CN101151764A - Immobilized enzymes in biocathodes - Google Patents

Immobilized enzymes in biocathodes Download PDF

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CN101151764A
CN101151764A CNA2004800383840A CN200480038384A CN101151764A CN 101151764 A CN101151764 A CN 101151764A CN A2004800383840 A CNA2004800383840 A CN A2004800383840A CN 200480038384 A CN200480038384 A CN 200480038384A CN 101151764 A CN101151764 A CN 101151764A
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biological
cathode
enzyme
fuel cell
anode
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S·D·明特尔
S·托普恰吉奇
B·特罗伊
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St Louis University
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    • Y02E60/30—Hydrogen technology
    • Y02E60/50—Fuel cells

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Abstract

Disclosed is an improved biofuel cell having a cathode comprising a dual function membrane, which contains an oxygen oxidoreductase enzyme immobilized within a buffered compartment of the membrane and an electron transport mediator which transfers electrons from an electron conducting electrode to the redox reaction catalyzed by the oxygen oxidoreductase enzyme. The improved biofuel cell also has an anode that contains an oxidoreductase enzyme that uses an organic fuel, such as alcohol, as a substrate. An electric current can flow between the anode and the cathode.

Description

Immobilised enzymes in the biological-cathode
Government supports
This working portion is subjected to the mandate (grant number N00014-03-0222) of Office of Naval Research (Office of Naval Research).U.S. government has some right in this invention.
Background of invention
The present invention relates generally to fuel cell (a.k.a. biological fuel cell) and their production method and purposes based on biology enzyme.More particularly, the present invention relates to biological-cathode and their production method and purposes.
Pertinent literature is described
Incorporate the list of references of quoting in this specification into this paper as a reference.The discussion of those lists of references only be intended to sum up they the author opinion and do not admit that any list of references constitutes prior art.The accuracy of the list of references of being quoted and the right of appropriateness are refuted in applicant's reservation.
Biological fuel cell is a kind of biological chemical device, wherein changes into electric energy from the energy of the chemical reaction catalytic activity by living cells and/or their enzyme.Biological fuel cell uses complicated molecule in the anode generation hydrogen reduction to be become the required hydrogen ion of water usually, is used for the free electron that electricity is used and produce.Biological-cathode is the electrode of biological fuel cell, and wherein electronics and the proton from anode used hydrogen reduction Cheng Shui by catalyst.Biological fuel cell is similar to conventional polymer dielectric film (" PEM ") fuel cell, because it is by by certain barrier or salt bridge, for example the negative electrode and the anode that separate of polymer dielectric film formed.Not existing together of biological fuel cell and conventional fuel battery is the material that is used for the catalytic electrochemical reaction.Biological fuel cell is not to use noble metal as catalyst, but depends on biomolecule such as enzyme reacts.Although early stage biological fuel cell technology is used the metabolic pathway of complete microbe, but the problem relevant with this method comprises low capacity catalytic activity and the output of unpractiaca power density [Palmore and the Whitesides of complete biology, 1994, ACS Symposium Series566:271-290].The enzyme isolation technics has stimulated biological fuel cell application and development [Palmore and Whitesides, 1994, ACS Symposium Series 566:271-290] by increase capacity activity and catalytic capability.The enzyme biological fuel cell that is separated has increased power density output by overcoming the interference relevant with the growth of microorganism that lacks consume fuel with the cell membrane impedance of electron transport.
Although enzyme is highly effective catalyst, they are mixed the fuel cell existing problems.In early days contain in solution rather than be fixed on enzyme on the electrode surface [Palmore and Whitesides, 1994, ACS Symposium Series 566:271-290 and list of references are wherein incorporated them into this paper as a reference] based on the fuel cell of enzyme.Enzyme in the solution is only stablized a couple of days, and immobilized enzyme can be stablized the several months.A major obstacle based on the biological fuel cell of enzyme is that enzyme is fixed in the film of electrode surface, and this film will prolong the life-span of enzyme and form machinery and chemically stable layer, and in electrode surface formation capacitive character district.At most H 2/ O 2In the fuel cell, the adhesive that catalyst is remained on electrode surface is Nafion .Nafion  is a perfluorinated ion-exchange polymer, and it has the outstanding character as ion conductor.Yet Nafion  does not also succeed on the surface that enzyme is fixed on the biological fuel cell electrode, because Nafion  forms acid film, it has reduced the life-span and the activity of enzyme.
Other people have carried out some and have attempted mixing with exploitation the biological fuel cell of immobilised enzymes.At U.S. Patent number 6,294,291, U.S. Patent number 6,531,239 and people such as Chen, J.Am.Chem.Soc.2001 has described the several different methods of the immobilised enzymes that is used for biological fuel cell among the vol.123:8630-8631, wherein enzyme demonstrates minimum at least activity and stable, with described reference citation as a reference.Those lists of references have been described multiple oxidoreducing enzyme have been fixed on the polymer sol gel-type vehicle, and this matrix is also mixed electron transfer mediator, as osmium, cobalt or ruthenium complex.Yet, being important to note that enzyme only is fixed to the surface of sol gel (that is, two dimension), it does not cushion.Thereby the enzyme of describing during those are open has very limited stability, and the maximum activity life-span is no more than 7 to 10 days usually.
People such as Minteer have developed a kind of biological fuel cell, and it comprises improved biological anode (open in patent application 60/429,829,60/486,076 and 10/617,452), and the active lifetime of this battery was greater than 45 days, and performance does not descend.A specific embodiments of this biological fuel cell is used dehydrogenase and NAD +As anode catalyst and use ethanol as anode fuel and ELAT electrode, it comprises about 20%Pt (E-Tek) on the Vulcan XC-72 as cathod catalyst, and dissolved oxygen is as negative electrode fuel.The open circuit voltage of this biological fuel cell is 0.82V under 20 ℃ and pH7.15, and maximum power density is 2.04mW/cm 2
Described improved biological anode comprises (modification) Nafion  film that the bromination quaternary ammonium salt is handled, and it provides stabilized enzyme immobilized ecotopia.Although the Nafion  film of being modified keeps the electrical properties of the Nafion  of unmodified, but showed in the past to compare for ion and neutral kind and had the mass transportation ability of increase with the Nafion  of unmodified, lower acid and cushion near neutral pH, (see people such as Schrenk with the aperture that increases with the immobilization that adapts to relative big molecule such as enzyme, 2002, J.Membr.Sci.205:3-10 quotes it as a reference).
(for example, people such as Chen, 2001) have described the other biological negative electrode that particular is unstable, efficient is lower and toxicity is bigger than disclosed biological-cathode in this application in the literature.For example, people's such as Chen biological-cathode utilizes aquagel membrane, it is not buffering and enzyme---cathode enzyme laccase (EC1.10.3.2) can only mating surface, its optimal pH be 5 and in the presence of chloride ion non-activity, and as electron transfer mediator, this osmium complex is poisonous with osmium complex.The improved biological-cathode that need comprise cathode enzyme, the influence that it is not subjected to chloride ion has the electron transfer mediator of low toxicity and modified amberplex, and it mixes cathode enzyme in the micella of buffering.
Summary of the invention
Of the present invention multiple aspect, biological-cathode is provided, it comprises (a) electronic conductor; (b) at least aly can produce the oxidised form of electron mediator and the enzyme of water with the reduction form of electron mediator and oxidant reaction; (c) comprise the enzyme immobilization material of eelctro-catalyst, described enzyme can be fixed and stablize to this enzyme immobilization material, described material is permeable for described oxidant, the oxidised form of described eelctro-catalyst can obtain electronics to produce the reduction form of eelctro-catalyst from electronic conductor, and it can react with the oxidised form of electron mediator with the reduction form that produces electron mediator and the oxidised form of eelctro-catalyst; Wherein eelctro-catalyst is enough to make the concentration of described enzyme immobilization conduct electronics exist.
Of the present invention is biological-cathode more on the one hand, and it comprises (a) electronic conductor; (b) at least aly can produce the oxidised form of electron mediator and the enzyme of water with the reduction form of electron mediator and oxidant reaction; (c) comprise the enzyme immobilization material of electron mediator, described enzyme can be fixed and stablize to this enzyme immobilization material, described material is permeable for described oxidant, and the oxidised form of described electron mediator can obtain electronics to produce the reduction form of electron mediator from electronic conductor; Wherein electron mediator is enough to make the concentration of described enzyme immobilization conduct electronics exist.
Of the present invention is biological-cathode more on the one hand, and it comprises (a) electronic conductor; (b) at least aly can produce the oxidised form of electron mediator and the enzyme of water with the reduction form of electron mediator and oxidant reaction; (c) comprise the enzyme immobilization material of electron mediator and eelctro-catalyst, described enzyme can be fixed and stablize to this enzyme immobilization material, described material is permeable for described oxidant, the oxidised form of described eelctro-catalyst can obtain electronics to produce the reduction form of eelctro-catalyst from electronic conductor, and it can react with the oxidised form of electron mediator with the reduction form that produces electron mediator and the oxidised form of eelctro-catalyst; Wherein eelctro-catalyst is enough to make the concentration of described enzyme immobilization conduct electronics exist.
Another aspect of the present invention is a biological-cathode, and it comprises (a) electronic conductor; (b) at least aly can produce the oxidised form of electron mediator and the enzyme of water with the reduction form of electron mediator and oxidant reaction; (c) comprise the enzyme immobilization material of electron mediator and eelctro-catalyst, described enzyme can be fixed and stablize to this enzyme immobilization material, described material is permeable for described oxidant, the oxidised form of eelctro-catalyst can obtain electronics to produce the reduction form of eelctro-catalyst from electronic conductor, and it can react with the oxidised form of electron mediator with the reduction form that produces electron mediator and the oxidised form of eelctro-catalyst; Wherein eelctro-catalyst is enough to make the concentration of described enzyme immobilization conduct electronics exist.
Of the present invention is the biological fuel cell that is used to produce electricity more on the one hand, and it comprises fuel liquid, electron mediator, can react the anode with oxygenated fuel liquid, and biological-cathode, and this biological-cathode comprises (a) electronic conductor; (b) at least aly can produce the oxidised form of electron mediator and the enzyme of water with the reduction form of electron mediator and oxidant reaction; (c) comprise the enzyme immobilization material of eelctro-catalyst, described enzyme can be fixed and stablize to this enzyme immobilization material, described material is permeable for described oxidant, the oxidised form of described eelctro-catalyst can obtain electronics to produce the reduction form of eelctro-catalyst from electronic conductor, and it can react with the oxidised form of electron mediator with the reduction form that produces electron mediator and the oxidised form of eelctro-catalyst; Wherein eelctro-catalyst is enough to make the concentration of described enzyme immobilization conduct electronics exist.
Another aspect of the present invention is the biological fuel cell that is used to produce electricity, and it comprises fuel liquid, can react the anode with oxygenated fuel liquid, and biological-cathode, and this biological-cathode comprises (a) electronic conductor; (b) at least aly can produce the oxidised form of electron mediator and the enzyme of water with the reduction form of electron mediator and oxidant reaction; (c) comprise the enzyme immobilization material of electron mediator, described enzyme can be fixed and stablize to this enzyme immobilization material, described material is permeable for described oxidant, and the oxidised form of described electron mediator can obtain electronics to produce the reduction form of electron mediator from electronic conductor; Wherein electron mediator is enough to make the concentration of described enzyme immobilization conduct electronics exist.
Of the present invention is the biological fuel cell that is used to produce electricity more on the one hand, and it comprises fuel liquid, can react the anode with oxygenated fuel liquid, and biological-cathode, and this biological-cathode comprises (a) electronic conductor; (b) at least aly can produce the oxidised form of electron mediator and the enzyme of water with the reduction form of electron mediator and oxidant reaction; (c) comprise the enzyme immobilization material of electron mediator and eelctro-catalyst, described enzyme can be fixed and stablize to this enzyme immobilization material, described material is permeable for described oxidant, the oxidised form of eelctro-catalyst can obtain electronics to produce the reduction form of eelctro-catalyst from electronic conductor, and it can react with the oxidised form of electron mediator with the reduction form that produces electron mediator and the oxidised form of eelctro-catalyst; Wherein eelctro-catalyst is enough to make the concentration of described enzyme immobilization conduct electronics exist.
Of the present invention is the biological fuel cell that is used to produce electricity more on the one hand, and it comprises fuel liquid, can react the anode with oxygenated fuel liquid, and biological-cathode, and this biological-cathode comprises (a) electronic conductor; (b) at least aly can produce the oxidised form of electron mediator and the enzyme of water with the reduction form of electron mediator and oxidant reaction; (c) comprise the enzyme immobilization material of electron mediator, described enzyme can be fixed and stablize to this enzyme immobilization material, described material is permeable for described oxidant, the oxidised form of eelctro-catalyst can obtain electronics to produce the reduction form of eelctro-catalyst from electronic conductor, and it can react with the oxidised form of electron mediator with the reduction form that produces electron mediator and the oxidised form of eelctro-catalyst; Wherein electron mediator is enough to make the concentration of described enzyme immobilization conduct electronics exist.
The invention still further relates to and use above-mentioned biological fuel cell to produce the method for electricity, it comprises the fuel liquid of (a) oxidation anode and the oxidant of reduction biological-cathode; (b) the reduction form of oxidation electron mediator between the oxidant reduction period of biological-cathode; (c) oxidation eelctro-catalyst; (d) eelctro-catalyst at reduction electronic conductor place.
The invention still further relates to and use above-mentioned biological fuel cell to produce the method for electricity, it comprises the fuel liquid of (a) oxidation anode and the oxidant of reduction biological-cathode; (b) the reduction form of oxidation electron mediator between the oxidant reduction period of biological-cathode; (c) electron mediator at reduction electronic conductor place.
Another aspect of the present invention is the fuel cell that comprises negative electrode and anode, and wherein negative electrode comprises electronic conductor, cathode enzyme, electron transfer mediator and film, and wherein said cathode enzyme is fixed in the interior compartment of buffering of film.
Another aspect of the present invention is the method that produces electrical power, it comprises (a) organic-fuel of oxidation anode in the presence of at least a anodic oxidation reductase that mixes anode, (b) by redox polymers electronics is transferred to the anode electrically conductive material from the organic-fuel of oxidation, (c) oxygen molecule of reduction negative electrode in the presence of the dioxygen oxidation reductase, described enzyme be fixed on modification negative electrode chloride ion exchange polymer membrane in buffered compartment, (d) electronics is transferred to the matrix of dioxygen oxidation reductase by electron transfer mediator from electrically conductive material, described electron transfer mediator be fixed on modified ion exchange polymer membrane in buffered compartment, thereby produce electric current.
Of the present invention is the biological-cathode that is used for accepting from circuit electronics more on the one hand, it comprises the electrically conductive material arranged side by side with the dual purpose film, with the dual purpose film, wherein said dual purpose film comprises modified amberplex, immobilization cathode enzyme, and electron transfer mediator.
Of the present invention is the biological-cathode that is used for accepting from circuit electronics more on the one hand, and it comprises electrically conductive material and the dual purpose film arranged side by side with the dual purpose film, and wherein said dual purpose film comprises modified amberplex, immobilization cathode enzyme, and eelctro-catalyst.
The invention still further relates to one or more previously described biological fuel cells, biological-cathode and produce electric method, wherein said enzyme immobilization material comprises micella or reversed micelle structure.
The invention still further relates to one or more previously described biological fuel cells, biological-cathode and produce electric method, wherein said enzyme immobilization material comprises the perfluorinated sulfonic acid-PTFE copolymer of modification.
The invention still further relates to one or more previously described biological fuel cells, biological-cathode and produce electric method, wherein said electronic conductor comprises material, metallic conductor, semiconductor, metal oxide or the modified conductor based on carbon; Especially based on the material of carbon.
The invention still further relates to one or more previously described biological fuel cells, biological-cathode, with the method that produces electricity, wherein said electronic conductor comprises charcoal cloth, carbon paper, the electrode of carbon screen printing (carbon screen printed electrode), carbon black, powdered carbon, carbon fiber, Single Walled Carbon Nanotube, double-walled carbon nano-tube, multi-walled carbon nano-tubes, carbon nano pipe array, the conductor of diamond-coating, vitreous carbon, mesoporous carbon, graphite, unpressed graphite worm (graphite worm), the purified flake graphite of layering, high-performance graphite, the pyrolytic graphite of high-sequential, pyrolytic graphite or polycrystalline graphite.
The invention still further relates to one or more previously described biological fuel cells, biological-cathode and produce electric method, wherein said enzyme immobilization material is used greater than NH 4 +Dewatering cationic modify; Preferably, described dewatering cationic comprises cation, quaternary ammonium cation, alkyl trimethyl ammonium cation, organic cation,  cation, triphenyl , pyridine  cation, imidazoles  cation, cetyl pyridinium , second ingot, purpurine (viologen), methyl viologen, benzyl viologen, two (triphenylphosphine) iminium metal complex, Bipyridine metal complexes, the metal complex based on the phenanthroline, [Ru (bipyridine) based on ammonium 3] 2+Perhaps [Fe (phenanthroline) 3] 3+Particularly, described dewatering cationic comprises the quaternary ammonium cation of formula 1 expression
Figure A20048003838400191
R wherein 1, R 2, R 3And R 4Be the alkyl or the heterocycle of hydrogen, alkyl, replacement, wherein R independently 1, R 2, R 3And R 4At least one be not hydrogen.In another embodiment, R 1, R 2, R 3And R 4Be hydrogen, methyl, ethyl, propyl group, butyl, amyl group, hexyl, heptyl, octyl group, nonyl, decyl, undecyl, dodecyl, tridecyl or myristyl, wherein R independently 1, R 2, R 3And R 4At least one be not hydrogen.Alternatively, R 1, R 2, R 3And R 4Identical and be methyl, ethyl, propyl group, butyl, amyl group or hexyl.Preferably, the quaternary ammonium cation of formula 1 is TBuA, triethyl group hexyl ammonium or dodecyl trimethyl ammonium.
The invention still further relates to one or more previously described biological fuel cells, biological-cathode and produce electric method, wherein said enzyme comprises oxidoreducing enzyme; Particularly, glucose oxidase, based on the oxidizing ferment of alcohol or based on the oxidizing ferment of cholesterol.
The invention still further relates to one or more previously described biological fuel cells, biological-cathode and produce the method for electricity, wherein said enzyme is included in the dioxygen oxidation reductase that about 6.5 to about 7.5 pH has optimal activity; Be specially laccase, cytochrome C oxidase, bilirubin oxidase or peroxidase; It more specifically is bilirubin oxidase.
Originally the invention still further relates to one or more previously described biological fuel cells, biological-cathode and produce electric method, wherein electron mediator comprises organic compound, sugar, sterol, aliphatic acid or the oxidasic coenzyme or the substrate of metalloprotein, conjugation; Particularly, the oxidised form of wherein said electron mediator comprises stellacyanin (stellacyanin), bilirubin, glucose or cholesterol; More specifically, wherein the oxidised form of electron mediator comprises bilirubin.
The invention still further relates to one or more previously described biological fuel cells, biological-cathode and produce the method for electricity, wherein the eelctro-catalyst of electron mediator comprises normal reduction potential greater than+0.4 volt organic metal cation; Particularly, wherein the eelctro-catalyst of electron mediator comprises osmium, ruthenium, iron, nickel, rhodium, rhenium or cobalt complex; More specifically, wherein the reduction form of the eelctro-catalyst of electron mediator comprises Ru (phen) 3 + 2, Fe (phen) 3 + 2, Ru (bpy) 3 + 2, Os (bpy) 3 + 2Perhaps Os (terpy) 3 + 2
The invention still further relates to one or more previously described biological fuel cells, biological-cathode and produce electric method, the reduction form of the eelctro-catalyst of wherein said electron mediator comprises Ru (bpy) 3 + 2
The invention still further relates to one or more previously described biological fuel cells, biological-cathode, with the method that produces electricity, the concentration of wherein said eelctro-catalyst arrives about 3M for about 100mM, more preferably, about 250mM is to about 2.25M, and more preferably, about 500mM is to about 2M, most preferably, about 1.0M is to about 1.5M.
The invention still further relates to one or more previously described biological fuel cells, biological-cathode and produce electric method, wherein said oxidant comprises oxygen or peroxide; Particularly, wherein said oxidant comprises oxygen.
The invention still further relates to one or more previously described biological fuel cells, biological-cathode, with the method that produces electricity, wherein said fuel liquid comprises ammonia, methyl alcohol, ethanol, propyl alcohol, isobutanol, butanols and isopropyl alcohol, allyl alcohol, aryl alcohol, glycerine, propylene glycol, sweet mellow wine, glucuronic acid, aldehyde, carbohydrate, glucose, glucose-1, D-glucose, L-glucose, G-6-P, lactic acid, 9--phosphoric acid, D-lactic acid, L-lactic acid, fructose, galactolipin-1, galactolipin, aldose, sorbose, mannose, glyceric acid, coacetylase, acetyl coenzyme A, malic acid, isocitric acid, formaldehyde, acetaldehyde, acetate, citric acid, the L-gluconic acid, beta-hydroxysteroid, alpha-hydroxysteroid, lactic aldehyde (lactaldehyde), testosterone, gluconic acid, aliphatic acid, lipid, phosphoglyceric acid, retinene, estradiol, cyclopentanol, hexadecanol, long-chain alcohol, coniferyl alcohol, cinnamyl alcohol, formic acid, long-chain aldehyde, pyruvic acid, butyraldehyde, acyl coenzyme A, steroids, amino acid, flavine, NADH, NADH 2, NADPH, NADPH 2Perhaps hydrogen; Particularly, wherein said fuel liquid comprises methyl alcohol, ethanol or propyl alcohol; More specifically, wherein said fuel liquid comprises ethanol.
The invention still further relates to one or more previously described biological fuel cells, biological-cathode and produce electric method, the perfluorinated sulfonic acid of wherein said modification-PTFE copolymer is modified with TBAB.
The invention still further relates to one or more previously described biological fuel cells and produce electric method, wherein said anode is biological anode.
Accompanying drawing is described
Fig. 1 is the sketch map of difunctional biological-cathode.
Fig. 2 is a voltammogram, and it has shown the current density of gained the best of the biological fuel cell of the biological-cathode that comprises the Nafion I preparation of describing as embodiment 1.
Fig. 3 is a voltammogram, and it has shown the intermediate range current density of the biological fuel cell of the biological-cathode that comprises the Nafion I preparation of describing as embodiment 1.
Fig. 4 is a voltammogram, and it has shown the poorest current density of gained of the biological fuel cell of the biological-cathode that comprises the Nafion I preparation of describing as embodiment 1.
Fig. 5 is a voltammogram, and it has shown the current density of gained the best of the biological fuel cell of the biological-cathode that comprises the Nafion II preparation of describing as embodiment 1.
Fig. 6 is a voltammogram, and it has shown the intermediate range current density of the biological fuel cell of the biological-cathode that comprises the Nafion II preparation of describing as embodiment 1.
Fig. 7 is a voltammogram, and it has shown the poorest current density of gained of the biological fuel cell of the biological-cathode that comprises the Nafion II preparation of describing as embodiment 1.
Fig. 8 is a voltammogram, and it has shown the current density of gained the best of the biological fuel cell of the biological-cathode that comprises the Nafion III preparation of describing as embodiment 1.
Fig. 9 is a voltammogram, and it has shown the intermediate range current density of the biological fuel cell of the biological-cathode that comprises the Nafion III preparation of describing as embodiment 1.
Figure 10 is a voltammogram, and it has shown the poorest current density of gained of the biological fuel cell of the biological-cathode that comprises the Nafion III preparation of describing as embodiment 1.
Figure 11 is the schematic diagram of prototype biological fuel cell, described biological fuel cell comprises biological anode, this anode contains immobilized alcohol dehydrogenase and the green redox film of poly-ethylidene Nafion  PEM, also comprises biological-cathode, and this negative electrode contains immobilized bilirubin oxidase and Ru (bpy) 3 + 2
Figure 12 is the power curve of the prototype biological fuel cell of Figure 11.
Figure 13 is the chemical action schematic diagram in the biological-cathode of ethanol/oxygen biological fuel cell and the generation of biological anode.
Figure 14 is the representative cyclic voltammogram of the 1.0mM PQQ/1.0mM ethanol in pH7.15 phosphate buffer during 100mV/s on the immobilized TBAB/Nafion  of the alcohol dehydrogenase film that relies on of PQQ-.
Figure 15 is under the room temperature in the 1mM ethanol in the pH7.15 phosphate buffer and the 1mM NAD+ solution, and the power output that the representativeness with biological anode that NAD-relies on does not have film ethanol/oxygen biological fuel cell is as the functional arrangement from time of assembling beginning.
Detailed Description Of The Invention
Multiple aspect of the present invention is biological-cathode, and it comprises for the immobilised enzymes of using, and the enzyme stability that wherein increases is favourable; Be particularly useful for biological fuel cell. For being used for biological-cathode, immobilization material has formed barrier, and it provides machinery and chemical stability, and by mix electron mediator or the eelctro-catalyst of enough concentration in immobilization material, described immobilization material works as electron mediator. Described enzyme is obtaining maximization than previously known stable longer time and the electronics transmission by immobilization material. For the present invention, if enzyme keep at least about 30 days to about 365 days its initial catalytic activity at least about 75%, this enzyme is subject to " stabilisation " so. In several different methods of the present invention is fuel cell on the other hand, and it utilizes organic-fuel (fuel liquid that perhaps comprises hydrogen, ammonia or hydrocarbon) to produce electricity by redox (oxidation/reduction) reaction of enzyme mediation. Another aspect of the present invention is biological fuel cell, and it comprises anode and biological-cathode. This biological-cathode comprises the enzyme immobilization material, and it is infiltration and fixing to oxidant and stablizes described enzyme, and can immobilization and stable electron mediator or eelctro-catalyst. Immobilized Enzymic stability allow biological fuel cell within least about 30 days to about 365 day time, produce initial current at least about 75% electric current.
Another aspect of the present invention disclosed herein is the improved biological fuel cell that comprises biological-cathode and biological anode, it mixes in the micella of the Nafion  film that quaternary ammonium salt is processed or " catching " dehydrogenase (is seen, U.S. Patent application 60/429,829,60/486,076 and 10/617,452, with the people such as Schrenk, Journal of Membrane Science 205 (2002) 3-10; With the people such as Thomas, Journal of Membrane Science 213 (2003) 55-66; It is quoted as a reference). Some advantages of improved biological anode are that enzyme mixes ion-exchange polymer in three-dimensional, this has increased power density and has increased Enzymic stability, and provide buffering for enzyme, thereby significantly increased the useful life of enzyme, it is considerably beyond any other biological fuel cell of developing so far.
I. biological fuel cell
Be biological fuel cell in many-side of the present invention, it utilizes fuel liquid to produce electricity by the redox reaction that the enzyme of the therein electrode generation of immobilized enzyme mediates. In the standard electric chemical cell, anode is the position that the oxidation reaction of fuel liquid discharges electronics simultaneously. Electronics arrives certain power consumption device by terminal box from anode. Electronics moves to another terminal box by this device, and it is delivered to the biological-cathode of biological fuel cell with electronics, is used to the reduction-oxidation agent to produce water at the described electronics of negative electrode. By this way, biological fuel cell of the present invention is as the energy (electricity) of its external electrical load. In order to promote the redox reaction of fuel liquid, electrode comprises electronic conductor, electron mediator, is used for eelctro-catalyst, enzyme and the enzyme immobilization material of electron mediator.
According to the present invention, electron mediator is the compound that can accept electronics or confess electronics. In current preferred biological fuel cell, the oxidised form of electron mediator and fuel liquid and enzyme reaction are to produce the oxidised form of fuel liquid and the reduction form of electron mediator at biological anode. Subsequently or side by side, the reaction of the reduction form of electron mediator and the oxidised form of eelctro-catalyst produces the oxidised form of electron mediator and the reduction form of eelctro-catalyst. Then the reduction form of eelctro-catalyst is oxidized and produce electronics to produce electricity at biological anode. Except the oxidation of fuel liquid, the redox reaction of biological anode can be reversible, thereby described enzyme, electron mediator and eelctro-catalyst are not consumed. Randomly, if adding electron mediator and/or eelctro-catalyst are to provide extra reactant, these redox reactions are irreversible so.
Alternatively, can use electronic conductor and enzyme, the electron mediator that wherein contacts with biological anode can be at not modified electrode it oxidation and the reduction form between metastatic electron. If electron mediator can be at not modified biological anode metastatic electron between oxidation and the reduction form, dispensable and the electron mediator of following reaction between eelctro-catalyst and the electron mediator is from oxidized and produce electronics in biological anode so, thereby produces electricity.
At biological-cathode, flow into from the electronics of biological anode in the electronic conductor of biological-cathode. There, the combination of the oxidised form of electronics and eelctro-catalyst, described eelctro-catalyst contacts with electronic conductor. This reaction produces the reduction form of eelctro-catalyst, and it reacts to produce the reduction form of electron mediator and the oxidised form of eelctro-catalyst with the oxidised form of electron mediator again. Then, the reaction of the oxidised form of the reduction form of electron mediator and oxidant produces oxidised form and the water of electron mediator. In one embodiment, have the permeable enzyme immobilization material of oxidant, it comprises eelctro-catalyst and randomly, electron mediator, and can immobilization and stablize described enzyme.
In the alternative embodiment of biological-cathode, there is not eelctro-catalyst. In this embodiment, the oxidised form of electronics and electron mediator makes up to produce the reduction form of electron mediator. Then, the reaction of the oxidised form of the reduction form of electron mediator and oxidant produces oxidised form and the water of electron mediator. In one embodiment, there is the enzyme immobilization material to described oxidant infiltration, its optional electron mediator that comprises, and can immobilization and stablize described enzyme.
Biological fuel cell of the present invention comprises biological-cathode and anode. In one embodiment, anode is biological anode. Usually, thus biological-cathode comprises realizes that fuel liquid oxidation electronics is released and imports the element of external electrical load. The gained electric current provides power for electric load, and electronics is imported biological-cathode subsequently, and here oxidant is reduced and produces water.
The inventor achieves success in the improved biological-cathode of exploitation now, and described biological-cathode can be used for actual electricity with the collaborative biological fuel cell that is used for of above-mentioned biological anode.
A. biological-cathode
Enzyme, electron mediator and eelctro-catalyst that biological-cathode according to the present invention comprises electronic conductor, fixes in the enzyme immobilization material. In one embodiment, these assemblies are mutually adjacent, represent they by the means that suit at physics or chemically connect.
1. electronic conductor
Electronic conductor (electrode) is the material of conduction electron. Electronic conductor can be organic or inorganic in nature, as long as it can pass through this material conduction electron. Electronic conductor can be based on material, stainless steel, stainless steel mesh, metallic conductor, semiconductor, the metal oxide of carbon, perhaps modified conductor. In preferred embodiments, electronic conductor is charcoal cloth.
Especially suitable electronic conductor is based on the material of carbon. Conductor, vitreous carbon and the mesoporous carbon of the electrode that representative material based on carbon is charcoal cloth, carbon paper, carbon screen printing, carbon paper (Toray), carbon paper (ELAT), carbon black (Vulcan XC-72, E-tek), carbon black, powdered carbon, carbon fiber, SWCN, double-walled carbon nano-tube, multi-walled carbon nano-tubes, carbon nano pipe array, diamond-coating. In addition, other representativenesses are purified flake graphite (Superior  graphite), high-performance graphite and powdered carbon (the Formula BT of graphite, unpressed graphite worm, layering based on the material of carbonTM, Superior  graphite), pyrolytic graphite, pyrolytic graphite and the polycrystalline graphite of high-sequential. Preferred electronic conductor (supporting film) is a slice charcoal cloth.
In another embodiment, electronic conductor can be by the metallic conductor manufacturing. Can and be suitable for the suitable electronic conductor of other preparation of metals that electrode is made from gold, platinum, iron, nickel, copper, silver, stainless steel, mercury, tungsten. In addition, can be by the nanoparticle manufacturing of cobalt, carbon and other suitable metal for the electronic conductor of metallic conductor. Other metal electron conductors can be that the nickel screen barrier of silver-coating prints electrode.
In addition, electronic conductor can be semiconductor. Suitable semi-conducting material comprises silicon and germanium, its other elements that can mix. Semiconductor can Doping Phosphorus, boron, gallium, arsenic, indium or antimony, perhaps their combination.
Other electronic conductors can be metal oxide, metal sulfide, main group compound (that is, transistion metal compound), and the material of modifying with electronic conductor. The aeroge that the representative electronic conductor of the type is the glass, cerium oxide particle, molybdenum sulfide, borazon nanotube of nano-pore titanium oxide, tin oxide coating, modify with conductive material such as carbon, solgels, the ruthenium carbon aerogels modified with conductive material such as carbon, and with the mesoporous silicate of conductive material such as carbon modification.
2. electron mediator
Electron mediator is the compound that can accept or confess electronics. In other words, electron mediator has oxidised form, and it can accept electronics to form the reduction form, and the form of wherein reducing also can be confessed electronics to produce oxidised form. Electron mediator is the compound that can be spread to immobilization material and/or be incorporated into immobilization material.
In one embodiment, the diffusion coefficient of electron mediator maximization. In other words, the mass transportation of the reduction form of electron mediator is fast as far as possible. The quick mass transportation of electron mediator allows to produce larger electric current and power density in used biological fuel cell.
The electron mediator of biological-cathode can be protein, such as stellacyanin, and the protein accessory substance, such as bilirubin, sugar, such as glucose, sterol, such as cholesterol, aliphatic acid, perhaps metalloprotein. Electron mediator can also be oxidasic coenzyme or substrate. In a preferred embodiment, the electron mediator of biological-cathode is bilirubin.
Technical staff in the present invention's practice will understand many different electron transfer mediators easily, and the transition metal complex that particularly has aromatic ligand can be used for practice of the present invention. In other words, have the transition metal complex of aromatic ligand and the interaction of polymer dielectric film (PEM) and changed the electronic property of PEM so that redox polymers to be provided.
3. the eelctro-catalyst of electron mediator
Usually, eelctro-catalyst (electron transfer mediator or redox polymers) is the material that promotes the electronics release of electronic conductor by reducing electron mediator place standard electrode potential.
Usually, eelctro-catalyst according to the present invention is standard electrode potential greater than+0.4 volt organic metal cation. Representative eelctro-catalyst is transition metal complex, such as osmium, ruthenium, iron, nickel, rhodium, rhenium and cobalt complex. Use the preferred organic metal cation of these complex compounds to comprise large organic aromatic ligand, it allows large electronics self exchange rate. The example of large organic aromatic ligand comprises 1,10-phenanthroline (phen), 2,2 '-bipyridyl (bpy) and 2,2 ', 2 " derivative of-three pyridines (terpy), such as Ru (phen)3 +2、Fe(phen) 3 +2、Ru(bpy) 3 +2、Os(bpy) 3 +2, and Os (terpy)3 +2 In preferred embodiments, eelctro-catalyst is ruthenium compound. Most preferably, the eelctro-catalyst of biological-cathode is Ru (bpy)3 +2(by formula 1 representative).
Figure A20048003838400271
Eelctro-catalyst exists with the concentration of effective transfer of promotion electronics. Preferably, eelctro-catalyst is so that the concentration of enzyme immobilization material conduction electron exists. Particularly, to about 3M, more preferably from about 250mM is to about 2.25M with about 100mM for eelctro-catalyst, and more preferably from about 500mM is to about 2M, and most preferably from about 1.0M exists to the concentration of about 1.5M.
Redox polymers can be modified amberplex, and it is further modified and contains electron transfer mediator (for example, osmium or ruthenium complex, perhaps aromatic series organic cation). The many electron transfer mediators or the redox polymers that are used for the present invention's practice are known in the art and are described in U.S. Patent number 5,262,035; 5,262,305; 5,320,725; 5,264,105; 5,356,786; 5,593,852; 5,665,222; 6,294,281; With 6,531,239, with described patent citation as a reference.
4. enzyme
According to the present invention, the oxidant of enzyme reduction biological-cathode. Usually, can utilize the enzyme, synthetic enzyme, artificial enzyme of natural generation and the enzyme of modified natural generation. In addition, can use by through engineering approaches enzyme natural or that orthogenesis is transformed. In other words, can use in embodiments of the invention the organic or inorganic molecule of the character of analogue enztme.
Especially, the representative enzyme that is used for biological-cathode is an oxidoreducing enzyme.Potential oxidoreducing enzyme comprises laccase and oxidizing ferment, as glucose oxidase, based on the oxidizing ferment of alcohol with based on the oxidizing ferment of cholesterol.In preferred embodiments, described enzyme is peroxidase or dioxygen oxidation reductase, and they are catalytic reduction hydrogen peroxide and oxygen respectively.Representative dioxygen oxidation reductase comprises laccase, cytochrome c oxidase, bilirubin oxidase and peroxidase.More preferably, described enzyme is the dioxygen oxidation reductase that has optimal activity at about 6.5 to about 7.5 pH.The oxidoreducing enzyme that pH about 6.5 to about 7.5 has an optimal activity is at physiological environment, is favourable as the application of plant or people or animal body.Most preferably, described enzyme is a bilirubin oxidase.
5. enzyme immobilization material
Biological anode and/or biological-cathode at biological fuel cell use the enzyme immobilization material.In one embodiment, the enzyme immobilization material of biological anode is permeable and fixing for fuel liquid and stablizes described enzyme.Described immobilization material is permeable for described fuel liquid, thereby immobilised enzymes can the fuel oxidation reaction of catalysis biological anode place.
Normally, use the redox reaction of enzymatic biological-cathode and/or biological anode.In electrode according to the present invention, enzyme is fixed in the enzyme immobilization material, and this enzyme immobilization material catalysis is also stablized described enzyme.Usually, the resolvase in the solution is lost its catalytic activity in a few hours to a couple of days, and correct immobilization and stable enzyme can keep its catalytic activity in about 30 days to about 365 days.With the reservation of catalytic activity be defined as have its initial activity at least about 75% enzyme, can measure initial activity by chemiluminescence, electrochemistry, UV-Vis, radiochemistry or fluorimetry.
Immobilised enzymes is physically to be confined to certain zone of enzyme immobilization material and to keep the enzyme of its catalytic activity.The plurality of enzymes process for fixation is arranged, comprise carrier combination, crosslinked and embedding.The carrier combination is that enzyme is attached to water insoluble carrier.Crosslinked is by the difunctional or poly functional reagent intermolecular cross-linking to enzyme.Embedding is the grid that enzyme is incorporated into semipermeable materials.The ad hoc approach of enzyme immobilization is not vital, as long as this enzyme of enzyme immobilization material (1) immobilization, (2) stablize this enzyme and (3) are permeable for fuel liquid or oxidant.
To the permeability of fuel liquid or oxidant and the immobilization of enzyme, in one embodiment, described material is permeable for the compound less than enzyme about the enzyme immobilization material.In other words, thus this enzyme immobilization material allows fuel liquid or oxidant compound can contact described enzyme by its this compound of motion.Can prepare the enzyme immobilization material in one way and make it contain internal holes, passage, opening or its combination, it allows compound by the enzyme immobilization material movement, but enzyme is constrained in the essentially identical space of enzyme immobilization material.This type of constraint makes enzyme keep its catalytic activity.In a preferred embodiment, enzyme is limited in having with this enzyme the space of essentially identical size and shape, and wherein this enzyme keeps its all catalytic activitys basically.Described hole, passage, perhaps opening has the satisfied physical size that requires above and depends on size and the shape for the treatment of immobilized certain enzyme.
In one embodiment, enzyme be preferably placed in the hole of enzyme immobilization material and compound by transport channel turnover enzyme immobilization material.The relative size of hole and transport channel can be for making the hole enough greatly with immobilized enzyme, but transport channel is too little and can not move by them for enzyme.In addition, transport channel preferably has the diameter at least about 10nm.In another embodiment, bore dia be at least about 2: 1 with the ratio of transport channel diameter, 2.5: 1,3: 1,3.5: 1,4: 1,4.5: 1,5: 1,5.5: 1,6: 1,6.5: 1,7: 1,7.5: 1,8: 1,8.5: 1,9: 1,9.5: 1,10: 1 or more than.In a further embodiment, preferably, transport channel have diameter and bore dia at least about 10nm be at least about 2: 1 with transport channel diameter ratio, 2.5: 1,3: 1,3.5: 1,4: 1,4.5: 1,5: 1,5.5: 1,6: 1,6.5: 1,7: 1,7.5: 1,8: 1,8.5: 1,9: 1,9.5: 1,10: 1 or more than.
About stablizing of enzyme, the enzyme immobilization material provides chemistry or the mechanical barrier that prevents or stop enzyme denaturation.For this reason, the enzyme immobilization material limits this enzyme physically, prevents that this enzymolysis is folding.From the process of folding three-dimensional structure unfoldase is a kind of mechanism of enzyme denaturation.In one embodiment, immobilization material is preferably stablized this enzyme, thereby this enzyme keeps its catalytic activity at least about 30 days to about 365 days.The maintenance of catalytic activity keeps the fate at least about 75% of its initial activity to define by this enzyme.Can measure enzymatic activity by chemiluminescence, electrochemistry, VU-Vis, radiochemistry or fluorimetry, wherein in the intensity of initial this character of time measurement.Usually, measure enzymatic activity with fluorimetry.Resolvase in the solution is lost its catalytic activity in a few hours to a couple of days.Thereby the stable aspect that is immobilized in of enzyme provides significant advantage.In another embodiment, preferably, immobilized enzyme at least about 30,45,60,75,90,105,120,150,180,210,240,270,300,330,365 days or above its initial catalytic activity of maintenance at least about 75%, preferably, at least about 30,45,60,75,90,105,120,150,180,210,240,270,300,330,365 days or above its initial catalytic activity of maintenance at least 80%, 85%, 90%, 95% or more than.
In one embodiment, the enzyme immobilization material is the colloidal materials that non-natural takes place.In another embodiment, the enzyme immobilization material is acellular colloidal materials, as liposome.Acellular material be can't help cell and is formed and do not contain cell.Colloidal materials is made up of the particle that is dispersed in fully in the another kind of material, and but described particle is too little and can not differentiate with ordinary optical microscope can not pass through pellicle.In addition, colloidal materials by obviously greater than atom or common molecule but too little and the sightless particle of naked eyes is formed.Their size can be about 10 -7To 10 -3Centimetre, and connect in many ways or combine.
In a further embodiment, the enzyme immobilization material has micella or reversed micelle structure.Usually, the molecule of forming micella is amphipathic, represents that they contain the hydrophilic radical and the nonpolar hydrophobic grouping of polarity.Molecule can be assembled to form micella, and wherein polar group is on the surface of aggregation and hydrocarbon, and non-polar group is isolated in the aggregation.Reversed micelle has rightabout polar group and non-polar group.Forming the amphiphile, amphiphilic molecule of aggregation can arrange in many ways, as long as polar group is approaching mutually and non-polar group is approaching mutually.And, molecule can with mutually towards non-polar group and mutually dorsad polar group form bilayer.Alternatively, can form bilayer, wherein polar group can be in bilayer mutually towards, and non-polar group is mutually dorsad.
Usually, micella or reversed micelle enzyme immobilization material can be polymer, pottery, liposome or by molecular any other material that forms micella or reversed micelle structure.Representative micella or reversed micelle enzyme immobilization material are perfluorinated sulfonic acid-polytetrafluoroethylene (PTFE) copolymer (perhaps perfluorinated ion-exchange polymer) (Nafion  or Flemion ), perfluorinated sulfonic acid-polytetrafluoroethylene (PTFE) copolymer (the perhaps perfluorinated ion-exchange polymer of Xiu Shiing) (the Nafion  of modification or the Flemion  of modification) of modifying, polysulfones, the micellar copolymerization thing, block copolymer based on poly-(ethylene oxide), polymer and methacrylate Arrcostab from microemulsion and/or micellar copolymerization effect formation, alkyl acrylate and cinnamic copolymer.Other representative micellas or reversed micelle immobilization material are pottery, two (2-ethylhexyl) sodium sulfo-succinate, dioctyl sodium sulfo-succinate, lipid, phosphatide, lauryl sodium sulfate, decyl trimethylammonium bromide, TTAB, (azo of 4-[(2-hydroxyl-1-naphthalenyl)] benzene sulfonic acid one sodium salt), linoleic acid, leukotrienes, colloid, liposome and micella network.
In a preferred embodiment, micella enzyme immobilization material is modified perfluorinated sulfonic acid-PTFE copolymer (perhaps modified perfluorinated ion-exchange polymer) (modified Nafion  or modified Flemion ) film.The perfluorinated ion-exchange polymer film is used greater than ammonium (NH 4 +) dewatering cationic of ion modifies.Dewatering cationic has difunctional: to help the pH level of retaining hole, two kinds of functions are all further done in order to stablize described enzyme as chemical buffer for the aperture of (1) decision film and (2).
First kind of function about dewatering cationic, provide the enzyme immobilization material with dewatering cationic mixture-casting perfluorinated sulfonic acid-PTFE copolymer (perhaps fluoridized ion-exchange polymer) to produce perfluorinated sulfonic acid-PTFE copolymer (the perhaps perfluorinated ion-exchange polymer of Xiu Shiing) (Nafion  or Flemion ) of modifying, wherein the size of dewatering cationic is depended in the aperture.Therefore, dewatering cationic is big more, and the aperture is big more.This function of dewatering cationic allows to make the aperture bigger or littler to be fit to specific enzyme by the size that changes dewatering cationic.
About second kind of function of dewatering cationic, by with dewatering cationic exchange as on perfluorinated sulfonic acid-PTFE copolymer (perhaps fluoridized ion-exchange polymer) film-SO 3-The proton of the equilibrium ion of group changes the character of perfluorinated sulfonic acid-PTFE copolymer (perhaps fluoridized ion-exchange polymer).The change of this equilibrium ion provides buffering effect to pH because dewatering cationic right-SO 3-Site comparison proton has bigger compatibility.This buffering effect of film causes the pH in hole to remain unchanged basically along with the change of pH value of solution; In other words, the change of the anti-pH value of solution of pH in hole.In addition, film provides mechanical barrier, and it further protects the enzyme of being fixed.
Following table has been illustrated the buffering effect of modified perfluorinated sulfonic acid-PTFE co-polymer membrane.The modified available proton exchange number of sites of perfluorinated sulfonic acid-PTFE co-polymer membrane of these value every grams of representative; Along with the available exchange number of sites of proton reduces, film increases the buffer capacity of immobilised enzymes.Film below the film abbreviation expression: NH4Br is the Nafion  film of ammonium bromide-modification, TMABr is the Nafion  film that 4 bromide is modified, TEABr is the Nafion  film that tetraethylammonium bromide is modified, TpropABr is the Nafion  film that 4-propyl bromide is modified, TBABr is the Nafion  film that TBAB is modified, and TpentABr is the Nafion  film that the four pentyl ammonium bromide is modified.
Film Mixture casting (* 10 -6mole/g) (* 10 of salt extraction -6 mole/g)
Nafion 907+68 -
NH 4Br 521+74 591±95
TMABr 171±19 458+27
TEABr 157±4 185±22
TPropABr 133±6 138±77
TBABr 8.68+2.12 96±23
TPentABr 2.71±0.6 1.78±1.66
For perfluorinated sulfonic acid-PTFE copolymer (perhaps perfluorinated ion-exchange polymer) film for preparing modification, the first step is the suspension with the solution casting perfluorinated sulfonic acid-PTFE copolymer (perhaps perfluorinated ion-exchange polymer) of dewatering cationic, and especially Nafion  is to form film.Extract the salt of excessive dewatering cationic and they from initial film after, film is cast again.When casting again, film contain with perfluorinated sulfonic acid-PTFE copolymer (perhaps perfluorinated ion-exchange polymer) film-SO 3 -The dewatering cationic of site combination.
In order to prepare the Nafion  film that more stable and reproducible quaternary ammonium salt is handled, must remove excessive bromide salt from casting solution.After extracting excessive quaternary ammonium bromides and HBr salt from initial film, form the film that this salt extracts by casting mixture-cast film again.The salt of film extracts and remains on sulfonic acid exchange site and have quaternary ammonium cation, but has eliminated the complexity of excess salt, and described salt can be trapped in the hole or can cause hole in the balance film.Before enzyme immobilization, characterize the chemistry and the physical property of the film of salt extraction by voltammetry, ion-exchange capacity measurement and fluorescence microscopy.Representative dewatering cationic is based on cation, quaternary ammonium cation, alkyl trimethyl ammonium cation, alkyl triethyl ammonium cation, organic cation,  cation, triphenyl , pyridine  cation, imidazoles  cation, cetyl pyridinium , second ingot, purpurine, methyl viologen, benzyl viologen, two (triphenylphosphine) iminium, metal complex, Bipyridine metal complexes, the metal complex based on the phenanthroline, [Ru (bipyridine) of ammonium 3] 2+[Fe (phenanthroline) 3] 3+
In a preferred embodiment, dewatering cationic is based on the cation of ammonium.Particularly, dewatering cationic is a quaternary ammonium cation.In another embodiment, quaternary ammonium cation is represented by formula (2):
Figure A20048003838400331
R wherein 1, R 2, R 3And R 4Be the alkyl or the heterocycle of hydrogen, alkyl, replacement, wherein R independently 1, R 2, R 3And R 4At least one be not hydrogen.In another embodiment, preferably, R 1, R 2, R 3And R 4Be hydrogen, methyl, ethyl, propyl group, butyl, amyl group, hexyl, heptyl, octyl group, nonyl, decyl, undecyl, dodecyl, tridecyl or myristyl, wherein R independently 1, R 2, R 3And R 4At least one be not hydrogen.In a further embodiment, R 1, R 2, R 3And R 4Identical and be methyl, ethyl, propyl group, butyl, amyl group or hexyl.In a further embodiment, preferably, R 1, R 2, R 3And R 4Be butyl.Preferably, quaternary ammonium cation is TBuA, triethyl group hexyl ammonium or dodecyl trimethyl ammonium.
Quaternary ammonium salt or surfactant are (for example, TBAB, triethyl group hexyl ammonium bromide, trimethyldodecane base ammonium bromide and phenyl trimethylammonium bromide) and mixture-cast film of Nafion  increased little analyte by film mass transportation and reduced the enzyme immobilization film to anionic selectivity.These enzyme immobilization films have the closely similar conductibility with the Nafion of unmodified, but they have higher preference to bromination quaternary ammonium comparison proton, as by in the titration enzyme immobilization film shown in the available exchange number of sites of proton.Therefore, these films have similar electrical property, but very different acid/alkaline matter.Handled enzyme immobilization film keeps their neutral pH in the pH of buffer of wide region.According to these advantages, preferred enzyme immobilization material is perfluorinated sulfonic acid-PTFE copolymer (the perhaps perfluorinated ion-exchange polymer of Xiu Shiing) (the Nafion  of modification or the Flemion  of modification) film that quaternary ammonium salt is handled.More preferably, described enzyme immobilization material is the Nafion  membrane material that TBAB-modifies.Even more preferably, the enzyme immobilization material is Nafion  membrane material, the Nafion  membrane material that the phenyl trimethylammonium bromide is modified, perhaps the Nafion  membrane material of trimethyl octyl ammonium bromide-modification that triethyl group hexyl ammonium bromide is modified.
In preferred embodiments, described film comprises the material that can form micella or reversed micelle, and described micella or reversed micelle can mix and stable oxidoreducing enzyme, and mix electron transfer mediator.Preferably, described membrane material is modified amberplex.More preferably, described membrane material is perfluorinated sulfonic acid-PTFE copolymer of handling of quaternary ammonium, surfactant or  salt (the perhaps perfluorinated ion-exchange polymer of Xiu Shiing, for example, the Nafion  of modification or the Flemion  of modification) film.Most preferably, described membrane material is the Nafion  membrane material that TBAB (TBAB) is handled.Described modification causes in the micella of ion-exchange polymer near neutral pH (～7.4).
6. biological-cathode embodiment
In one embodiment, biological-cathode comprises the enzyme immobilization material, the enzyme of its fixed negative pole and promoting in biological-cathode generation redox reaction.Described enzyme, eelctro-catalyst and electron mediator are preferably placed in the pocket or micella of enzyme immobilization material.In preferred embodiments, described enzyme immobilization material comprises the material that can form micella or reversed micelle, described micella or reversed micelle can mix again and stabilized enzyme, and form other zones, as hole, passage, opening or their combination, they can mix eelctro-catalyst and electron mediator.Preferably, micella also has buffer capacity, and promptly micellar structure comprises buffer portion.The micellar structure of this buffering of enzyme immobilization material promotes electronics directly to transfer to electrode and eelctro-catalyst or electron mediator or from the transfer of described electrode and eelctro-catalyst or electron mediator.
Advantageously, in multiple embodiments, the concentration of eelctro-catalyst or electron mediator enough makes enzyme immobilization conduct electronics in the enzyme immobilization material.The concentration of eelctro-catalyst or electron mediator arrives about 3M for about 100mM in the enzyme immobilization material, and more preferably, about 250mM is to about 2.25M, and more preferably from about 500mM is to about 2M, and most preferably from about 1.0M is to about 1.5M.This concentration of eelctro-catalyst or electron mediator promotes the speed of electron transport, and it allows the maximization current density.When the enzyme immobilization material was polymer, preferably, the electronic property of the enzyme immobilization material of the top concentration change polymerization of eelctro-catalyst or electron mediator was so that it is a redox polymers.
In a further embodiment, the present invention relates to fuel cell, it comprises biological-cathode and anode, and wherein biological-cathode comprises eelctro-catalyst, enzyme immobilization material, and enzyme.Described enzyme is incorporated in the micella compartment of enzyme immobilization material.Preferably, the enzyme immobilization material is the perfluorinated ion-exchange polymer of the quaternary ammonium processing of salt-extraction.Comprise Nafion  (DuPont) and Flemion  (Asahi Glass) by the available perfluorinated ion-exchange polymer of commercial sources.Preferably, perfluorinated ion-exchange polymer is Nafion  polymer or Flemion  polymer.Preferred quaternary ammonium salt comprises TBAB.Preferred eelctro-catalyst is poly-methylene green (polymethylene green).Biological-cathode can comprise more than one different enzymes.
The schematic diagram of the redox reaction that takes place about biological-cathode is in preferred embodiments seen Fig. 1.Wherein, be used for being positioned at the eelctro-catalyst (Ru (bipyridine) of (15) from the electronics of electronic conductor (electrode) (13) 3 + 2), the redox reaction between electron mediator (bilirubin), enzyme (bilirubin oxidase) (14) and the oxidant (11) to be to form the water accessory substance.Enzyme (14) is stabilisation in the micellar structure (12) of enzyme immobilization material (10).
In one embodiment, the present invention relates to improved biological-cathode, it utilizes amberplex, and this film comprises one or more oxidoreducing enzyme and one or more electron transfer mediators, thereby makes things convenient for the effective manufacturing and the effective efficiency performance of biological-cathode.This type of film is called " difunctional film " in this article.This difunctional film can be applied to electrically conductive material.
In another embodiment, the present invention relates to improved biological-cathode, it utilizes amberplex, and this film comprises one or more oxidoreducing enzyme and one or more eelctro-catalysts, thereby makes things convenient for the effective manufacturing and the effective efficiency performance of biological-cathode.This type of film is called " difunctional film " in this article.This difunctional film can be applied to electrically conductive material.
The present invention relates to comprise the biological-cathode of difunctional film, described film works as enzyme immobilization film and redox film, this biological-cathode also comprises cathode enzyme, and it is preferably placed in the pocket or micella of difunctional film, and this biological-cathode also comprises electron transfer mediator.
Biological-cathode of the present invention has useful power density and stable enzyme, and described enzyme works and at room temperature for a long time at physiology or near neutral pH, promptly keeps active in 10 days.Usually, biological-cathode comprises oxidoreducing enzyme, as the dioxygen oxidation reductase, and the electron reduction oxygen that it uses anode (from complete circuit) to provide.Biological-cathode of the present invention can combine the part as fuel cell with the anode of any kind.Preferably, biological-cathode is used in combination with biological anode in the biological fuel cell.
In preferred embodiments, the difunctional film of biological-cathode comprises cathode enzyme, it (for example is preferably the dioxygen oxidation reductase, laccase, bilirubin oxidase), more preferably, in neutral pH or near the dioxygen oxidation reductase that has optimal activity under the neutral pH, as bilirubin oxidase, it is fixed in the micella with buffer capacity.Preferably, cathode enzyme is fixed in the perfluorinate amberplex, this film modified with in micellar structure, hold enzyme and wherein this micellar structure comprise buffer portion, as ammonium or  ion.Most preferably, cathode enzyme be fixed on the Nafion  film that TBAB (TBAB), triethyl group hexyl ammonium halide (TEHA) or trimethyldodecane base ammonium halide (TMDA) that salt extracts handle (as U.S. Patent application 10/617,452 and hereinafter description be used for preferred biological anode).In addition, the micella film of this buffering contains electron transfer mediator, as comprises ruthenium, nickel, rhenium, rhodium, iron, cobalt or the osmium complex of aromatic ligand, to make things convenient for electronics to the direct transfer of electrode and enzyme catalyst with from the direct transfer of described electrode and enzyme catalyst.Alternatively, the micella film of this buffering contains eelctro-catalyst, as comprises ruthenium, nickel, rhenium, rhodium, iron, cobalt or the osmium complex of aromatic ligand, to make things convenient for electronics to the direct transfer of electrode and enzyme catalyst with from the direct transfer of described electrode and enzyme catalyst.
B. biological anode
In one embodiment, biological anode comprises electronic conductor and the enzyme that is fixed in the enzyme immobilization material.In another embodiment, the optional eelctro-catalyst that also comprises electron mediator of biological anode.Can be when electronic conductor experience the electron mediator of reversible redox reaction when biological anode contact, biological anode can lack eelctro-catalyst.The assembly of identifying above of biological anode can be adjacent mutually; Represent that they connect by suitable means physically or chemically.Other embodiments are described among the I.A.6 hereinafter.Because described assembly is usually identical with the biological-cathode assembly, so following discussion relates to difference in the component of element separately and different (if suiting) of function.
1. electronic conductor
As biological-cathode, the electronic conductor of biological anode can be an organic or inorganic in nature, as long as it can pass through this conduct electronics.In one embodiment, biological anode electronic conductor is a charcoal cloth.
2. electron mediator
Biological anode electron mediator is used for accepting or confessing electronics, easily changes to the form of reduction from oxidation.Electron mediator is the compound that can be spread to immobilization material and/or be incorporated into immobilization material.As biological-cathode, the maximization of the diffusion coefficient of preferred electron amboceptor.
Representative electron mediator is nicotinamide adenine dinucleotide (NAD +), flavin adenine dinucleotide (FAD) (FAD), nicotinamide-adenine dinucleotide phosphate (NADP), perhaps PQQ (PQQ), perhaps every kind equivalent.Other representative electron mediators are phenazine methosulfate, dichloropheno-lindophenol, short chain ubiquinone, the potassium ferricyanide, protein, metalloprotein, and stellacyanin.In a preferred embodiment, the electron mediator of biological anode is NAD +
Send out at once when electron mediator can not self experience redox at the electronic conductor place, biological anode comprises the eelctro-catalyst of electron mediator, and it promotes the release of electronic conductor place electronics.Alternatively, has the reversible redox of normal reduction potential of 0.0V ± 0.5V to as electron mediator.In other words, can use the electron mediator that reversible electrochemical is provided on the electronic conductor surface.Electron mediator and the enzyme that depends on the natural generation of this electron mediator, modified and depend on the enzyme of this electron mediator perhaps depend on the synzyme coupling of this electron mediator.The example that the electron mediator of reversible electrochemical is provided on the electronic conductor surface is PQQ (PQQ), phenazine methosulfate, dichloropheno-lindophenol, short chain ubiquinone and the potassium ferricyanide.In this embodiment, the preferred electron amboceptor that is used for biological anode is PQQ.Because electron mediator can provide reversible electrochemical on the electronic conductor surface, in this embodiment needn't electricity consumption catalyst redox reaction.
Comprise the adenine-dinucleotide of reduction as the preferred compound of the substrate of the electro-catalysis of the redox polymers of biological anode, as NADH, FADH 2And NADPH.
3. the eelctro-catalyst of electron mediator
Usually, eelctro-catalyst is the material that promotes that electronics discharges on the electronic conductor.In other words, thus eelctro-catalyst has improved the reduction of electron mediator or electron mediator oxidation or reduction can take place under lower standard electrode potential the dynamics of oxidation.Eelctro-catalyst can biological anode reversibly oxidation with produce electronics and, thereby produce.When eelctro-catalyst was adjacent with electronic conductor, eelctro-catalyst and electronic conductor electrically contacted mutually, but physics contact mutually.In one embodiment, electronic conductor is the part of the eelctro-catalyst of electron mediator, combines with described eelctro-catalyst or adjacent.
Usually, eelctro-catalyst can be azine, conducting polymer or electroactive polymer.The ligand transition metal, 1 of the terpyridyl that representative eelctro-catalyst is methylene green, methylenum careuleum, luminol, nitro-fluorenone derivatives, azine, phenanthroline dione osmium, catechol-side group, toluene blue, cresyl blue, Nile blue, dimethyl diaminophenazine chloride, azophenlyene derivative, tionin, sky blue (azure) A, aB, blutene, acetophenone, metal phthalocyanine, Nile blue A, modification, 10-phenanthroline-5,6-diketone, 1,10-phenanthroline-5, the 6-glycol, [Re (phendione) (CO) 3Cl], [Re (phendione) 3] (PF 6) 2Poly-(metal phthalocyanine), poly-(thionine), quinone, diimine, diaminobenzene, diamino-pyridine, phenthazine, fen  piperazine, toluidine blue, brilliant cresyl blue, 3, the 4-4-dihydroxy benzaldehyde, poly-(acrylic acid), poly-(sky blue I), poly-(Nile blue A), poly-(methylene green), poly-(methylenum careuleum), polyaniline, polypyridine, polypyrrole, polythiophene, poly-(thieno [3,4-b] thiophene), poly-(3-hexyl thiophene), poly-(3,4-ethylenedioxy pyrroles), poly-(isothianaphthene), poly-(3,4-ethylenedioxy thiophene), poly-(difluoro acetylene), poly-(4-dicyano methylene-4H-ring penta [2,1-b; 3,4-b '] two thiophene), poly-(3-(4-fluorophenyl) thiophene), poly-(dimethyl diaminophenazine chloride), protein, metalloprotein or stellacyanin.In a preferred embodiment, the eelctro-catalyst of electron mediator is poly-(methylene green).
4. enzyme
The oxidation of the biological anode of enzymatic place fuel liquid.Because enzyme reduces the oxidant of biological-cathode, so they are more generally described in I.A.4 above.Usually, can utilize the enzyme, synthetic enzyme, artificial enzyme of natural generation and the enzyme of modified natural generation.In addition, can use through engineering approaches enzyme by natural or orthogenesis through engineering approaches.In other words, can use the organic or inorganic molecule of the character of analogue enztme in embodiments of the invention.
Especially, the representative enzyme that is used for biological anode is an oxidoreducing enzyme.In a preferred embodiment, oxidoreducing enzyme acts on the CH-OH base or the CH-NH base of fuel (alcohol, ammoniate, carbohydrate, aldehyde, ketone, hydrocarbon, aliphatic acid or the like).
In another preferred embodiment, described enzyme is a dehydrogenase.Representative enzyme in this embodiment comprises alcohol dehydrogenase, aldehyde dehydrogenase, hydrogenlyase, formaldehyde dehydrogenase, glucose dehydrogenase, glucose oxidase, lactic dehydrogenase, lactose dehydrogenase or pyruvic dehydrogenase.Preferably, described enzyme is alcohol dehydrogenase (ADH).
In currently preferred embodiments, described enzyme is the alcohol dehydrogenase that relies on PQQ.PQQ be rely on PQQ ADH coenzyme and keep electrostatic adherence to the ADH that relies on PQQ and therefore this enzyme will remain in the film, cause the life-span and active the increasing of biological fuel cell.The alcohol dehydrogenase that relies on PQQ extracts from gluconobacter sp (gluconobacter).When extract relying on the ADH of PQQ, it can be two kinds of forms: (1) PQQ static be attached to the ADH that relies on PQQ or (2) PQQ not static be attached to the ADH that relies on PQQ.For PQQ not static be attached to second kind of form of the ADH that relies on PQQ, when the biological anode of assembling, PQQ is added ADH.In currently preferred embodiments, extract the ADH of the dependence PQQ of PQQ static combination from gluconobacter sp.
5. enzyme immobilization material
Mention at I.A and I.B as mentioned, use the enzyme immobilization material at biological anode and/or the biological-cathode of biological fuel cell.About can the see before I.A.5 of face of the further details of the composition of enzyme immobilization material and immobilization mechanism.In one embodiment, the enzyme immobilization material of biological anode is permeable for fuel liquid and immobilization and stablize described enzyme.Immobilization material is that fuel liquid is permeable, thereby the oxidation of the fuel liquid of biological anode can be by immobilized enzymatic.Preferably, the enzyme immobilization material is perfluorinated sulfonic acid-PTFE copolymer (the perhaps perfluorinated ion-exchange polymer of Xiu Shiing) (the Nafion  of modification or the Flemion  of modification) film that quaternary ammonium salt is handled.More preferably, the enzyme immobilization material is the Nafion  membrane material that TBAB (TBAB) is handled.Even more preferably, the enzyme immobilization material is Nafion  membrane material, the Nafion  membrane material of trimethyl octyl ammonium bromide-processing or the Nafion  membrane material that the phenyl trimethylammonium bromide is handled that triethyl group hexyl ammonium bromide is handled.
6. biological anode embodiment
In another embodiment, preferably, biological anode is made up of electronic conductor, by modifying this electronic conductor to this electronic conductor absorption, polymerization or covalent bond eelctro-catalyst.This embodiment has the advantage of the surface area that increases electronic conductor.Chemistry by absorption eelctro-catalyst and eelctro-catalyst subsequently on the surface of electronic conductor before assembling or electrochemical polymer are handled electronic conductor and are compared with untreated electronic conductor and cause higher catalytic activity.
In another embodiment, electron mediator desmoenzyme physically.Physical bond can be covalency or the ions binding between electron mediator and the enzyme.In a further embodiment, if electron mediator can carry out reversible electrochemical reaction at electronic conductor, physically desmoenzyme and this electron mediator can also be physically in conjunction with electronic conductors for this electron mediator so.
In a further embodiment, electron mediator is fixed in the immobilization material.In preferred embodiments, electron mediator is the NAD of oxidation fixing in cation modified perfluorinated sulfonic acid-PTFE copolymer (cation modified Nafion ) film +In this embodiment, in battery, add fuel liquid after, NAD +Being reduced into NADH and NADH can be by cation modified perfluorinated sulfonic acid-PTFE copolymer (cation modified Nafion ) film diffusion.
In another embodiment, dehydrogenase is fixed on TBuA/perfluorinated ion-exchange polymer film (for example, Nafion  film or Flemion  film [Asahi Glass Co., Tokyo]) that salt extracts.The polymer suspension that salt extracts is neutral, can add the enzyme solutions of buffering to this suspension.The biological anode that the mixture casting can be modified with formation at biological anode, wherein enzyme is fixed on the near surface of biological anode.
In another embodiment, biological anode comprises modified enzyme immobilization material, and it causes interior neutral pH and one or more enzymes of micella of this material, and described enzyme is incorporated in the micella of modified enzyme immobilization material.Preferred enzyme immobilization material is a Nafion  polymer.Preferred enzyme is an oxidoreducing enzyme, as dehydrogenase, and the oxidation of its catalysis organic-fuel and the reduction of electron mediator.
In a further embodiment, the present invention relates to comprise the fuel cell of biological anode and biological-cathode, wherein biological anode comprises eelctro-catalyst, enzyme immobilization material, and enzyme.Described enzyme is incorporated in the micella compartment of enzyme immobilization material.Preferably, the enzyme immobilization material is the perfluorinated ion-exchange polymer of the quaternary ammonium processing of salt extraction.Comprise Nafion  (DuPont) and Flemion  (Asahi Glass) by the obtainable perfluorinated ion-exchange polymer of commercial sources.Preferably, perfluorinated ion-exchange polymer is Nafion  polymer or Flemion  polymer.Preferred quaternary ammonium salt comprises TBAB.Preferred eelctro-catalyst is poly-methylene green.Biological anode can comprise more than one different enzymes, as alcohol dehydrogenase and aldehyde dehydrogenase.
It is known in the art preparing and using the method for biological anode, and described method can be used to make and use the biological fuel cell that comprises biological-cathode of the present invention.Preferred biological anode is described in U.S. Patent application 10/617,452, quotes as a reference described patent application is complete.Other biological anodes that come in handy are described in U.S. Patent number 6,531, and 239 and 6,294,281, its also complete quoting as a reference.
In brief, in an embodiment of preferred biological anode, described biological anode can be used to make and use the biological fuel cell that comprises top disclosed biological-cathode, and described biological anode comprises the anodic oxidation reductase of the oxidation of catalysis organic-fuel.Usually, anode provides the electronics source of circuit and current potential.Representative preferred biological anode comprises supporting film or structure, as carbon cloth or carbon felt, itself and redox polymer film are arranged side by side, and the ion exchange polymer membrane of this film and modification is arranged side by side, this ion exchange polymer membrane comprises the micella of buffering, fixed anode oxidoreducing enzyme in this micella.
Current preferred biological anode comprises the carbon electronic conductor, the alcohol dehydrogenase coating that it relies on the PQQ-that is fixed on cation modified perfluorinated sulfonic acid-PTFE copolymer.The carbon electronic conductor can be glassy carbon electrode, carbon felt, carbon paper or the like.
C. fuel liquid and oxidant
Can be the component of biological fuel cell of the present invention with the fuel liquid that produces electronics and the oxidant that can produce water in biological anodic oxidation in the biological-cathode reduction.
The fuel liquid of biological anode consumes in the oxidation reaction of electron mediator and immobilised enzymes.The molecular size of fuel liquid is enough little, thereby very big by the diffusion coefficient of enzyme immobilization material.Representative fuel liquid is a hydrogen, ammonia, alcohol is (as methyl alcohol, ethanol, propyl alcohol, isobutanol, butanols and isopropyl alcohol), allyl alcohol, aryl alcohol, glycerine, propylene glycol, sweet mellow wine, glucuronic acid, aldehyde, carbohydrate is (as glucose, glucose-1, D-glucose, L-glucose, G-6-P, lactic acid, 9--phosphoric acid, D-lactic acid, L-lactic acid, fructose, galactolipin-1, galactolipin, aldose, sorbose, mannose), glyceric acid, coacetylase, acetyl coenzyme A, malic acid, isocitric acid, formaldehyde, acetaldehyde, acetate, citric acid, the L-gluconic acid, beta-hydroxysteroid, alpha-hydroxysteroid, lactic aldehyde, testosterone, gluconic acid, aliphatic acid, lipid, phosphoglyceric acid, retinene, estradiol, cyclopentanol, hexadecanol, long-chain alcohol, coniferyl alcohol, cinnamyl alcohol, formic acid, long-chain aldehyde, pyruvic acid, butyraldehyde, acyl coenzyme A, steroids, amino acid, flavine, NADH, NADH 2, NADPH, NADPH 2, hydrocarbon, and amine.In preferred embodiments, fuel liquid is an alcohol, more preferably, and methyl alcohol and/or ethanol; Most preferred ethanol.
The oxidant of biological-cathode is consumed in the reduction reaction that the electronics that electron mediator and immobilized enzyme use biological anode to provide carries out.The molecular size of oxidant is enough little, thereby very big by the diffusion coefficient of enzyme immobilization material.Can utilize the several different methods that oxidant source known in the art is provided.
In preferred embodiments, oxidant is a gas oxygen, and it is transported to biological-cathode by diffusion.In another preferred embodiment, oxidant is a peroxide compound.
II. biological fuel cell embodiment
In another embodiment, the present invention relates to comprise the biological fuel cell of improved biological-cathode.Usually, biological fuel cell utilizes the oxidation of organic-fuel (hydrocarbon, amine, alcohol, sugar or the like) as the energy and redox enzymatic organic-fuel.Biological fuel cell of the present invention can be used for the application of needs electricity supply, for example, but is not limited to the vehicles of electronic installation and equipment, toy, small item, inner medical instruments and electric energy supply.Fuel cell of the present invention can also be implanted living organism, and wherein organic-fuel is the device energy supply of implanting this living organism from this biology and fuel cell.
People such as Minteer are further improving patent application 60/429 by the method for research and development bioelectrode and production bioelectrode, 829,60/486,076 and 10/617, the biological fuel cell of disclosed reality has been obtained success in 452, the perfluorinate amberplex that described bioelectrode is not only handled at quaternary ammonium (perhaps season ) salt (for example, Nafion  and Flexion ) in (for example mix cathode enzyme, laccase, oxidizing ferment, peroxidase or the like) or the anode enzyme (for example, oxidizing ferment, dehydrogenase or the like), and in the micella of the perfluorinate amberplex that quaternary ammonium (perhaps season ) salt is handled, mix electron transfer mediator, as the ruthenium that forms with the aromatic series part, iron, cobalt, osmium, nickel, chromium, the complex compound of rhenium or rhodium.Thereby described polymer/enzyme complex compound is also as redox polymers (" difunctional film ").This innovation has increased the efficient of electron transport between enzyme and the electrode.
In another embodiment, the present invention relates to biological fuel cell, it is used in physiological environment and the non-physiological environment and produces electric current, and described biological fuel cell comprises biological-cathode (describing as mentioned) and anode.Preferably, anode is biological anode, and it comprises the film that can form micella with the inside of buffering and contain immobilized anode enzyme.Described biological anode can comprise independent electrical conductivity film (redox film), as the polymer of methylene green.Alternatively, the micella film that contains the buffering of immobilized anode enzyme can also be as the redox film, and wherein said micella film also contains the electron transfer mediator of as above biological-cathode being described.
Biological fuel cell of the present invention can comprise the polymer dielectric film that is used to separate anodal compartment and cathodic compartment (" PEM " or salt bridge, for example, Nafion  117).Yet, consider the anode that has in the amberplex separately that is fixed on them and the innovation of cathode enzyme, another embodiment of biological fuel cell does not comprise the PEM (" no film biological fuel cell ") that is used to separate anodal compartment and cathodic compartment.In order to make no film biological fuel cell, use biological-cathode and biological anode.The preferential selectivity of the used enzyme that is used for catalytic oxidant or fuel fluid reaction allows anodal compartment not separate with cathodic compartment physically in biological anode and the biological-cathode.
In currently preferred embodiments, biological fuel cell comprises biological-cathode, and this biological-cathode comprises the charcoal cloth of the Nafion  film coating of modifying with the quaternary ammonium that contains bilirubin and bilirubin oxidase.The charcoal cloth that is coated with is at 1mM Ru (bpy) 3 + 2The middle immersion to allow redox mediators Ru (bpy) 3 + 2Pre-concentration in film.In addition, biological fuel cell comprises biological anode, and it comprises the charcoal cloth of the alcohol dehydrogenase coating that relies on the PQQ-that is fixed in cation modified perfluorinated sulfonic acid-PTFE copolymer.The sketch map of the chemical reaction that takes place at biological-cathode and biological anode shows in Figure 13.As described in example 5 above, biological-cathode and biological anode are placed contain NAD +With in the beaker of ethanol and be exposed in the air to finish this biological fuel cell.
The anode embodiment The negative electrode embodiment Separate embodiment
Standard anode The biological-cathode of in I.A., describing Salt bridge or PEM
The biological anode of in I.B., describing Standard cathode Salt bridge or PEM
The biological anode of in I.B., describing The biological-cathode of in I.A., describing No film
Anode embodiment, negative electrode embodiment and the top combination that separates embodiment are within the scope of the invention.
III. produce the method for electricity
In a further embodiment, the present invention relates to use biological-cathode of the present invention, produce the method that proton and electronics produce electric energy in conjunction with biological anodic oxidation organic-fuel with oxygen reduction Cheng Shui.
In a further embodiment, the present invention includes and use a kind of and multiple above-mentioned biological fuel cell embodiment to produce the method for electricity, described method comprises the fuel liquid of (a) oxidation anode and reduces the oxidant of biological-cathode; (b) the reduction form of oxidation electron mediator between the reduction period of biological-cathode oxidant; (c) oxidation eelctro-catalyst; (d) eelctro-catalyst of reduction electronic conductor.
In another embodiment, the present invention includes and use a kind of and multiple biological fuel cell embodiment to produce the method for electricity, wherein said biological-cathode comprises the enzyme immobilization material, this material comprises above-mentioned electron mediator, and described method comprises the fuel liquid of (a) oxidation anode and reduces the oxidant of biological-cathode; (b) the reduction form of oxidation electron mediator between the reduction period of biological-cathode oxidant; (c) reduction electronic conductor.
In another embodiment, the present invention relates to produce the method for electrical power, it comprises (a) organic-fuel of oxidation anode in the presence of at least a anodic oxidation reductase that mixes anode; (b) by redox polymers electronics is transferred to the anode electrically conductive material from the organic-fuel of oxidation; (c) oxygen molecule of reduction negative electrode in the presence of the dioxygen oxidation reductase, described enzyme is fixed in the cushion compartments of modified cathode ion exchange polymer film; (d) by electron transfer mediator electronics is transferred to the substrate of dioxygen oxidation reductase from electrically conductive material, described electron transfer mediator is fixed in the buffered compartment of modified ion exchange polymer membrane, thereby produces electric current.
Biological fuel cell of the present invention can be used for multiple pH environment, comprises in the physiological environment.Use biological fuel cell that enzyme replaces metallic catalyst catalytic oxidation/reduction (" redox ") reaction through optimization with near neutral pH environmental work.
Definition
Term used herein " redox polymers ", " redox polymer film " or " redox polymers film " refer to and can accept or confess electronics from compound, causes the oxidation of compound and reduction respectively and produce available free electron in order to transfer to the polymer in the circuit.
Term used herein " quaternary ammonium " or " quaternary ammonium salt " refer to comprise the nitrogen compound of four organic groups of covalent bond, as illustrating among the eq.2.N is a nitrogen, R 1-R 4It is organic group.Preferably, R 1, R 2, R 3And R 4Be selected from propyl group, butyl, amyl group or the like.Preferably, R 1, R 2, R 3And R 4It is identical organic group.In alternative embodiment, R 1, R 2And R 3Be methyl or ethyl, R 4Be hexyl, heptyl, octyl group, nonyl or decyl.In another alternative embodiment, can use season  salt, wherein this salt can be season , thus the N+ of Eq.2 is replaced by phosphonium ion.The equilibrium ion of quaternary ammonium (perhaps ) can be any anion, as bromide ion (Br -).
" fuel cell " used herein comprises anode and negative electrode, and they are separately to avoid electrical short.Preferably, anode and negative electrode separate by polymer dielectric film.Biological fuel cell utilizes the enzyme of fuel liquid and this fuel liquid oxidation of catalysis.In one embodiment, " biological fuel cell " utilizes organic-fuel as the energy with the oxidation of redox enzymatic organic-fuel.Term " fuel cell " and " biological fuel cell " can exchange use in disclosure full text.In one embodiment, fuel cell of the present invention can be used for the application of needs electricity supply, as, but be not limited to the vehicles of electronic installation and equipment, toy, inner medical science apparatus and electric energy supply.In another embodiment, fuel cell of the present invention can be implanted biology alive, and wherein organic-fuel is the device energy supply of implanting this biology of living from this biology and fuel cell.
Term used herein " organic-fuel " refers to any based on carbon compound of storage power.Organic-fuel includes but not limited to nucleic acid, carbohydrate (as glucose), alcohol, aliphatic acid and other hydrocarbon, ketone, aldehyde, amino acid and protein.Organic-fuel can be biological interior biologic artifact.Preferred fuel is alcohol, and it comprises methyl alcohol, ethanol, butanols and isopropyl alcohol, and sugar, particularly glucose or its polymer.Preferred alcohol is ethanol and methyl alcohol.
Term used herein " biological anode " is the anode that comprises the enzyme of catalytic fuel liquid oxidation.In one embodiment, term " biological anode " refers to comprise the anode of the oxidoreducing enzyme of catalysis organic-fuel oxidation.Anode provides the electronics source for circuit or voltage.Term used herein " biological-cathode " refers to comprise the negative electrode of the oxidoreducing enzyme of catalytic oxidant reduction.
Term used herein " supporting film " refers to rigidity or semi-rigid inert material, and it can conduction current and the polymer film that is used for the biological support fuel cell electrode.Supporting film can comprise any conductive material, as stainless steel, stainless steel mesh, carbon, carbon nano-tube, platinum or semi-conducting material.Preferred supporting film is a slice carbon felt.Term " carbon felt ", " charcoal cloth " and " charcoal cloth supporting film " can exchange use.
Term used herein " ion-exchange polymer " or " ion exchange polymer membrane " refer to allow the polymer by its conducting ion.Preferred ion-exchange polymer is fluoridized ion-exchange polymer, as Nafion  (DuPont, Wilmington, DE).The invention still further relates to the fluoridized ion-exchange polymer that comprises modification, it is included in the quaternary ammonium ion at sulfonic acid exchange position.Described modification causes the interior neutral pH of micella of ion-exchange polymer.According to the present invention, one or more oxidoreducing enzyme mix or are captured in the micella (perhaps " micella compartment ") of the fluoridized ion-exchange polymer that quaternary ammonium that salt extracts handles.
In one embodiment, term " enzyme " or " oxidoreducing enzyme " refer to the protein as catalyst in the chemical reaction.
Term used herein " hydrocarbon " and " alkyl " are described organic compound or the group of only being made up of carbon and protium.These parts comprise alkyl, thiazolinyl, alkynyl and aryl moiety.These parts also comprise alkyl, thiazolinyl, alkynyl and the aryl moiety that replaces with other aliphatic series or cyclic hydrocarbon group, as alkaryl, alkene aryl and alkynes aryl.Unless otherwise noted, these parts preferably comprise 1 to 20 carbon atom.
" alkyl of replacement " described herein part is the hydrocarbyl portion that is different from the atom replacement of carbon with at least one, comprises the part that wherein the carbochain atom is replaced with hetero-atom such as nitrogen, oxygen, silicon, phosphorus, boron, sulphur or halogen atom.These substituting groups comprise hydroxyl, ketone, acyl group, acyloxy, nitro, amino, acylamino-, nitro, cyano group, thiol, ketal, acetal, ester and the ether of halogen, heterocycle, alkoxyl, alkene oxygen base, alkynyloxy group, aryloxy group, hydroxyl, protection.
Unless otherwise noted, alkyl described herein is low alkyl group preferably, and it contains 1 to 8 carbon atom and contains maximum 20 carbon atoms in main chain.They be straight chain or side chain or ring-type and comprise methyl, ethyl, propyl group, isopropyl, butyl, hexyl or the like.
Unless otherwise noted, thiazolinyl described herein is low-grade alkenyl preferably, and it contains 2 to 8 carbon atoms and contains maximum 20 carbon atoms in main chain.They be straight chain or side chain or ring-type and comprise vinyl, acrylic, isopropenyl, cyclobutenyl, isobutenyl, hexenyl or the like.
Unless otherwise noted, alkynyl described herein is low-grade alkynyl preferably, and it contains 2 to 8 carbon atoms and contains maximum 20 carbon atoms in main chain.They be straight chain or side chain and comprise acetenyl, propinyl, butynyl, isobutyl alkynyl, hexin base or the like.
Term used herein " aryl " or " ar " refer to the optional isocyclic aryl that replaces separately or as the part of another group, contain the monocycle or the bicyclic groups of 6 to 12 carbon at loop section, as the phenyl of phenyl, xenyl, naphthyl, replacement, the xenyl of replacement or the naphthyl of replacement.The phenyl of phenyl and replacement is preferred aryl.
Term used herein " halogen " or " halogen " refer to chlorine, bromine, fluorine and iodine separately or as the part of another group.
Term used herein " acyl group " refers to remove the part that hydroxyl forms by the group-COOH from organic carboxyl acid separately or as the part of another group, for example, RC (O)-, wherein R is R 1, R 1O-, R 1R 2N-, perhaps R 1S-, R 1Be alkyl, the assorted alkyl that replaces, perhaps heterocycle, R 2It is the alkyl of hydrogen, alkyl or replacement.
Term used herein " acyloxy " separately or as the part of another group refer to logical peroxide bridge (--O--) combination as above-mentioned acyl group, for example, RC (O) O-, wherein R is as the definition about term " acyl group ".
Term " hetero-atom " is different from expression in the atom of carbon and hydrogen.
This paper refers to optional that replace, saturated fully or undersaturated, monocycle or two rings, aromatic series or non-aromatic group separately or as the used term of the part of another group " heterocycle " or " heterocycle ", it has at least 1 hetero-atom at least one ring, preferably in each ring 5 or 6 atoms are arranged.Heterocyclic group preferably has 1 or 2 oxygen atom in ring, 1 or 2 sulphur atom, and/or 1 to 4 nitrogen-atoms, and can be attached to the remainder of molecule by carbon or hetero-atom.Representative heterocycle comprises heteroaromatic, as furyl, thienyl, pyridine radicals,  azoles base, pyrrole radicals, indyl, quinolyl or isoquinolyl, or the like.Representative substituting group comprises the group below one or more: the hydroxyl of the alkyl of alkyl, replacement, ketone, hydroxyl, protection, acyl group, acyloxy, alkoxyl, alkene oxygen base, alkynyloxy group, aryloxy group, halogen, acylamino-, amino, nitro, cyano group, thiol, ketal, acetal, ester and ether.
Term used herein " hydroxyl protecting group " expression can be protected the group of free hydroxyl (" hydroxyl and protected "), and it can be removed after the reaction that is used to protect and the remainder of disturbing molecule not.Synthetic " the Protective Groups inOrganic Synthesis " that can see T.W.Greene of the kinds of protect base of hydroxyl and its, John Wiley and Sons, 1981, perhaps Fieser﹠amp; Fieser.Representative hydroxyl protecting group comprises methoxy, 1-ethoxyethyl group, benzyloxymethyl, (β-trimethylsilylethoxy)) methyl, THP trtrahydropyranyl, 2; 2; 2-trichlorine ethoxy carbonyl, the tert-butyl group (diphenyl) silicyl, trialkylsilkl, trichlorine methoxycarbonyl and 2; 2,2-trichlorine ethoxyl methyl.
The following examples are illustrated the present invention.
Embodiment
Experimental technique
The inventor has has researched and developed can be with the dual plasma membrane of the biological-cathode that acts on fuel cell.Dual plasma membrane refers to that this film is as the catalyst that comprises cathode enzyme with as comprising electron transfer mediator, as Ru (bpy) 3 + 2The electrical conductivity film.Carry out electron transfer mediator (" amboceptor ") Ru (bpy) according to several diverse ways 3 + 2The importing of the Nafion  film of handling to the bromination quaternary ammonium salt, described method was promptly mixed electron transfer mediator by mixture casting or ion-exchange before or after mixing cathode enzyme (for example, bilirubin oxidase).Three kinds of schemes of preparation biological-cathode in this research, have been studied.No matter used concrete scheme, show in the film～Ru (bpy) of 1.0-1.5M 3 + 2The concentration Nafion film central hole structure that allows to be modified in Ru (bpy) 3 + 2Molecule closely approaching, this allows between enzyme and the electrode based on the electrical conductivity of exchange certainly.This causes having the biological-cathode of single film, and it is caught and stablize cathode enzyme and as the redox polymers electronics of round transporting between enzyme and electrode.
Embodiment 1
The salt of enzyme immobilization extracts the preparation of film
Ru (bpy) 3 + 2The preparation of/Nafion I
By in Nafion suspension, directly adding Ru (bpy) 3 + 2Preparation Ru (bpy) 3 + 2/ NafionI (mixture casting).
In order to prepare Ru (bpy) 3 + 2The film (" Nafion I ") that/Nafion I salt extracts is with 0.15 mM Ru (bpy) 3 + 2Add 4ml Nafion , use ultrasonoscope fully to mix～3-4 hour by vortex and in water bath with thermostatic control.Then mixture is poured into the weighing boat with dried overnight.In case dry, by with Ru (bpy) 3 + 2/ Nafion  mixture immerses deionized water, uses vortex salt to extract, and is centrifugal then.When all salt all were extracted, the solution that is extracted became clarification from orange.The film flushing that salt extracts is also dry, and then be dissolved in 4ml 80% ethanol (can be dissolved in again in the mixture that contains the lower aliphatic alcohols that reaches 30% water).
By following step cathode enzyme is fixed on Ru (bpy) 3 + 2In the film that/Nafion I salt extracts.(Solution Technologies, common casting Inc.) forms the Nafion  film that mixes the bromination quaternary ammonium with 5%Nafion  suspension by weight by the bromination quaternary ammonium.Prepare mixture casting solution by in 5% suspension by weight, adding the bromination quaternary ammonium.Preparing all mixture casting solution makes the concentration of bromination quaternary ammonium surpass the concentration in sulfonic acid site in the Nafion  suspension.After the optimization, determine bromination quaternary ammonium concentration the most stable and reproducible film 3 times for exchange position concentration.
1 milliliter of casting solution is placed the weighing boat and allows dry.Research has in the past shown that all bromide ions that import film all penetrate from film when soaking in water.Therefore, add the 7.0mL 18M Ω water and the immersion that allows to spend the night to the weighing boat.Remove and to anhydrate and with abundant flushing membrane of 18M Ω water and drying.Then, film is resuspended in the 1.0mL methyl alcohol.Subsequently, to 100ml Ru (bpy) 3 + 2Add about mg electron mediator and about 0.5 in the film (above) that/NafionIII salt extracts and also fully mix (in this case, vortex is 20 minutes) to the 1mg cathode enzyme.
Preparation Ru (bpy) 3 + 2/ Nafion II:
Ru (bpy) 3 + 2/ Nafion II is immersed in 1mM before resuspension and bilirubin oxidase are fixing
Ru (bpy) 3 + 2TBAB/Nafion film in the solution.
In order to prepare Ru (bpy) 3 + 2The film (" Nafion II ") that/Nafion II salt extracts adds 0.3 mM TBAB (TBAB) among every 1ml Nafion  in (0.09672g), then by vortex mixed 10 minutes.Then mixture is poured into dried overnight in the weighing boat.(at this moment, the film mixture is light yellow).In case dry, the Nafion  that TBAB is handled is immersed in the deionized water 24 hours, then with deionized water rinsing 3 times and allow dry.(at this moment, the film mixture is clarified).Then with the salt extract layer of drying at Ru (bpy) 3 + 2Solution (is dissolved in the 1mM Ru (bpy) in buffer solution, water or the electrolyte 3 + 2) in the immersion of spending the night, to allow drying, be dissolved in again in the 1ml ethanol then.According to the scheme that provides above cathode enzyme is fixed in the modified film.
Ru (bpy) 3 + 2The preparation of/Nafion III
Ru (bpy) 3 + 2/ Nafion III is immersed in 1mMRu (bpy) after the fixing and electrode assembling of bilirubin oxidase 3 + 2In TBAB/Nafion  film.
In order to prepare Ru (bpy) 3 + 2The film (" Nafion III ") that/Nafion III salt extracts adds 0.3 mM TBAB (TBAB) among every 1ml Nafion  in (0.09672g), then by vortex mixed 10 minutes.Then mixture is poured into dried overnight in the weighing boat.(at this moment, the film mixture is light yellow).In case dry, the Nafion  that TBAB is handled is immersed in the deionized water 24 hours, then with deionized water rinsing 3 times and allow dry.(at this moment, the film mixture is clarified).Salt extract layer with drying dissolves in 1ml ethanol more then.According to the scheme that provides above cathode enzyme is fixed in the modified film.To contain Nafion  casting that the TBAB-of immobilized bilirubin oxidase modifies to electrode, the permission drying is immersed in Ru (bpy) then 3 + 2Solution reaches 48 hours in (above); Preferably, test is preceding 2 to 3 hours.
Embodiment 2
The preparation of electrode
Ru on the glass carbon electrode (bpy) 3 + 2The preparation of/Nafion III
In order to prepare cathode enzyme/film casting solution, general～1mg bilirubin and～the 0.5-1mg bilirubin oxidase adds 100ml Ru (bpy) 3 + 2In the film (above) that/Nafion III salt extracts and abundant mix (in this case, vortex is 20 minutes).Glass carbon electrode (3mm diameter) and permission that the 2ml cathode enzyme/film casting solution is applied to polish is dry.In case dry, with cathode enzyme/film/carbon electrode at N 2The Ru of-degasification (bpy) 3 + 2Soaked 3 hours in the solution.TBAB and Ru (bpy) 3 + 2After the exchange, carbon electrode is imported N 2Also allow in the phosphate buffer of the pH7.4 of-degasification to soak 1 hour.After the balance, by cyclic voltammetry with 0.05 and the sweep speed of 0.1V/s test negative electrode.Then, also as above tested negative electrode in 10 minutes with the saturated buffer solution of oxygen.
1cm 2Ru on the carbon felt (bpy) 3 + 2The preparation of/Nafion III
In order to prepare cathode enzyme/film casting solution, general～1mg bilirubin and～the 0.5-1mg bilirubin oxidase adds 100ml Ru (bpy) 3 + 2In the film (above) that/Nafion III salt extracts and abundant mix (in this case, vortex is 20 minutes).Be applied to 10ml cathode enzyme/film casting solution on per 1 square centimeter of carbon felt electrode and the permission drying.In case dry, with cathode enzyme/film/carbon felt electrode at Ru (bpy) 3 + 2Soak in the solution.TBAB and Ru (bpy) 3 + 2After the exchange, will import in the phosphate buffer of pH7.4 and allow in the U-shaped cylindricality glass tube of the phosphate buffer that contains pH7.4, to soak 1 hour based on the electrode of carbon felt.Anode side immerses 1mM NAD for being fixed on +With the dehydrogenase in the TBAB/Nafion film of 1.0mM ethanol.
Experimental result
Characterize each with cyclic voltammetry according to the multiple prototype biological-cathode of " Nafion I " method, " Nafion II " method or " NafionIII " method (above) preparation.
The representative voltammogram of best, the medium and the poorest current density of every kind of biological-cathode film preparation thing is described.See Fig. 2 to 10, it has described these voltammograms.In order to summarize these results, these data are pointed out TBAB-Nafion/Ru (bpy) 3 + 2III film (" Nafion III ") is immobilization bilirubin oxidase and this enzyme of consistency or inactivation effectively, and can produce 2.0mA/cm at least under current catalysis load 2Current density.
Embodiment 3
Biological fuel cell
Manufacturing comprises the prototype biological fuel cell (Figure 11) of biological anode, this biology anode comprises and is fixed on Nafion  that TBAB-modifies (as patent application 60/429,829,60/486,076 and 10/617, description in 452) alcohol dehydrogenase in, and this Nafion III film comprises bilirubin oxidase, bilirubin and Ru (bpy) 3 + 2(Fig. 1 is seen in the description about difunctional biological-cathode film).The biological fuel cell of this non-optimum have PEM (the Nafion  117) film of separately anode and cathode solution and wherein catalyst loading be film of describing in the voltammogram experiment (above) only～28%, preliminary test shows open circuit voltage to this biological fuel cell is the 0.4179-0.819 volt, and the maximum density of ionization is 0.224mA/cm 2To 2.23mAmps/cm 2And maximum power is 0.951mW/cm 2(see Figure 12, it has described the power curve of this prototype).
Embodiment 4
Rely on the anode of PQQ
Form the Nafion  film of modifying with two-stage process.The first step is the suspension of the Nafion  of casting dissolving tetrabutyl phosphonium bromide ammonium salt.Second step was that these initial films are moulded in double teeming after extracting excessive TBAB and HBr salt from initial film.Modified Nafion  film mixes tetrabutyl phosphonium bromide ammonium salt and 5%Nafion  suspension by weight in the weighing boat.Preparation mixture casting solution makes that the concentration of tetrabutyl phosphonium bromide ammonium salt is that three times of sulfonic acid position concentration are excessive in the Nafion  suspension.Research has in the past shown that all bromide ions all penetrated from film when film soaked in water.Therefore, in the weighing boat, add the 18M Ω water and the immersion that allows to spend the night.This step is necessary for removing all excessive HBr and quaternary ammonium salt.After film spends the night and soaks, except that anhydrating and washing film also dry with 18M Ω water.Then film is resuspended in the ethanol.The film that is suspended is used to form enzyme/film casting solution then.
For the alcohol dehydrogenase (ADH) that obtains relying on PQQ, 30 ℃ of following aerobics are cultivated about 1 week of gluconobacter sp sp.33 of buying by commercial sources in the GYC culture medium.Stick with paste centrifugal cell with the 0.9%NaCl washed twice and be kept at-20 ℃ standby.The cell that thaws is stuck with paste to be suspended in and is contained 1mMCaCl 20.2M phosphate buffer pH7.0 in and by sonicated in ice bath 1 minute to prevent the sample heating.Remove intact cell by centrifugal 20 minutes, and add 10% deoxycholic aicd sodium solution (to final concentration 0.5%).This solution was hatched 1 hour at 4 ℃ under gentle agitation, and centrifugal then 1 hour to remove insoluble matter.In clarified supernatant, add 10%CaCl 2Solution to 0.5% final concentration.Be suspended among the 0.3M kaliumphosphate buffer pH7.2 and gentle agitation 10-20 minute by centrifugal collection gained calcium phosphate gel and with it.Abandon insoluble matter after centrifugal 30 minutes.To supernatant add solid ammonium sulfate and centrifugal after abandon the sediment of formation.Repeat this step and the gained supernatant be dissolved in to contain 1mM CaCl 2With in the 20mM Tris-HCl pH of buffer 7.2 of 1% sucrose and to identical buffer solution dialyzed overnight.
The enzyme of being dialysed is by the centrifugal DEAE Toyo-pear 650M post that is applied to after removing insoluble precipitate with dialysis buffer liquid balance.Contain 1% sucrose and 1mM CaCl by same buffer and two bed volumes that pass two bed volumes 275mM Tris-HCl pH of buffer 7.2 washing pillars.Rely on the ADH of PQQ with the same buffer wash-out that contains 0.2%TritonX-100.Collect and concentrate the fraction of ADH activity and precipitate enzyme with Macrogol 6000 with dependence PQQ.Be dissolved in by centrifugal 15 minutes collecting precipitation things and with it and contain 1mM CaCl 25mM kaliumphosphate buffer pH7.2 in and be applied to containing 1mM CaCl 2CM-Sepharose post with the 5mM kaliumphosphate buffer pH7.2 balance of 1% sucrose.Collection is closed the fraction of the ADH activity that relies on PQQ and is adsorbed excessive water according to the method in the list of references 3 with carboxymethyl cellulose.
Is that 2: 20 fixed ratio is in TBAB/Nafion  film and be coated on the surface of glass carbon electrode with the enzyme of purifying with enzyme and 5% film suspended matter by weight.Also, then two electrodes were placed drier dry 15 minutes by with the glassy carbon electrode preparation of 2 μ lTBAB/Nafion  casting solution coat reference electrode.Allow dry electrode balance 1 hour in 1.0mM PQQ/1.0mM ethanol/pH7.15 phosphate buffer.Study the electrochemistry of biological anode with cyclic voltammetry.
The anode 1 of the Nafion  film preparation of modifying with ADH that relies on PQQ and TBAB-has following performance.The life-span of making back anode 1 is 152 days.Make and obtained 2.47mW/cm in back 8 days 2Maximum power and the maximum current of 7.05mA.Make the maximum open circuit voltage that obtained 1.08V in back 100 days.Another anode of the Nafion  film preparation of modifying with ADH that relies on PQQ and bromination triethyl group hexyl ammonium provides 3.01mW/cm 2Maximum power, the maximum current of 7.50mA and 0.62 is to the open circuit voltage of 1.005V.The life-span of this anode is 35 days.
Embodiment 5
The biological fuel cell of no film
Biological anode/biological-cathode battery with no film carries out preliminary experiment, except not using PEM (Nafion 117) film that separates negative electrode and anodic dissolution, this battery comprises all aspects (therefore being called " no film ") of describing in the prototype biological fuel cell of Figure 11 (above).The open circuit voltage that prototype with the biological anode that relies on NAD does not have the film biological fuel cell is the 0.4063-0.7385 volt, and maximum current density is 0.288mAmps/cm 2To 5.38mAmps/cm 2, maximum power is 0.46mW/cm 2
With TBAB (Sigma) add by weight 5% Nafion suspension (1100EW, Aldrich) in and by vortex mixed～10 minute.With with Nafion go up sulfonic group compare three times of excessive adding 4-butyl ammoniums with guarantee all protons all with the TBuA cation exchange.Then with mixture casting solution casting and allow dried overnight in the weighing boat.After the drying, mixture-cast film is soaked 24 hours to remove all excessive bromide salt in 18M Ω water.After salt extracts, fully wash film three times and allow dry with 18M Ω water.Film is resuspended in the absolute ethyl alcohol then and is used for enzyme immobilization to prepare them.
The Nafion film suspension of modifying to the 100ml 4-butyl ammonium add 1 milligram of bilirubin (Sigma) and 0.5mg bilirubin oxidase (from Myrothecium verrucaria, units activity=10 units/mg, Sigma) and vortex 20 minutes.10 microlitres enzyme/film casting solution is pipetted into 1cm 2(Ballard Material Product Inc.) goes up and allows drying to carbon fiber paper.In case dry, with them at 1.0mM Ru (bpy) 3 + 2With 0.1M NaSO 4Immersion (Ru (bpy) spends the night in the solution 3 + 2Ion-exchange TBA +).Wash electrode with 18M Ω water before using then.
Ru (bpy) 3 + 2Be exchanged into TBA +After, biological-cathode is assembled to is used for data collection in the battery at once.Use two types battery.With U-shaped glass battery testing conventional fuel battery, the U-shaped glass battery middle-jiao yang, function of the spleen and stomach utmost point and cathodic compartment are by Nafion 117 PEM films (Alfa Aesar) separately.Anode and cathodic compartment keep about 50mL solution.Anodal compartment is equipped with the 1mM solution that contains in the pH7.15 buffer solution; Randomly, if used enzyme is independent of NAD, so also the anode compartment adds 1mM NAD +Cathodic compartment is equipped with the solution that contains the pH7.15 buffer solution that is exposed to air.At experimental session, unique oxygen source form and solution are to the exposure of air.Biological-cathode and the biological anode (according to the method manufacturing in the US application serial No. 10/617,452) finished are imported compartment and test.For second type the fuel cell (no membrane cell) of research, biological-cathode and the importing of biological anode are contained in the 50mL beaker of fuel solution.Fuel solution by the 1.0mM ethanol in the phosphate buffer of pH7.15 and randomly, if the enzyme that uses is to rely on NAD, the so also optional 1.0mM NAD that contains +Allow solution in air balance with oxygen dissolution before guaranteeing to test in buffer solution.Electrode is placed to guarantee that they are not in contact with one another at a distance of about 1cm.
All electrochemical measurements all carry out under room temperature (20-25 ℃).On the CHInstruments potentiostat model 810 that connects the PC computer, measure.This potentiostat is used to measure open circuit voltage and the load different to fuel cells applications, measures electric current simultaneously and keeps current potential.
In Figure 15, shown power output curve diagram as the function of time of no film ethanol/oxygen fuel cell.In following table, shown electrochemical data from the multiple embodiments of biological fuel cell.Unless otherwise noted, as the biological-cathode in the embodiment below the preparation of describing among the top embodiment 2.Embodiment 1 is to have biological anode, biological-cathode that relies on NAD and the biological fuel cell that separates the film of anode and cathodic compartment.Embodiment 2 is to have biological anode, biological-cathode that relies on NAD and the biological fuel cell that does not have film.Embodiment 3 is to have biological anode, biological-cathode that relies on NAD and the biological fuel cell that does not have film, collects electrochemical data in the temperature of appointment.Embodiment 4 is to have biological anode, biological-cathode that relies on PQQ and the biological fuel cell that does not have film.Embodiment 5 be have the biological anode that relies on PQQ, the biological-cathode casting that obtains from the 70 microlitre 1mM enzyme solutions that mix with 50 microlitre TBAB/Nafion and the biological fuel cell that does not have film.Embodiment 6 be have the biological anode that relies on PQQ, based on the biological-cathode of Fe (bpy) and be the biological fuel cell of no film.Embodiment 7 is the biological fuel cells that have the beer of biological anode, the biological-cathode that relies on PQQ, the liquid that acts as a fuel and do not have film.Embodiment 8 is the biological fuel cells with Nafion  film of TBAB-modification, has the ADH that relies on PQQ at biological anode, has the Nafion film of trimethyl hexyl ammonium bromide-modification at biological-cathode.Embodiment 1-7 contains the biological anode and the biological-cathode of the Nafion  film with TBAB-modification.
Embodiment Temperature (℃) Open-circuit potential (V) Electric current (A) Power (W/cm 2)
1 0.8190 2.23×10 -3 9.51×10 -4
2 0.7385 5.38×10 -3 4.60×10 -4
3 37.5 0.5903 1.85×10 -3 5.26×10 -4
3 24.0 0.5660 1.00×10 -3 4.38×10 -4
3 6.0 0.6172 1.43×10 -3 5.57×10 -4
4 1.0453 8.47×10 -3 1.41×10 -3
5 0.7840 4.19×10 -3 4.82×10 -4
6 0.1465 2.46×10 -4 1.71×10 -5
7 0.7200 1.19×10 -3 1.44×10 -4
8 1.0613 1.66×10 -3 1.32×10 -3
Come from above, will see and realize purposes more of the present invention and obtained other favourable results.
Above method in can carry out multiple modification and not deviate from scope of the present invention, with contain in the specification above estimating or accompanying drawing in all situations that shows all be interpreted as the property illustrated and unrestricted.
From understanding and the practice of the present invention disclosed herein to specification, other embodiments in this paper claim scope will be conspicuous for those skilled in the art.Estimate that it only is exemplary that this specification and embodiment will be considered to, scope and spirit of the present invention are pointed out by embodiment following claim book.

Claims (110)

1. biological-cathode, it comprises:
(a) electronic conductor;
(b) at least a enzyme, oxidised form and water that it can produce electron mediator with the reduction form and the oxidant reaction of electron mediator; With
(c) enzyme immobilization material, it comprises eelctro-catalyst, described enzyme can be fixed and stablize to this enzyme immobilization material, this material is permeable to described oxidant, the oxidised form of eelctro-catalyst can obtain electronics to produce the reduction form of eelctro-catalyst from electronic conductor, and the reduction form of this eelctro-catalyst can produce the reduction form of electron mediator and the oxidised form of eelctro-catalyst with the oxidised form reaction of electron mediator;
Wherein eelctro-catalyst is enough to make the concentration of enzyme immobilization conduct electronics exist.
2. biological-cathode, it comprises:
(a) electronic conductor;
(b) at least a enzyme, oxidised form and water that it can produce electron mediator with the reduction form and the oxidant reaction of electron mediator; With
(c) enzyme immobilization material, it comprises described electron mediator, described enzyme can be fixed and stablize to this enzyme immobilization material, and this material is permeable to oxidant, and the oxidised form of electron mediator can obtain electronics to produce the reduction form of electron mediator from electronic conductor;
Wherein electron mediator is enough to make the concentration of enzyme immobilization conduct electronics exist.
3. biological-cathode, it comprises:
(a) electronic conductor;
(b) at least a enzyme, oxidised form and water that it can produce electron mediator with the reduction form and the oxidant reaction of electron mediator; With
(c) enzyme immobilization material, it comprises electron mediator and eelctro-catalyst, described enzyme can be fixed and stablize to this enzyme immobilization material, described material is permeable for described oxidant, the oxidised form of described eelctro-catalyst can obtain electronics to produce the reduction form of eelctro-catalyst from electronic conductor, and the reduction form of this eelctro-catalyst can be reacted with the oxidised form of electron mediator with the reduction form that produces electron mediator and the oxidised form of eelctro-catalyst;
Wherein eelctro-catalyst is enough to make the concentration of described enzyme immobilization conduct electronics exist.
4. biological-cathode, it comprises:
(a) electronic conductor;
(b) at least a enzyme, oxidised form and water that it can produce electron mediator with the reduction form and the oxidant reaction of electron mediator; With
(c) enzyme immobilization material, it comprises electron mediator and eelctro-catalyst, described enzyme can be fixed and stablize to this enzyme immobilization material, described material is permeable for described oxidant, the oxidised form of eelctro-catalyst can obtain electronics to produce the reduction form of eelctro-catalyst from electronic conductor, and the reduction form of this eelctro-catalyst can produce the reduction form of electron mediator and the oxidised form of eelctro-catalyst with the oxidised form reaction of electron mediator;
Wherein electron mediator is enough to make the concentration of described enzyme immobilization conduct electronics exist.
5. be used to produce the biological fuel cell of electricity, it comprises:
Fuel liquid;
Electron mediator;
Can react anode with oxygenated fuel liquid; With
The biological-cathode of claim 1.
6. be used to produce the biological fuel cell of electricity, it comprises:
Fuel liquid;
Can react anode with oxygenated fuel liquid; With
The biological-cathode of claim 2.
7. be used to produce the biological fuel cell of electricity, it comprises:
Fuel liquid;
Can react anode with oxygenated fuel liquid; With
The biological-cathode of claim 3.
8. be used to produce the biological fuel cell of electricity, it comprises:
Fuel liquid;
Can react anode with oxygenated fuel liquid; With
The biological-cathode of claim 4.
9. the biological-cathode of claim 1, wherein said enzyme immobilization material comprises micella or reversed micelle structure.
10. the biological-cathode of claim 1, wherein said enzyme immobilization material comprises the perfluorinated sulfonic acid-PTFE copolymer of modification.
11. the biological-cathode of claim 1, wherein said electronic conductor comprise material, metallic conductor, semiconductor, metal oxide or modified conductor based on carbon.
12. the biological-cathode of claim 1, wherein said electronic conductor comprises the material based on carbon.
13. the biological-cathode of claim 1, wherein said electronic conductor comprise purified flake graphite, the high-performance graphite of conductor, vitreous carbon, mesoporous carbon, the graphite of electrode, carbon black, powdered carbon, carbon fiber, Single Walled Carbon Nanotube, double-walled carbon nano-tube, multi-walled carbon nano-tubes, carbon nano pipe array, the diamond-coating of charcoal cloth, carbon paper, carbon screen printing, unpressed graphite worm, layering, pyrolytic graphite, pyrolytic graphite or the polycrystalline graphite of high-sequential.
14. the biological-cathode of claim 1, wherein said enzyme immobilization material is used greater than NH 4 +Dewatering cationic modify.
15. the biological-cathode of claim 14, wherein said dewatering cationic comprise cation, quaternary ammonium cation, alkyl trimethyl ammonium cation, organic cation,  cation, triphenyl , pyridine  cation, imidazoles  cation, cetyl pyridinium , second ingot, purpurine, methyl viologen, benzyl viologen, two (triphenylphosphine) iminium metal complex, Bipyridine metal complexes, the metal complex based on phenanthroline, [Ru (bipyridine) based on ammonium 3] 2+Perhaps [Fe (phenanthroline) 3] 3+
16. the biological-cathode of claim 14, wherein said dewatering cationic comprise the quaternary ammonium cation of formula 1 expression
Figure A2004800383840004C1
R wherein 1, R 2, R 3And R 4Be the alkyl or the heterocycle of hydrogen, alkyl, replacement, wherein R independently 1, R 2, R 3And R 4At least one be not hydrogen.
17. the biological-cathode of claim 16, wherein R 1, R 2, R 3And R 4Be hydrogen, methyl, ethyl, propyl group, butyl, amyl group, hexyl, heptyl, octyl group, nonyl, decyl, undecyl, dodecyl, tridecyl or myristyl, wherein R independently 1, R 2, R 3And R 4At least one be not hydrogen.
18. the biological-cathode of claim 16, wherein R 1, R 2, R 3And R 4Identical and be methyl, ethyl, propyl group, butyl, amyl group or hexyl.
19. the biological-cathode of claim 10, wherein modified perfluorinated sulfonic acid-PTFE copolymer is modified with tetrabutyl ammonium halide, triethyl group hexyl ammonium halide or trimethyldodecane base ammonium halide.
20. the biological-cathode of claim 10, wherein modified perfluorinated sulfonic acid-PTFE copolymer is modified with tetrabutyl ammonium halide or triethyl group hexyl ammonium halide.
21. the biological-cathode of claim 10, wherein modified perfluorinated sulfonic acid-PTFE copolymer is modified with triethyl group hexyl ammonium halide.
22. the biological-cathode of claim 1, wherein said enzyme comprises oxidoreducing enzyme.
23. the biological-cathode of claim 1, wherein said enzyme comprise glucose oxidase, based on the oxidizing ferment of alcohol or based on the oxidizing ferment of cholesterol.
24. the biological-cathode of claim 1, wherein said enzyme comprises laccase, cytochrome C oxidase, bilirubin oxidase or peroxidase.
25. the biological-cathode of claim 1, wherein said enzyme are included in the dioxygen oxidation reductase that about 6.5 to about 7.5 pH has optimal activity.
26. the biological-cathode of claim 1, wherein said enzyme comprises bilirubin oxidase.
27. the biological-cathode of claim 1, wherein said electron mediator comprise organic compound, sugar, sterol, aliphatic acid or the oxidasic coenzyme or the substrate of metalloprotein, conjugation.
28. the biological-cathode of claim 1, the oxidised form of wherein said electron mediator comprises stellacyanin, bilirubin, glucose or cholesterol.
29. the biological-cathode of claim 1, the oxidised form of wherein said electron mediator comprises bilirubin.
30. the biological-cathode of claim 1, the eelctro-catalyst of wherein said electron mediator comprise normal reduction potential greater than+0.4 volt organic metal cation.
31. the biological-cathode of claim 1, the eelctro-catalyst of wherein said electron mediator comprises osmium, ruthenium, iron, nickel, rhodium, rhenium or cobalt complex.
32. the biological-cathode of claim 1, the reduction form of the eelctro-catalyst of wherein said electron mediator comprises Ru (phen) 3 + 2, Fe (phen) 3 + 2, Ru (bpy) 3 + 2, Os (bpy) 3 + 2Perhaps Os (terpy) 3 + 2
33. the biological-cathode of claim 1, the reduction form of the eelctro-catalyst of wherein said electron mediator comprises Ru (bpy) 3 + 2
34. the biological-cathode of claim 1, the concentration of wherein said eelctro-catalyst arrives about 3M for about 100mM.
35. the biological-cathode of claim 1, the concentration of wherein said eelctro-catalyst arrives about 2.25M for about 250mM.
36. the biological-cathode of claim 1, the concentration of wherein said eelctro-catalyst arrives about 2M for about 500mM.
37. the biological-cathode of claim 1, the concentration of wherein said eelctro-catalyst arrives about 1.5M for about 1M.
38. the biological fuel cell of claim 5, the oxygenous or peroxide of wherein said oxidant package.
39. the biological fuel cell of claim 5, wherein said oxidant package is oxygenous.
40. the biological fuel cell of claim 5, wherein said fuel liquid comprises ammonia, methyl alcohol, ethanol, propyl alcohol, isobutanol, butanols and isopropyl alcohol, allyl alcohol, aryl alcohol, glycerine, propylene glycol, sweet mellow wine, glucuronic acid, aldehyde, carbohydrate, glucose, glucose-1, D-glucose, L-glucose, G-6-P, lactic acid, 9--phosphoric acid, D-lactic acid, L-lactic acid, fructose, galactolipin-1, galactolipin, aldose, sorbose, mannose, glyceric acid, coacetylase, acetyl coenzyme A, malic acid, isocitric acid, formaldehyde, acetaldehyde, acetate, citric acid, the L-gluconic acid, beta-hydroxysteroid, alpha-hydroxysteroid, lactic aldehyde, testosterone, gluconic acid, aliphatic acid, lipid, phosphoglyceric acid, retinene, estradiol, cyclopentanol, hexadecanol, long-chain alcohol, coniferyl alcohol, cinnamyl alcohol, formic acid, long-chain aldehyde, pyruvic acid, butyraldehyde, acyl coenzyme A, steroids, amino acid, flavine, NADH, NADH 2, NADPH, NADPH 2Perhaps hydrogen.
41. the biological fuel cell of claim 5, wherein said fuel liquid comprises methyl alcohol, ethanol or propyl alcohol.
42. the biological fuel cell of claim 5, wherein said fuel liquid comprises ethanol.
43. the biological fuel cell of claim 5, wherein said anode are biological anodes.
44. the biological fuel cell of claim 43, wherein said biological anode comprises modified perfluorinated sulfonic acid-PTFE copolymer.
45. the biological fuel cell of claim 44, wherein modified perfluorinated sulfonic acid-PTFE copolymer is modified with tetrabutyl ammonium halide, triethyl group hexyl ammonium halide or trimethyldodecane base ammonium halide.
46. the biological fuel cell of claim 44, wherein modified perfluorinated sulfonic acid-PTFE copolymer is modified with triethyl group hexyl ammonium halide.
47. the biological fuel cell of claim 43, wherein biological anode comprises the alcohol dehydrogenase that relies on PQQ, and the alcohol dehydrogenase of this dependence PQQ has the PQQ molecule that combines with its static.
48. the biological fuel cell of claim 47, wherein biological anode and biological-cathode do not separate by salt bridge or polymer dielectric film.
49. use the biological fuel cell of claim 5 to produce the method for electricity, it comprises
(a) at the anodic oxidation fuel liquid and in biological-cathode reduction-oxidation agent;
(b) the reduction form of oxidation electron mediator between the reduction period of biological-cathode oxidant;
(c) oxidation eelctro-catalyst; With
(d) at electronic conductor reduction eelctro-catalyst.
50. use the biological fuel cell of claim 6 to produce the method for electricity, it comprises
(a) at the anodic oxidation fuel liquid and in biological-cathode reduction-oxidation agent;
(b) the reduction form of oxidation electron mediator between the reduction period of biological-cathode oxidant; With
(c) at electronic conductor reduction electron mediator.
51. the method for claim 49, wherein said oxidant are oxygen or peroxide.
52. the method for claim 49, wherein said oxidant is an oxygen.
53. the method for claim 49, wherein said fuel liquid comprises ammonia, methyl alcohol, ethanol, propyl alcohol, isobutanol, butanols and isopropyl alcohol, allyl alcohol, aryl alcohol, glycerine, propylene glycol, sweet mellow wine, glucuronic acid, aldehyde, carbohydrate, glucose, glucose-1, D-glucose, L-glucose, G-6-P, lactic acid, 9--phosphoric acid, D-lactic acid, L-lactic acid, fructose, galactolipin-1, galactolipin, aldose, sorbose, mannose, glyceric acid, coacetylase, acetyl coenzyme A, malic acid, isocitric acid, formaldehyde, acetaldehyde, acetate, citric acid, the L-gluconic acid, beta-hydroxysteroid, alpha-hydroxysteroid, lactic aldehyde, testosterone, gluconic acid, aliphatic acid, lipid, phosphoglyceric acid, retinene, estradiol, cyclopentanol, hexadecanol, long-chain alcohol, coniferyl alcohol, cinnamyl alcohol, formic acid, long-chain aldehyde, pyruvic acid, butyraldehyde, acyl coenzyme A, steroids, amino acid, flavine, NADH, NADH 2, NADPH, NADPH 2Perhaps hydrogen.
54. the method for claim 49, wherein said fuel liquid comprises methyl alcohol, ethanol or propyl alcohol.
55. the method for claim 49, wherein said fuel liquid comprises ethanol.
56. the method for claim 49, wherein said enzyme comprises oxidoreducing enzyme.
57. the method for claim 49, wherein said enzyme comprise glucose oxidase, based on the oxidizing ferment of alcohol or based on the oxidizing ferment of cholesterol.
58. the method for claim 49, wherein said enzyme comprises laccase, cytochrome c oxidase, bilirubin oxidase or peroxidase.
59. the method for claim 49, wherein said enzyme comprises bilirubin oxidase.
60. the method for claim 49, wherein said electron mediator comprise organic compound, sugar, sterol, aliphatic acid or the oxidasic coenzyme or the substrate of metalloprotein, conjugation.
61. the method for claim 49, wherein said electron mediator comprises stellacyanin, bilirubin, glucose or cholesterol.
62. the method for claim 49, the oxidised form of wherein said electron mediator comprises bilirubin.
63. the method for claim 49, the eelctro-catalyst of wherein said electron mediator comprise normal reduction potential greater than+0.4 volt organic metal cation.
64. the method for claim 49, the eelctro-catalyst of wherein said electron mediator comprises osmium, ruthenium, iron, nickel, rhodium, rhenium or cobalt complex.
65. the method for claim 49, the reduction form of the eelctro-catalyst of wherein said electron mediator comprises Ru (phen) 3 + 2, Fe (phen) 3 + 2, Ru (bpy) 3 + 2, Os (bpy) 3 + 2Perhaps Os (terpy) 3 + 2
66. the method for claim 49, the reduction form of the eelctro-catalyst of wherein said electron mediator comprises Ru (bpy) 3 + 2
67. comprise the fuel cell of negative electrode and anode, wherein negative electrode comprises electronic conductor, cathode enzyme, electron transfer mediator, and film, wherein cathode enzyme is immobilized in the interior compartment of buffering of film.
68. the fuel cell of claim 67, wherein said cathode enzyme are the dioxygen oxidation reductases.
69. the fuel cell of claim 68, wherein said cathode enzyme is a bilirubin oxidase.
70. the fuel cell of claim 68, wherein said cathode enzyme is a laccase.
71. the fuel cell of claim 67, wherein electron transfer mediator is immobilized in the interior compartment of buffering of film.
72. the fuel cell of claim 71, wherein said electron transfer mediator is a transistion metal compound.
73. the fuel cell of claim 71, wherein said electron transfer mediator are the compounds that comprises ruthenium.
74. the fuel cell of claim 71, wherein said electron transfer mediator are Ru (bpy) 3 + 2
75. the fuel cell of claim 67, wherein said film are modified ion exchange polymer membrane.
76. the fuel cell of claim 75, wherein said film are perfluorinated sulfonic acid-PTFE copolymers that quaternary ammonium salt is handled.
77. the fuel cell of claim 75, wherein said film are the Nafion  films that TBAB is handled.
78. the fuel cell of claim 67, wherein said electronic conductor is based on the material of carbon, and it is selected from purified flake graphite, high-performance graphite, the high-performance powdered carbon of conductor, vitreous carbon, mesoporous carbon, the graphite of electrode, carbon paper, carbon black, powdered carbon, carbon fiber, Single Walled Carbon Nanotube, double-walled carbon nano-tube, multi-walled carbon nano-tubes, carbon nano pipe array, the diamond-coating of charcoal cloth, carbon felt, carbon paper, carbon screen printing, unpressed graphite worm, layering, pyrolytic graphite, pyrolytic graphite and the polycrystalline graphite of high-sequential.
79. the fuel cell of claim 78, wherein said electronic conductor are the carbon felts.
80. the fuel cell of claim 67, wherein said anode are biological anodes.
81. the fuel cell of claim 80, wherein said biological anode comprise electronic conductor, redox polymer film, modified ion exchange polymer membrane and are fixed on anode enzyme in the interior compartment of buffering of modified ion exchange polymer membrane.
82. the fuel cell of claim 81, wherein said redox polymer film are poly-methylene greens.
83. the fuel cell of claim 81, wherein said modified amberplex are perfluorinated sulfonic acid-PTFE copolymers that quaternary ammonium salt is handled.
84. the fuel cell of claim 83, wherein said film are the Nafion  films that TBAB is handled.
85. the fuel cell of claim 81, wherein said anode enzyme is the oxidoreducing enzyme with organic compound substrate.
86. the fuel cell of claim 85, wherein said anode enzyme is an alcohol dehydrogenase.
87. the fuel cell of claim 86, wherein said biological anode comprise second kind of biological anode enzyme.
88. the fuel cell of claim 87, wherein said second kind of biological anode enzyme is aldehyde dehydrogenase.
89. the fuel cell of claim 81, wherein said electronic conductor is based on the material of carbon, and it is selected from purified flake graphite, high-performance graphite, the high-performance powdered carbon of conductor, vitreous carbon, mesoporous carbon, the graphite of electrode, carbon paper, carbon black, powdered carbon, carbon fiber, Single Walled Carbon Nanotube, double-walled carbon nano-tube, multi-walled carbon nano-tubes, carbon nano pipe array, the diamond-coating of charcoal cloth, carbon felt, carbon paper, carbon screen printing, unpressed graphite worm, layering, pyrolytic graphite, pyrolytic graphite and the polycrystalline graphite of high-sequential.
90. the fuel cell of claim 89, wherein said electronic conductor are the carbon felts.
91. the fuel cell of claim 80, wherein said fuel cell comprises the polymer dielectric film that is used to separate anodal compartment and cathodic compartment.
92. the fuel cell of claim 80, wherein said fuel cell do not comprise salt bridge or the polymer dielectric film that is used to separate anodal compartment and cathodic compartment.
93. produce the method for electrical power, it comprises (a) organic-fuel of oxidation anode in the presence of at least a anodic oxidation reductase that mixes anode, (b) by redox polymers electronics is transferred to the anode electrically conductive material from the organic-fuel of oxidation, (c) oxygen molecule of reduction negative electrode in the presence of the dioxygen oxidation reductase, described dioxygen oxidation reductase is fixed in the buffered compartment of modified cathode ion exchange polymer film, (d) electronics is transferred to the substrate of dioxygen oxidation reductase from electrically conductive material, thereby produce electric current by the electron transfer mediator in the cushion compartments that is fixed on modified ion exchange polymer membrane.
94. the method for claim 93, wherein the dioxygen oxidation reductase is that bilirubin oxidase and substrate are oxygen.
95. the method for claim 93, wherein the anodic oxidation reductase is that pure reductase and organic-fuel are alcohol.
96. the method for claim 93, wherein said redox polymers are poly-methylene greens.
97. the method for claim 93, wherein said modified cathode ion exchange polymer are the Nafion  polymer that the bromination quaternary ammonium is handled.
98. the method for claim 93, wherein said electron transfer mediator comprise transition metal and aromatic series ligand complex.
99. the method for claim 96, wherein said electron transfer mediator are Ru (bpy) 3 + 2
100. the method for claim 93, wherein anode is biological anode, and it comprises modified anode amberplex and at least a anodic oxidation reductase, and this anodic oxidation reductase is fixed in the buffered compartment of modified anode amberplex.
101. the method for claim 100, wherein said modified anode amberplex are the Nafion  polymer that the bromination quaternary ammonium is handled.
102. the method for claim 100, wherein the anodic oxidation reductase is an alcohol dehydrogenase.
103. the method for claim 100, wherein said biological anode comprises first kind of anodic oxidation reductase and second kind of anodic oxidation reductase, these enzymes are fixed in the buffered compartment of modified anode amberplex, and wherein said first kind of anodic oxidation reductase is that alcohol dehydrogenase and described second kind of anodic oxidation reductase are aldehyde dehydrogenases.
104. be used for accepting from circuit the biological-cathode of electronics, it comprises electrically conductive material and the double duty film arranged side by side with the film of double duty, wherein this double duty film comprises modified amberplex, immobilized cathode enzyme, and electron transfer mediator.
105. the biological-cathode of claim 104, wherein modified amberplex are perfluorinated sulfonic acid-PTFE copolymers that quaternary ammonium salt is handled.
106. the biological-cathode of claim 105, wherein modified amberplex are the Nafion  films that TBAB (TBAB) is handled.
107. the biological-cathode of claim 104, wherein said cathode enzyme (a) are fixed in the micella of buffering of modified amberplex and (b) be the dioxygen oxidation reductase.
108. the biological-cathode of claim 107, wherein said cathode enzyme is a bilirubin oxidase.
109. the biological-cathode of claim 104, wherein said electron transfer mediator comprise transition metal and aromatic series part together as complex compound.
110. the biological-cathode of claim 109, wherein said electron transfer mediator are Ru (bpy) 3 + 2
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