in the art, with exemplary counter ions including halogen ions, BF 4 " and PF 6 " , with halogen ions being preferred.
  • the diazonium salts are suitably used in the invention in the form of a solution comprising the cation RN 2 + and the counter ion. Suitable methods for preparing diazonium solutions are found in Langmuir, 2005, 21(8), 3362.
  • the electrode which can be modified according to the invention may be selected from a multitude of materials which will be apparent to the skilled person.
  • the material In the case of an electrode, the material must of course be capable of functioning as an electrode in an electrochemical cell.
  • the material In the case of an electrode precursor, the material must be one which can be used to manufacture an electrode. In either case, the material must also be capable of modification with an organic residue R, preferably by participating in an electroreduction reaction of a diazonium salt in order to form an electrode surface which is modified with organic residue R.
  • preferred electrodes comprise carbon, a metal, a semiconductor or a composite material containing carbon, metal or plastic onto which a coating of carbon has been deposited, as well as combinations thereof.
  • Suitable forms of carbon include glassy or amorphous carbon, highly oriented pyrolytic graphite, pyrolized photoresists, pyrolized Teflon (RTM), carbon fibers, carbon blacks, carbon nanotubes and diamond.
  • Particularly preferred forms of carbon include glassy or amorphous carbon, as well as carbons that have a high surface area, either naturally or through pore engineering.
  • Suitable metals include noble metals such as gold and platinum, as well as industrial metals such as iron, zinc, nickel, cobalt, copper and palladium.
  • Particularly preferred metals include gold.
  • Suitable semiconductors include silicon and GaAs, more preferably silicon.
  • the electrode comprises carbon in one of its many forms outlined above, gold or silicon, more preferably carbon or gold.
  • the electrode comprises carbon, for example it may be a pyrolytic graphite electrode (PGE).
  • PGE pyrolytic graphite electrode
  • this may be advantageous over other materials because carbon electrodes are often significantly cheaper than other modified electrodes which have been employed with the aim of improving stability of enzymes or of improving the length of time on which they can be immobilised on the electrode.
  • preferred materials include carbon, a metal or a semi-conductor. Preferred types of carbon, metal or semi-conductor are those described above for the preferred electrodes.
  • the electrode precursor is a material which can be used to manufacture the preferred electrodes described above.
  • the electrode precursor is a powder, solution or cloth suitable for coating onto a base material to form an electrode.
  • Particularly preferred electrode precursors include carbon cloth and carbon powder.
  • the electrodes prepared according to the invention which bear an oxidase catalyst will be useful as cathodes in an electrochemical cell.
  • Suitable enzymes for use in the invention are blue copper oxidase enzymes capable of acting as electrocatalysts on the electrodes of the invention and in the electrochemical cells of the invention. These oxidases are also known as blue multi-copper oxidases, and are a group of copper-containing enzymes that oxidize organic substrates and concomitantly reduce O 2 to water. Multi-copper oxidases are generally described in Solomon et al, Chem. Rev. 96, 2563-2605, and the blue copper active site is investigated in Solomon et al, Pure Appl. Chem. 70, 799-808. To fall into this class, an enzyme must contain at least one of each type (Tl, T2 and T3) of the spectroscopically defined copper centres.
  • laccases which are the simplest in the class since they contain only four Cu atoms
  • analogues of laccase in particular human analogues to laccase (e.g. ceruloplasmin) and ascorbate oxidase, which occurs in higher plants.
  • oxoreductases such as phenoxazinone synthase ⁇ Streptomyces antibioticus), bilirubin oxidase (Myrothecium verrucaria and Trachyderma tsudondae), dihydrogeodin oxidase, sulochrin oxidase (fungal enzymes involved in the synthesis of grisans such as griseofulvin and geodin) and fet3 (an extracellular membrane-bound protein in Saccharomyces cerevisiae).
  • oxoreductases such as phenoxazinone synthase ⁇ Streptomyces antibioticus), bilirubin oxidase (Myrothecium verrucaria and Trachyderma tsudondae), dihydrogeodin oxidase, sulochrin oxidase (fungal enzymes involved in the synthesis of grisans such as griseofulvin and geodin) and fet3 (an extracellular
  • Bilirubin oxidase in particular, as with laccase, this enzyme is a monomeric glycoprotein and has a highly stable structure which can therefore withstand a reasonable amount of variation in temperature and pH allowing more flexibility in the operation of the fuel cell.
  • the invention also encompasses the use of other blue copper oxidases which act as reductases for oxidants other than oxygen which may be employed in the fuel cell, for example fumarate reductase.
  • Fragments, variants, functional mimetics and derivatives of the above described blue copper oxidases may also be used provided they maintain oxidase activity.
  • glycosylated, hydroxylated or phosphorylated oxidases or those modified by the addition of an affinity tag such as a histidine stretch, a strep-tag or a FLAG-tag.
  • Preferred blue copper oxidases for use in the invention include laccases, human ceruloplasmin, plant ascorbate oxidase, Bilirubin oxidase, fumarate reductase and analogues thereof, more preferably laccases and analogues thereof.
  • laccases these are typically blue, multi-copper oxidases widely found in fungi, higher plants, and (more recently) prokaryotes.
  • blue copper oxidases which are laccases are 11 amino acid residues which bind the four catalytic copper ions. Of these residues, 10 are histidine and the other is cysteine.
  • laccase catalyses the oxidation of a range of aromatic and phenolic substrates with the accompanying four- electron reduction of molecular oxygen to water.
  • Laccases have been employed in technological applications such as delignification in the paper industry, bioremediation, bleaching in washing powders and decolourisation of dyes, wine clarification and as biosensors for detection of drugs and phenols. Recently, Trametes versicolor laccase III (TvL III) has been used as the cathode catalyst for enzymatic hydrogen-oxygen biological fuel cells.
  • Preferred laccases are fungal laccases (Claus, Micron 35, 93-96; Baldrian, FEMS Microbiol. Rev. 30, 215-242; Thurston, Microbiology, 140, 19-26).
  • the natural co-substrates for fungal laccases are phenolic products of lignin degradation that are oxidised to radicals.
  • these enzymes therefore catalyse the reduction of oxygen to water with no intermediate.
  • these enzymes typically operate at potentials of 0.6V or greater, although lower-potential variants are also envisaged.
  • fungal laccases include those secreted by the white rot fungus Coriolus hirsutus, and the fungus Trametes versicolor.
  • Other useful fungi as the source of the laccase include Pycnoporus cinnabarinus, as well as laccase from Rhus vernicifera. These fungal laccases are distinguished among other laccases and so-called 'tree' laccases, by having very high reduction potentials for all copper sites. This makes them extremely useful for technological application as oxidation catalysts.
  • Laccases can be divided into low, medium and high potential enzymes depending on the potential of the Tl copper (Shleev et al, Biochem. J., 385, 745-754).
  • High potential laccases are able to reduce O 2 at a potential close to the thermodynamic potential for O 2 reduction.
  • T. versicolor laccase reduces O 2 at a potential (0.79 V) which is ca. 400 mV from the thermodynamic potential for O 2 reduction.
  • Tree laccases such as Rhus vernicifera laccase have typically low potentials for all sites (Tl Cu 0.43 V, T2 Cu 0.39 V, T3 Cu 0.48 V vs. standard hydrogen electrode, SHE).
  • fungal laccases Compared to other laccases, fungal laccases have higher reduction potentials for the Cu sites and generally fall into either the middle or high potential class. Preferably high potential laccases are employed in the invention.
  • a pH of around 4 to 6 can mean that both fuel cell catalysts can work in the same buffer in the fuel cell (which is essential for a 'membraneless' fuel cell).
  • electrodes of the invention which employ laccase are improved over known electrodes because the laccase is retained on the electrode surface for a greater length of time, and also has an improved activity. Furthermore, it has surprisingly been found that the laccase is actually more stable when attached to the electrode than when in solution, particularly where the electrode is kept hydrated and cool (e.g. between about 0 and 10 0 C, such as between 2 and 5 0 C.
  • Fragments, variants, functional mimetics and derivatives of the above described oxidases may also be used provided they maintain oxidase activity.
  • oxidase activity For example, also included are glycosylated, hydroxylated or phosphorylated oxidases, or those modified by the addition of an affinity tag such as a histidine stretch, a strep-tag or a FLAG-tag.
  • oxidases such as laccases which have been genetically modified, provided that the genetic modification does not prevent the modified oxidase from acting as an electrocatalyst.
  • laccases could be modified by changing a reasonably small number of residues, such that they still have a relatively high degree of homology with the unmodified laccase.
  • laccases such as Pycnoporus cinnabarinus laccase which are genetically modified by the addition of a peptide such as a 12-residue peptide that is known to stick to graphite. All such genetically modified oxidases are included provided their oxidase capabilities are not altered and that they are still suitable for use in the fuel cells of the invention.
  • the enzymes for use in the invention can generally be obtained from suitable fungi, yeast or bacteria which are available commercially.
  • the fungi, yeast or bacteria may be cultured to provide a sufficient quantity of enzymes to use in the fuel cell. This may be carried out, for example, by culturing the enzyme in accordance with known techniques. Cells may then be harvested, isolated and purified by any known technique.
  • this may be carried out for example by applying the enzyme or a concentrated solution thereof to the electrode surface, e.g. by pipette.
  • a potential may be applied to the electrode during this period if desired.
  • the potential enables the degree of coating with the enzyme to be easily monitored.
  • the potential will be increased and then subsequently decreased within a range of from approximately +45OmV to +100OmV vs. SHE and the potential cycled in this manner for up to 10 minutes at a rate of 10m V/s, typically for about 5 or 6 minutes.
  • the film of enzyme is simply allowed to grow without application of a potential.
  • the modified surface can be rotated under oxygen, with a film of enzyme forming as the enzyme gradually adsorbs onto the modified surface.
  • the enzyme may be applied in a sub-monolayer, a monolayer, or as multiple layers, for example 2, 3, 4 or more layers.
  • the enzyme need not be applied to the entire surface of the electrode.
  • at least 10% of the available surface of the electrode is coated with enzyme.
  • at least 25%, 50%, 75% or 90% of the available surface is coated with enzyme.
  • the "available surface" of the electrode is the surface which is in contact with the fuel or with the oxidant respectively.
  • the 'available surface' is the surface which is in contact with the electrolyte.
  • typically sufficient modified electrode precursor is used in the subsequent manufacture of an electrode to provide an electrode in which at least 10%, preferably at least 25%, 50%, 75% or 90% of the available surface of the electrode is coated with enzyme.
  • an electrode precursor such as carbon powder may be modified with a residue R to provide a surface-modified electrode precursor.
  • the surface-modified electrode precursor is then used to manufacture a surface-modified electrode, for example by coating a surface-modified carbon powder onto a base material to form a surface-modified electrode.
  • An enzyme may then be adsorbed onto the modified surface of the electrode to provide an enzyme-modified electrode.
  • the enzyme-adsorption may be carried out prior to manufacture of the electrode to provide an enzyme-modified electrode precursor.
  • the enzyme-modified electrode precursor may then be used to manufacture an electrode.
  • the electrode (or electrode precursor) surface is modified using a combination of a diazonium salt and Nafion.
  • Nafion is a proprietary proton-conducting ionomer consisting of a fluorinated polyethylene backbone with sulfonic acid side groups that has been shown to stabilise enzymes, particularly in a fuel cell context. This embodiment has been found to further enhance the electrocatalytic stability and lifetime of the electrodes of the invention.
  • the Nafion may be a derivatised form of Nafion, or may be ion-exchanged Nafion.
  • tetrabutyl ammonium-exchanged Nafion has been used with success.
  • the electrode may be modified with Nafion using the same techniques as modification with a diazonium salt described above. For example, electroreduction can be used by contacting the electrode with Nafion and applying a potential. Alternatively, the electrode can be contacted with Nafion and heated. The Nafion may be added at the same time as, or separately from, the diazonium salt. For example, the Nafion and diazonium salt may be mixed prior to application to the electrode.
  • the residue R formed from the diazonium salt extends away from the surface of the electrode and interacts with a binding pocket or cleft in the enzyme. While other residues could possibly form an interaction with the enzyme, the residues R used in the present invention appear to effectively form a connection between the electrode surface and the binding pocket or cleft of the enzyme.
  • the residue R forms a connection between the electrode surface and a copper atom present within the laccase binding pocket. This "plugs" the enzyme to the carbon-containing surface and provides a physical and/or electrical connection between the two. This can result in improved stability of the enzyme, preventing the enzyme from becoming detached from the surface or becoming deactivated. It can also result in improved efficiency of a fuel cell employing the new electrode.
  • the electrode (excluding electrode precursor) provided by the present invention may be used as a cathode in combination with any fuel cell, as long as the operating conditions are sufficiently mild that the enzymes used in the fuel cell are not denatured.
  • fuel cells which operate at very high temperatures, or which require extreme pH conditions, may well cause the enzymes to denature.
  • a fuel cell comprising an electrode according to the invention, as well as the use of an electrode or electrode precursor as described above in the manufacture of a fuel cell.
  • Conventional fuel cells which are currently used include alkaline, proton exchange membrane, phosphoric acid, molten carbonate and solid oxide fuel cells. Of these, the most suitable for use with the present invention are those utilising hydrogen as a fuel, for example the proton exchange membrane cell or a similar membrane-less cell. These cells typically operate at temperatures of from 50 to 90 0 C and at substantially neutral pH, or at slightly acidic pH (around pH 5).
  • FIG. 5 An example of a fuel cell according to the invention is depicted in Figure 5.
  • the anode and cathode are separated physically but are electrically connected via the external circuit and the electrolyte. Electrons flow from the anode to the cathode via the external load. Ions flow between the electrodes through the electrolyte.
  • a membrane may be present between the anode and the cathode.
  • the electrons produced are transferred via the conductor to the cathode and, similarly, the protons are transferred to the cathode via the electrolyte.
  • the source of hydrogen may be hydrogen gas itself. If desired, the hydrogen may be derived from a source such as an alcohol, including methanol and ethanol, or from fossil fuels such as natural gas. The hydrogen may be in a crude form and thus may contain impurities, or purified hydrogen may be used.
  • the fuel source is typically a gas which comprises hydrogen and which is provided to the anode. It is also conceivable that the fuel may be provided in liquid form. Generally, the fuel source also comprises an inert gas, although substantially pure hydrogen may also be used. For example, a mixture of hydrogen with one or more gases such as nitrogen, helium, neon or argon may be used as the fuel source.
  • the hydrogen fuel source may optionally comprise further components, for example other additives.
  • hydrogen is present in the fuel source in an amount of at least 0.5%, e.g. at least 2% by volume, preferably at least 5% and more preferably at least 10% by volume, for example 25%, 50%, 75% or 90% by volume.
  • the remainder of the fuel source is typically an inert gas, although it may be air.
  • Provision of hydrogen to the anode encompasses supplying hydrogen to the electrode directly, to the electrolyte and/or to a space in the fuel cell to which the electrolyte is exposed.
  • the fuel source is supplied from an optionally pressurised container of the fuel source in gaseous or liquid form.
  • the fuel source is supplied to the electrode via an inlet, which may optionally comprise a valve.
  • An outlet is also provided which enables used or waste fuel source to leave the fuel cell.
  • the oxidant is a material which can be reduced at the cathode.
  • the oxidant is oxygen, although other oxidants which will be known to a person skilled in the art are also suitable.
  • other oxidants include fumarate and those disclosed in WO-A-2006/109057.
  • the oxygen fuel source may optionally comprise further components, for example other additives.
  • oxygen is present in the fuel source in an amount of at least 0.5%, e.g. at least 2% by volume, preferably at least 5% and more preferably at least 10% by volume, for example 25%, 50%, 75% or 90% by volume.
  • the remainder of the fuel source is typically an inert gas, although it may be air.
  • the reduction of the oxidant preferably has the same stoichiometry as oxidation of hydrogen.
  • the electrons/protons produced at the anode and cathode should balance overall. This avoids, for example, a build up of protons and a pH change of the electrolyte.
  • the reaction which occurs at the anode can typically be described according to the following equation (2):
  • the product is the reduced form of the oxidant.
  • the overall reaction converts hydrogen and oxygen into water and generates an electric current.
  • the electrolyte comprises the oxidant.
  • the oxidant may be separately supplied in gaseous or liquid form.
  • the cathode in a fuel cell is an enzyme-modified electrode (excluding electrode precursor) according to the invention.
  • the anode may be made of any conducting material, for example stainless steel, brass or carbon, e.g. graphite.
  • the surface of the anode may, at least in part, be coated with a different material which facilitates adsorption of a hydrogenase catalyst.
  • the surface onto which the catalyst is adsorbed should be of a material which does not cause the hydrogenase to denature. Suitable surface materials include graphite, for example a polished graphite surface or a material having a high surface area such as carbon cloth, carbon sponge or porous carbon. Materials with a rough surface and/or with a high surface area are generally preferred.
  • the fuel cells of the present invention comprise an electrolyte suitable for conducting ions between the two electrodes.
  • the electrolyte should preferably be one which does not require the fuel cell to be operated under extreme conditions which would cause the enzymes to denature. Thus, electrolytes which rely on high temperature or extreme p ⁇ should be avoided.
  • the electrolyte is typically an aqueous solution containing salts such as alkali metal halides, e.g. NaCl or KCl. Appropriate concentrations are in the range of 0.05 to 0.5 M, e.g. about 0.1 M.
  • a p ⁇ buffer may also be present in the electrolyte, e.g. a phosphate, citrate or acetate buffer.
  • Other additives may also be present as desired, including glycerol, polymyxin B sulphate or other attachment means which may help to stabilise the enzymes.
  • the electrolyte is typically a non-chloride containing electrolyte since chloride inhibits laccase. In such a case, citrate, acetate or phosphate may be used to act as both the electrolyte and buffer.
  • the conditions under which the fuel cell is operated must be controlled so that the enzymes do not denature. Furthermore, the conditions can be optimised to provide a maximum amount of the enzymes in the active state and thereby increase the efficiency of the system.
  • the fuel cell is operated at a temperature of from 10 to 65°C, preferably from 15 to 55 °C, more preferably from 20 to 45 °C.
  • the preferred pH of the cell is from 4 to 9, e.g. 5 to 9.
  • a pH of around 5 is particularly suitable, for example from 4 to 6, more preferably from 4.5 to 5.5.
  • a pH of from 4 to 9, more preferably from 5 to 9, more preferably from 6 to 8, is suitable.
  • hydrogen is supplied directly to the anode, it is typically supplied at such a rate as to provide a partial pressure of from IxIO 3 to 1x10 5 Pa. Hydrogenases have been found to show hydrogen oxidation activity within this pressure range.
  • the partial pressure may be at least 1x10 4 , 2x10 4 or 5x10 4 Pa.
  • the potential at the anode when working at pH 7 is typically maintained at -40OmV or greater (i.e. at -40OmV or a less negative potential).
  • Preferred potentials at the anode are from -40OmV to + 40OmV, preferably from -20OmV to + 30OmV, for example from 0 to +30OmV. Each of these potentials is measured against a standard hydrogen electrode.
  • the preferred potential ranges when working at different pH may vary from the ranges stated above. The skilled person would be able to determine suitable ranges for use at a chosen pH.
  • a fuel cell as described above, may be operated under the conditions described above, to produce a current in an electrical circuit.
  • the fuel cell is operated by supplying hydrogen to the anode and supplying oxidant to the cathode, for example by using an electrolyte which comprises the oxidant.
  • hydrogen and/or oxidant may be provided via diffusion through the electrolyte from a surrounding environment containing the hydrogen and/or oxidant.
  • two or more cells of the invention may be used either in series or in parallel. If the cells are connected in parallel, the same electrolyte may be employed for each cell. If series cells are employed, a separate electrolyte is required for each individual cell.
  • 2-aminoanthracene was purchased from Aldrich and used as received without further purification. Diazonium solutions were prepared following the method disclosed in Langmuir, 2005, 21(8), 3362.
  • the PB 94 strain of Pycnoporus cinnabarinus fungus was purchased from the American Type Culture Collection (ATCC No. 200478). Small squares of fungus were plated onto malt extract agar and grown at 28 0 C for 7 d. Washings from nine plates were used to inoculate 3 1 modified Dodson media which were grown for 5 d at 28 0 C with shaking at >130 rpm and laccase expression induced after 24 h. A substrate mimic, 2,5-xylidine, is used to induce the extracellular expression of laccase.
  • Extracellular protein was harvested by ammonium sulphate precipitation (500 g I "1 ), resuspended in 10 mM pH 4.6 sodium acetate buffer, and dialysed overnight into the same buffer.
  • the dialysate was concentrated to approximately 30 ml and loaded onto a DE-52 anion exchange column previously equilibrated with pH 4.6 acetate buffer. Protein was washed with acetate buffer pH 4.6 and eluted with a stepwise increasing gradient (0-100 mM) of ammonium sulphate.
  • the purest laccase active fractions by SDS-PAGE were pooled and dialysed into approximately 10 ml of 100 tnM potassium phosphate, pH 6 before addition of 3g ammonium sulphate.
  • pyrolytic graphite plates measuring 50.8 mm x 50.8 mm x 3.2 mm (2" x 2" x 1/8", GE Quartz Europe) were cut into strips approximately 3 mm wide and were turned on a lathe to 2 mm diameter, electrically connected with silver-loaded epoxy (RS Components) to a stainless steel rod mounted in a plastic tube, embedded in two-part Araldite epoxy resin (CY 1300 and HYl 300, 3:1 mass ratio, Robnor Resins) and cured overnight.
  • RS Components silver-loaded epoxy
  • Abrasives used for polishing the graphite surface were P800 Tufbak Durite sandpaper (Norton). All water used was purified by reverse osmosis and ion exchange to a resistivity of 18.2 M ⁇ cm.
  • Example 1 Preparation of a modified carbon-containing surface
  • Electrode surfaces were modified by scanning once from 0.5 V (vs. SCE) to -0.3 V (vs. SCE) and back again at 50 mV s "1 .
  • Trametes versicolor laccase III was purified from the crude powder (Fluka, 23.7 units rng "1 ).
  • a suspension of the powder (2 mg ml "1 ) was made by stirring with 10 mM sodium acetate buffer (pH 5.5) at room temperature.
  • the crude suspension was centrifuged for 1 h at 17,000 rpm at 4 0 C to remove solids.
  • the enzyme was applied to a Toyopearl DEAE 650M (Toya Soda; 1.5 cm diameter and 5 cm height; ⁇ 10 ml resin) anion exchange column (previously equilibrated with buffer) at 4 °C and washed with buffer (ca. 10 column volumes) to remove unbound material.
  • Laccase was then released from the column by stepping salt concentration in the elution buffer to 100 mM ammonium sulphate. The fractions containing laccase were eluted as a dark green/blue band, while a brown/yellow band with no laccase activity remained bound. Fractions collected were tested for laccase activity with ABTS. Fractions showing laccase activity were combined, reapplied to an anion exchange column and eluted as previous. This led to a laccase active fraction >95% pure by SDS-PAGE. The laccase was desalted into pH 4.0, 10 mM acetate buffer and stored as 20 ⁇ l samples at -80 0 C. To the surface of a modified electrode, 50-100 ⁇ l of the concentrated laccase is added per square centimetre of carbon. The laccase solution can be withdrawn from the surface for reuse.
  • the surface of the embedded graphite was abraded with sandpaper, rinsed with water, then sonicated in water for 10 min.
  • the surface of the electrode (modified or unmodified) was spotted with 4 ⁇ l of 15 ⁇ M laccase in acetate buffer.
  • the electrode was connected to the rotator and placed in ⁇ 2 ml of 200 mM pH 4.0 citrate buffer.
  • the cell was maintained at 25 °C by a continual flow of water through the cell jacket.
  • a flowing atmosphere of oxygen (industrial grade, Air Products) at atmospheric pressure was maintained over the liquid while the electrode was rotated at 2500 rpm.
  • the potential was swept from 0.7 V vs. SCE to 0.2 V vs. SCE and back again at 5 mV s "1 .
  • the cell solution was replaced with fresh buffer, reoxygenated and the same potential range scanned to observe catalysis with no enzyme in solution.
  • the electrodes were tested periodically at intervals over 1 month. When the protein films were not being analysed, the electrode tips were kept hydrated, wrapped in paraffin film, and stored at 4 0 C. Cyclic voltammetry data (including deconvolution, peak fitting, Fourier-filtering and baseline correction) was analysed quantitatively using an in-house software program.
  • this provides a comparison of the change in electrocatalytic activity with time of a sub-monolayer protein film of Trametes versicolor laccase III on (a) a PGE electrode modified with anthracene-2-diazonium (A) with enzyme still in solution, (B) with no enzyme in solution, (C) after 2 d, (D) after 3 d, (E) after 17 d, and (F) after 28 d; and (b) an unmodified PGE electrode (A) with enzyme still in solution, (B) with no enzyme in solution, (C) after 2 d, (D) after 3 d, (E) after 17 and 28 d (overlaid). In all cases the electrode area was 0.03 cm 2 .
  • Example 4 Epifluorescence Data
  • the laccase was labelled with fluorescein-5-EX, succinimidyl ester (F5EX, Molecular Probes).
  • the laccase was dialysed against 0.1 M carbonate buffers in a 2 ml Amicon with a YM 10 Diaflo ultrafiltration membrane, to remove ammonium ions that would interfere with the labelling and to raise the pH to facilitate the labelling.
  • the pH was stepped from 4.0 to 7.0 in steps of one pH unit and the laccase was finally concentrated to ca. 12 mg ml "1 .
  • the degree of labelling was determined spectrophotometrically based on absorption at 280 nm and the maximum visible absorption for the dye, and was calculated to be 0.6 dye molecules per laccase molecule.
  • the laccase-containing fraction was diluted tenfold with the eluent before it was used on graphite.
  • Graphite samples for microscopy were abraded and rinsed with ultrahigh purity water and air dried. The surface was spotted with 2 ⁇ l of the dilute labelled laccase solution and the samples were left on ice for 30 minutes. Laccase coverage was estimated from the histograms of the images captured using ImageJ 1.33.
  • lighter colours represent a greater concentration of laccase present.
  • the scale is the same for both images, with the exposure time for (a) being 63 s and the exposure time for (b) being 17.3 s.
  • the epifluorescence microscopy pictures clearly show an increased amount of laccase on the surface of the modified electrode compared to the unmodified.
  • the coverage of the laccase on the surface of the modified electrode appears to be more uniform compared to the patchy coverage of the laccase on the unmodified surface.
  • Cloth A had a geometric area of 7.75 cm “2 ; Cloth D was 5.52 cm “2 . Both cloths were soaked in the same manner as cloths 3 and 4 above, first in anthracene solution followed by a nitrite solution. The soaked cloths were then placed in a 65 0 C oven for 60 min, then washed in ethanol, water, then buffer. Cloth A was coated with 116 ⁇ l of a mixture of 227 ⁇ l laccase solution, 24 ⁇ l TBA-Nafion solution and 49 ⁇ l buffer.
  • Cloth D was coated with 83 ⁇ l of a mixture of 189 ⁇ l laccase solution and 61 ⁇ l buffer (no TBA-Nafion). The volumes and concentrations were chosen to give a constant area loading of laccase on each cloth. Each cloth sat for 3 min before lifetime measurements were carried out. Oxygen reduction current was recorded over more than two months using a custom-built cathode testing rig (e.g. that described in Example 3) at room temperature. The cloths sat in well-aerated (rather than oxygenated) buffer and were held at 0.2 V versus the calomel reference electrode potential. The solution level and pH were held constant through controlled inputs of water and citric acid, respectively. Oxygen reduction current was drawn continuously; current flow was recorded automatically at least once per minute.
  • Electrochemical cell a jacketed glass vessel held at ⁇ 4 °C with a platinum mesh counter electrode and a saturated calomel reference electrode connected to the main vessel by a Luggin capillary; the carbon cloth served as the working electrode.
  • TSA-Nafion Tetrabutylammonium-exchanged Nafion

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Abstract

L'invention concerne un procédé de préparation d'une électrode modifiée par une enzyme ou d'un précurseur d'électrode modifiée par une enzyme consistant à : (i) mettre en contact une électrode ou un précurseur d'électrode avec un sel de diazonium afin de modifier une surface de ladite électrode ou dudit précurseur d'électrode avec un résidu R, le résidu R étant un résidu organique du sel de diazonium et le sel de diazonium comprenant (a) un cation aromatique comprenant : soit i. de deux à quatre cycles aromatiques qui sont fusionnés ensemble et/ou liés les uns aux autres soit directement soit via un groupe de liaison -CR=CR-, -C≡C- ou -N=N- où R représente un hydrogène ou un alkyle en C1-4, l'un desdits cycles portant un substituant -N2+ et les cycles aromatiques étant identiques ou différents et sélectionnés chacun parmi un phényle substitué ou non substitué et des cycles hétéroaryliques à 5 ou 6 chaînons; soit ii. un cycle aromatique monocyclique sélectionné parmi un phényle substitué ou non substitué et un cycle hétéroarylique à 5 ou 6 chaînons, ledit cycle aromatique portant un substituant -N2+ et un autre substituant conjugué sélectionné parmi un alcényle en C2-6 et un alcynyle en C2-6; et (b) un contre-ion; et (ii) exposer la surface modifiée contenant du carbone à une enzyme de type oxydase à cuivre bleu de façon à ce que l'enzyme se lie au résidu R. L'invention concerne également une électrode modifiée par une enzyme ou un précurseur d'électrode modifiée par une enzyme.
PCT/GB2007/004499 2006-11-24 2007-11-26 Électrode Ceased WO2008062216A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006030196A1 (fr) * 2004-09-13 2006-03-23 Isis Innovation Limited Pile a combustible biochimique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006030196A1 (fr) * 2004-09-13 2006-03-23 Isis Innovation Limited Pile a combustible biochimique

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DELAMAR M ET AL: "Modification of carbon fiber surfaces by electrochemical reduction of aryl diazonium salts: application to carbon epoxy composites", CARBON, ELSEVIER, OXFORD, GB, vol. 35, no. 6, 1997, pages 801 - 807, XP004073601, ISSN: 0008-6223 *
WANG ET AL: "Covalent immobilization of glucose oxidase on conducting ultrananocrystalline diamond thin films", DIAMOND AND RELATED MATERIALS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 15, no. 2-3, February 2006 (2006-02-01), pages 279 - 284, XP005333774, ISSN: 0925-9635 *
ZILLE A ET AL: "Laccase kinetics of degradation and coupling reactions", JOURNAL OF MOLECULAR CATALYSIS. B, ENZYMATIC, ELSEVIER, AMSTERDAM,, NL, vol. 33, no. 1-2, 3 May 2005 (2005-05-03), pages 23 - 28, XP004862512, ISSN: 1381-1177 *

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