CN101351913A

CN101351913A - Direct electron transfer using enzymes in bioanodes, biocathodes, and biofuel cells

Info

Publication number: CN101351913A
Application number: CNA2006800502177A
Authority: CN
Inventors: S·D·明特尔; B·L·特鲁; R·杜马
Original assignee: St Louis University
Current assignee: St Louis University
Priority date: 2005-11-02
Filing date: 2006-11-02
Publication date: 2009-01-21

Abstract

Bioanodes, biocathodes, and biofuel cells comprising an electron conductor, at least one anode enzyme or cathode enzyme, and an enzyme immobilization material. The anode enzyme is capable of reacting with a fuel fluid to produce an oxidized form of the fuel fluid, and capable of releasing electrons to the electron conductor. The cathode enzyme is capable of reacting with an oxidant to produce water, and capable of gaining electrons from the electron conductor. The enzyme immobilization material for both the anode enzyme and the cathode enzyme is capable of immobilizing and stabilizing the enzyme, and is permeable to the fuel fluid and/or the oxidant.

Description

In biological anode, biological-cathode and biological fuel cell, use the direct electron of enzyme to shift

[0001] the subsidy project 3-00487 that authorizes according to the subsidy project 3-00475 that authorizes by Office of Naval Research, by senior defence project mechanism of the present invention and under the support of government, carry out by the subsidy project 300477 that Central Intelligence Agency authorizes.U.S. government has some right in the present invention.

Background of invention

[0002] generally speaking, the present invention relates to fuel cell (biological fuel cell) and their preparation method and application based on biology enzyme.More specifically, the present invention relates to comprise can the enzyme that direct electron shifts between fuel fluid and electronic conductor biological anode, biological-cathode and biological fuel cell and their preparation method and application.

[0003] biological fuel cell is a kind of electrochemical appliance, and wherein the mode of the catalytic activity by living cells and/or their enzyme will be electric energy derived from the Conversion of energy of chemical reaction.Biological fuel cell uses complex molecule to produce hydrogen reduction to be the required hydrogen ion of water usually on anode, produces simultaneously to be used for the free electron that electricity is used.Biological anode is for wherein discharging the electrode of the biological fuel cell of electronics when oxidized, and biological-cathode be wherein use electronics by catalyst and from the proton of anode with peroxide or hydrogen reduction electrode as water.The difference of biological fuel cell and conventional fuel cell is to be used for the material of catalytic electrochemical reaction.Biological fuel cell depends on biomolecule such as enzyme and is not to use porous metals to react as catalyst.

[0004] most biological anodes and biological-cathode comprise electron mediator.But some comprise the biological anode of electron mediator and life-span, the stability of reduction, disadvantageous thermokinetics and the low electron mediator activity that biological-cathode can have reduction.Thus, exist for not existing and the biological anode that comprises the problem that electron mediator is relevant and the demand of biological-cathode.

Summary of the invention

[0005] one of many aspects of the present invention are for comprising electronic conductor; At least a anode enzyme; Biological anode with enzyme immobilization material.This anode endonuclease capable and fuel fluid react the fuel fluid with the generation oxidised form, and electronics can be discharged to electronic conductor.This enzyme can be fixed and stablize to this enzyme immobilization material, and the fuel permeable fluid.

[0006] on the other hand for comprising electronic conductor; At least a cathode enzyme; Biological-cathode with enzyme immobilization material.This cathode enzyme can with oxidant reaction generating water, and can from electronic conductor, obtain electronics.This enzyme can be fixed and stablize to this enzyme immobilization material, and permeable oxidation agent.

[0007] still is the biological fuel cell that comprises fuel fluid, above-mentioned biological anode and above-mentioned biological-cathode on the other hand.Be the biological fuel cell that comprises fuel fluid, above-mentioned biological anode and negative electrode on the other hand.In addition, be the biological fuel cell that comprises fuel fluid, anode and above-mentioned biological-cathode on the other hand.

[0008] use the method for described biological fuel cell generating to comprise herein, with fuel fluid oxidation and oxidant is reduced on negative electrode or biological-cathode on anode or biological anode.

Description of drawings

[0009] Figure 1A has shown the chemical reaction that takes place on the bilirubin oxidase biological-cathode that shifts based on direct electron, and Figure 1B has shown the chemical reaction that takes place on the biological-cathode of electron mediator comprising.

[0010] Fig. 2 has shown single, functional living being anode or biological-cathode.

[0011] Fig. 3 has shown microfluidic biofuel cell.

[0012] Fig. 4 (a)-(d) has shown the process that forms single microelectrode.

[0013] Fig. 5 has shown the microfluidic biofuel cell heap.

[0014] Fig. 6 is that (mediated) the biological anode with mediation (comprises TBuA-modification

And NAD ⁺-dependence (NAD ⁺-dependent) alcohol dehydrogenase) and direct electron shift biological-cathode and (comprise TBuA-modification

The power curve of no film biological fuel cell and bilirubin oxidase).

[0015] Fig. 7 shows that the biological anode with mediation (comprises TBuA-modification

And NAD ⁺-dependence alcohol dehydrogenase) and direct electron shift biological-cathode and (comprise TBuA-modification The time dependent chart of power of no film biological fuel cell and bilirubin oxidase).

[0016] Fig. 8 shows that the biological anode with mediation (comprises TBuA-modification And NAD ⁺-dependence alcohol dehydrogenase) and direct electron shift biological-cathode and (comprise TBuA-modification

The temperature variant chart of power of no film biological fuel cell and bilirubin oxidase).

[0017] Fig. 9 is that the biological anode with mediation (comprises TBuA-modification

And NAD ⁺-dependence alcohol dehydrogenase) and direct electron shift biological-cathode and (comprise TBuA-modification

The power curve of biological fuel cell and bilirubin oxidase).

[0018] Figure 10 is that the biological anode with mediation (comprises butyl-chitosan and NAD ⁺-dependence alcohol dehydrogenase) and direct electron shift the power curve of the biological fuel cell of biological-cathode (comprising butyl-chitosan and bilirubin oxidase).

[0019] Figure 11 is the fluorescence micrograph of the low molecular weight seaweed acid esters of employing four pentyl ammonium ion modification.

[0020] Figure 12 has shown and has comprised trimethyl octyl group ammonium (TMOA)-modification

Shift the power curve of biological-cathode with the direct electron of hepatocuprein.

Detailed Description Of The Invention

[0021] the present invention relates to biological anode, biological-cathode and biological fuel cell, it comprises enzyme, its can with the electronic conductor Direct electron transfer. Described another way, this biology anode and comprise the biological fuel cell of this biology anode, contain the anode enzyme that electronics can be discharged to electronic conductor, and this biological-cathode and comprise the biological fuel cell of this biological-cathode contains the cathode enzyme that can obtain electronics from electronic conductor. The ability that electronics shifts between this kind of enzyme and the electronic conductor, it is the remarkable advantage that is better than inefficient such electronics mediation system, in this electronics mediation system, electron mediator must be transferred near the of redox reaction and cannot have appropriate local concentration to promote the efficient oxidation reduction reaction. By getting rid of the electron mediator in this system, this reaction power can not learn is subjected to the mass transfer limit of electron mediator, and thus, can be more effective. In addition, when being fixed on enzyme on the electronic conductor, can make the dynamics of oxidation-reduction maximization by having reactant (enzyme and fuel fluid) near the place very much at electronic conductor (it can collect the electronics of generation subsequently).

[0022] still in another embodiment, bioelectrode assembly of the present invention has the enzyme stability of enhancing. For using in biological anode or biological-cathode, this curing materials forms the obstruct that machinery and chemical stability are provided. Thus, with respect to previously known, the stable longer time of enzyme. For purposes of the present invention, if enzyme keeps its initial catalytic activity at least about 75% when continued operation was at least about 7 days～about 730 days in biological fuel cell, it is exactly " stable ".

I. biological fuel cell

[0023] one of many aspects of the present invention are for utilizing the biological fuel cell of fuel fluid generating, by means of the enzyme mediation redox reaction that occurs at the electrode with fixing therein enzyme. As in the standard electric chemical cell, anode is the place of the oxidation reaction of fuel fluid, discharges simultaneously electronics. This electronics by electric connector from anode some current consuming apparatus that lead. To another electric connector, it transfers an electron to the biological-cathode of biological fuel cell to electronics by this equipment moving, is used for oxidant is reduced to produce water at this this electronics. In this way, biological fuel cell of the present invention has served as the external energy source (electric energy) of electric load that is used for to this. In order to promote the redox reaction of fuel fluid, this electrode comprises electronic conductor, enzyme and enzyme immobilization material.

[0024] on biological-cathode, the electronics that results from biological anode flows in the electric conductor of biological-cathode.At this, the electronics contact can obtain the cathode enzyme of electronics from electric conductor.In the various execution modes, have the enzyme immobilization material of permeable oxidation agent, and it can fix and stablize this enzyme.

[0025] biological fuel cell of the present invention comprises biological-cathode and/or biological anode.Usually, this biology anode comprises the element of the oxidation that realizes fuel fluid, discharges electronics and directed outwards electric loading thus.The electric current that obtains provides power for this electric loading, and electronics is directed to biological-cathode subsequently, wherein with oxidant reduction and generation water.

A. biological-cathode

[0026] comprises electronic conductor according to biological-cathode of the present invention and be fixed on enzyme in the enzyme immobilization material.In a kind of execution mode, these components are adjacent one another are, and promptly they link to each other by suitable way physics or chemistry.

1. electronic conductor

[0027] electronic conductor is the material of conduction electron.This electronic conductor can be an organic or inorganic in nature, as long as can pass through this conduct electronics.This electronic conductor can be based on material, stainless steel, stainless (steel) wire, metallic conductor, semiconductor, metal oxide or the modification conductor of carbon.In the preferred implementation, this electronic conductor is the material based on carbon.

[0028] electronic conductor that suits especially is the material based on carbon.The electrode that exemplary material based on carbon is carbon cloth, carbon paper, carbon filament reticulated printing, carbon paper (Toray), carbon paper (ELAT), carbon black (VulcanXC-72, E-tek), carbon black, carbon dust, carbon fiber, Single Walled Carbon Nanotube, double-walled carbon nano-tube, multi-walled carbon nano-tubes, carbon nano pipe array, diamond coated conductor, vitreous carbon and mesoporous carbon.In addition, other exemplary material based on carbon be graphite, not graphite worm (worm), the layering of compacting the purifying flake graphite (

Graphite), high-performance graphite and carbon dust (Formula BT ^TM, Graphite), pyrolytic graphite, pyrolytic graphite and the polycrystalline graphite of high rule (highly ordered).Preferred electronic conductor (carrier film) is the carbon cloth sheet.

[0029] in another execution mode, this electronic conductor can be made by metallic conductor.Suitable electronic conductor can be to be applicable to the metal of electrode structure by gold, platinum, iron, nickel, copper, silver, stainless steel, mercury, tungsten and other.In addition, be the electronic conductor of metallic conductor, can constitute by the nano particle of making of cobalt, carbon and other suitable metal.Other metal electron conductor can be silver-plated nickel wire reticulated printing electrode.

[0030] in addition, this electronic conductor can be a semiconductor.Suitable semi-conducting material comprises silicon and germanium, its other element that can mix.This semiconductor can be used phosphorus, boron, gallium, arsenic, indium or antimony, or it makes up and mixes.

[0031] other electronic conductor can be a metal oxide, metal sulfide, the material of main group compound (that is transistion metal compound) and the modification of employing electronic conductor.The mesoporous silica of the aeroge (aerogel) of glass, cerium oxide particles, molybdenum sulfide, boron nitride nano-tube, employing conductor material such as carbon modification that this exemplary electron-like conductor is nano porous titanium dioxide, titanium dioxide coating, the collosol and gel (solgel) that adopts conductor material such as carbon modification, ruthenium carbon aerogels and employing conductor material such as carbon modification.

[0032] in the various preferred implementations, this electronic conductor is carbon cloth, carbon nano-tube, expanded graphite worm, carbon paste and its combination.More preferably, this electronic conductor is a carbon nano-tube.

2. enzyme

[0033] according to the present invention, enzyme reduces oxidant on biological-cathode.Usually, the enzyme that contains more than one redox center is applicable to biological-cathode of the present invention and biological fuel cell.For example, bilirubin oxidase contains 4 atom copper centers, and wherein T1 copper center is used to accept be used for oxygen reduction from electronics and the T2-T3 electronation bundle of supplying with matrix.Many enzymes that bound by theory not, suggestion contain more than one redox center can be used as they self interior amboceptor and are used for and electron transfer from electronic conductor.The exemplary enzyme that is applicable to biological-cathode is bilirubin oxidase, laccase, superoxide dismutase, peroxidase or its combination.In the various preferred implementations, when oxidant was oxygen, this enzyme was a bilirubin oxidase.In some execution modes, when oxidant was oxide, this enzyme was a hepatocuprein.

3. enzyme immobilization material

[0034] on biological anode and/or biological-cathode, used enzyme immobilization material in this biological fuel cell.In a kind of execution mode, the enzyme immobilization material fuel permeable fluid of this biology anode and fixing and stablize this enzyme.This immobilization material fuel permeable fluid makes and can react in biological anode place fuel oxidation by fixing enzymatic.

[0035] common, enzyme is used for the redox reaction of catalysis at biological-cathode and/or biological anode place.According in biological-cathode of the present invention and/or the biological anode, be fixed on enzyme fixing and stablize in the enzyme immobilization material of this enzyme.Typically, the free enzyme in the solution was lost its catalytic activity in several hours to several days, and still suitably fixing and stable enzyme can keep its catalytic activity at least about 7 days to about 730 days.The maintenance of catalytic activity is defined as the enzyme that has at least about its initial activity of 75%, and its initial activity can be measured by chemiluminescence, electrochemistry, UV-Vis, radiation chemistry or fluorescence assays.This enzyme keeps its initial activity at least about 75%, and this biological fuel cell uninterruptable power generation simultaneously was at least about 7 days to about 730 days.

[0036] Gu Ding enzyme is that physics is limited to the enzyme that keeps its catalytic activity in certain zone of enzyme immobilization material, simultaneously.There is the multiple fixing method of enzyme that is used for, comprises carrier-combination, crosslinked and seizure.Carrier-be combined into is incorporated into water insoluble carrier with enzyme.Crosslinked for using the intermolecular cross-linking enzyme of difunctionality or multifunctional reagent.Capture as enzyme is attached in the grid of semipermeable materials.The ad hoc approach that enzyme is fixing is also non-key important, as long as this enzyme immobilization material (1) is fixed this enzyme, (2) stablize this enzyme and (3) fuel permeable fluid or oxidant.

[0037] about the fuel fluid infiltration of enzyme immobilization material or fixing of oxidant performance and enzyme, in various execution modes, enzyme immobilization material is allowed fuel fluid or oxidant compound by its motion, thereby this compound can contact this enzyme.This enzyme immobilization material can be so that its mode that contains endoporus, passage, perforate or its combination prepares, and it allows this compound by this enzyme immobilization material motion, but it retrains in the same space in fact of this enzyme in enzyme immobilization material.This constraint allows that enzyme keeps its catalytic activity.In the various preferred implementations, enzyme is limited in the space of identical in fact size with enzyme and shape, wherein this enzyme keeps in fact all its catalytic activitys.This hole, passage or perforate have the physical size that satisfies above-mentioned requirements and depend on the size and dimension of certain enzyme to be fixed.

[0038] in the various execution modes, this enzyme is preferably located in the hole of enzyme immobilization material and this compound passes in and out in this enzyme immobilization material by transmitting passage.The hole and transmit passage relative size can so that the hole enough greatly with immobilized enzyme, to such an extent as to but transmit passage and can not pass it and move for enzyme is too small.In addition, the diameter of transmission passage is preferably at least about 10nm.Still in another execution mode, hole dimension with transmit the channel radius ratio be at least about 2: 1,2.5: 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 bigger.Still in another execution mode, preferably, the diameter that transmits passage be at least about 10nm and hole dimension with transmit the channel diameter ratio be at least about 2: 1,2.5: 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 bigger.

[0039] stable about enzyme, enzyme immobilization material provide chemistry and machinery to intercept to prevent or to stop enzyme denaturation.For this reason, enzyme immobilization material limits this enzyme physically, prevents that this enzyme from stretching.Enzyme is a kind of mechanism of enzyme denaturation from the process that folding three-dimensional structure stretches.In a kind of execution mode, this immobilization material preferably, is fixed this enzyme, makes this enzyme keep its catalytic activity at least about 7 days～about 730 days.The confining force of catalytic activity is defined as the fate that enzyme keeps producing continuously as the part of biological fuel cell simultaneously at least about its initial activity of 75% electric power.Enzymatic activity can be measured by chemiluminescence, electrochemistry, UV-Vis, radiochemistry or fluoremetry, wherein measures original performance intensity.Typically, adopt fluoremetry to measure enzymatic activity.Enzyme in the solution was lost its catalytic activity in several hours to several days.Thus, the stable aspect that is fixed on of enzyme provides significant advantage.In other execution mode, preferably, fixing enzyme keeps at least about its initial catalytic activity of 75% at least about 5,10,15,20,25,30,45,60,75,90,105,120,150,180,210,240,270,300,330,365,400,450,500,550,600,650,700,730 days or longer, more preferably keep at least about 80%, 85%, 90%, 95% or bigger its initial catalytic activity at least about 5,10,15,20,25,30,45,60,75,90,105,120,150,180,210,240,270,300,330,365,400,450,500,550,600,650,700,730 or longer.

[0040] in some execution modes, this enzyme immobilization material has micella or reversed micelle structure.Usually, the molecule that constitutes micella is amphipathic, show they contain polarity, hydrophilic radical and nonpolar, lipophile group.This molecule can be built up to form micella, and wherein polar group is on the surface of building up body and hydrocarbon, non-polar group is completely cut off is building up body inside.Reversed micelle has the polar group and the non-polar group of opposed orientation.Constituting the amphipathic molecule of building up body can arrange in many ways, as long as polar group is close to each other and non-polar group is close to each other.In addition, this molecule can form bilayer, and wherein non-polar group points to and polar group sensing privately mutually each other relative to one another.Replaceability ground can form bilayer, and wherein polar group can point in bilayer relative to one another, and non-polar group points to each other mutually privately simultaneously.

[0041] some enzyme immobilization materials, and the perfluorinated sulfonic acid of micella enzyme immobilization material and modification-PTFE copolymer particularly, be described in U.S. Patent application 10/931147 (publishing) with U.S. Patent Application Publication 2005/0095466, with U.S. Patent application 10/,617 452 (publishing) with U.S. Patent Application Publication 2004/0101741, during the two all is incorporated herein with it as a reference.

[0042] in a kind of preferred implementation, the micella enzyme immobilization material be modification perfluorinated sulfonic acid-PTFE copolymer (the perhaps perfluorinated ion-exchange polymer of modification) (modification

Or modification

) film.Employing is greater than ammonium ion (NH ₄ ⁺) dewatering cationic modification perfluorinated ion-exchange polymer film.This dewatering cationic plays (1) domination membrane pore size and (2) double action as the chemical buffer of the pH level that helps retaining hole, and the two all makes enzyme stable.

[0043] about first kind of function of dewatering cationic, with dewatering cationic mixture casting perfluorinated sulfonic acid-PTFE copolymer (or perfluorinated ion-exchange polymer) with the perfluorinated sulfonic acid PTFE copolymer (the perhaps perfluorinated ion-exchange polymer of modification) that produces modification (

Or ) film provides the enzyme immobilization material, its intermediate pore size depends on the size of dewatering cationic.Therefore, dewatering cationic is big more, and hole dimension is big more.The above-mentioned functions of dewatering cationic allows to make hole dimension greater or lesser to be fit to specific enzyme by the size that changes dewatering cationic.

[0044] about second kind of effect of dewatering cationic, by exchange conduct-SO on perfluorinated sulfonic acid-PTFE copolymer (or perfluorinated ion-exchange polymer) film with dewatering cationic ₃ ^-The proton of the counter ion of group, the performance of change perfluorinated sulfonic acid-PTFE copolymer (or perfluorinated ion-exchange polymer) film.The change of this counter ion provides the cushioning effect to pH, because dewatering cationic has right-SO than proton ₃ ^-The affinity that the site is bigger.The sort buffer effect of film makes that the pH in hole keeps constant in fact when changing pH value of solution; In other words, the variation of the pH in hole opposing pH value of solution.In addition, this film provides mechanical obstruct, and it is the fixing enzyme of protection further.For perfluorinated sulfonic acid-PTFE copolymer (or perfluorinated ion-exchange polymer) film for preparing modification, the first step be casting with perfluorinated sulfonic acid-PTFE copolymer (or perfluorinated ion-exchange polymer) film of the solution of dewatering cationic, particularly

Suspension to form film.From this film, extract the salt of excessive dewatering cationic and they subsequently, and cast membrane once more.When casting once more, this film contain with perfluorinated sulfonic acid-PTFE copolymer (or perfluorinated ion-exchange polymer) film-SO ₃ ^-The dewatering cationic that the site links mutually.The salt of removing dewatering cationic from this film causes more stable and reproducible film, because excessive salt can become trapped in the hole or cause hole in the film.

[0045] in a kind of execution mode, modification

Film is by the solution of casting and dewatering cationic salt such as bromination quaternary ammonium

The suspension of polymer prepares.It being cast once more, from this film, remove excessive bromination quaternary ammonium or hydrogen bromide with before forming the film that extracts salt.The salt of film extracts the existence that has kept quaternary ammonium cation on sulfonic acid exchange site, but has eliminated the problem that may be trapped in the hole or may cause the excess salt of the hole in the balance film.Chemistry and the physical property of extracting the film of salt characterize by the voltammetry before enzyme is fixing, ion exchange capacity measurement and fluorescence spectrum.Exemplary dewatering cationic is the cation based on ammonium, quaternary ammonium cation, alkyl trimethyl ammonium cation, alkyl triethyl ammonium cation, organic cation phosphonium cation, triphenyl phosphonium, pyridylium, glyoxaline cation, cetyl pyridinium, second ingot (ethidium), purpurine (viologen), methyl viologen, benzyl viologen, two (triphenylphosphine) imines (iminium), metal complex, bipyridine (bipyridine) metal complex, metal complex based on phenanthroline (phenanthroline), [Ru (bipyridine) ₃] ²⁺[Fe (phenanthroline) ₃] ³⁺

[0046] in a kind of preferred implementation, this dewatering cationic is the cation based on ammonium.Especially, this dewatering cationic is a quaternary ammonium cation.In another execution mode, this quaternary ammonium cation is by shown in the formula 4:

Wherein, R 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 ₄In at least one be not hydrogen.In another execution mode, 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 ₄In at least one be not hydrogen.Still in another execution mode, R ₁, R ₂, R ₃And R ₄Identical and be methyl, ethyl, propyl group, butyl, amyl group or hexyl.Still in another execution mode, preferably, R ₁, R ₂, R ₃And R ₄Be butyl.Preferably, quaternary ammonium cation is tetrapropyl ammonium (T3A), four pentyl ammonium (T5A), tetrahexyl ammonium (T6A), four heptyl ammoniums (T7A), trimethyl eicosyl ammonium (TMICA), trimethyl octyl-decyl ammonium (TMODA), trimethyl hexyl decyl ammonium (TMHDA), trimethyl myristyl ammonium (TMTDA), trimethyl octyl group ammonium (TMOA), trimethyldodecane base ammonium (TMDDA), trimethyl decyl ammonium (TMDA), trimethyl hexyl ammonium (TMHA), TBuA (TBA), triethyl group hexyl ammonium (TEHA) and its combination.

[0047] exemplary micella or reversed micelle enzyme immobilization material are, the polysaccharide of hydrophobically modified, and these sugar are selected chitosan, cellulose, chitin, starch, amylose, alginates and combination thereof.In the various execution modes, micella or reversed micelle enzyme immobilization material are polycationic polymer, particularly, and the chitosan of hydrophobically modified.Chitosan is poly-[β-(1-4)-2-amino-2-deoxidation-D-glucopyranose].Chitosan is deacetylated preparation the by chitin (poly-[β-(1-4)-2-acetamido-2-deoxidation-D-glucopyranose]) usually.Typical commodity chitosan have about 85% deacetylated.These deacetylated or free amino groups can further use alkyl, and particularly, alkyl carries out functionalized.Thus, in the various execution modes, the chitosan of this micella hydrophobically modified is corresponding to the structure of formula 1:

Wherein, n is an integer; R ₁₀Be the alkyl of hydrogen, alkyl or replacement independently; And R ₁₁Be the alkyl of hydrogen, alkyl or replacement independently.In the some embodiments of the present invention, n be make polymer molecular weight be about 21000～about 500000, be preferably about 90000～about 500000, about 150000～about 350000, about integer of 225000～about 275000 more preferably more preferably.In some execution modes, R ₁₀Be hydrogen or alkyl independently, and R ₁₁Be hydrogen or alkyl independently.In addition, R ₁₀Be hydrogen or hexyl independently, and R ₁₁Be hydrogen or hexyl independently.Replaceability ground, R ₁₀Be hydrogen or octyl group independently, and R ₁₁Be hydrogen or octyl group independently.

[0048] in addition, in the various execution modes, the chitosan of micella hydrophobically modified is the modification chitosan corresponding to formula 1B

Wherein, R ₁₁, R ₁₂, with n as the definition relevant with formula 1.In some execution modes, R ₁₁And R ₁₂Be hydrogen or straight chain or branched-alkyl independently; Preferably, hydrogen, butyl, amyl group, hexyl, heptyl, octyl group, nonyl, decyl, undecyl or dodecyl.In the various execution modes, R ₁₁And R ₁₂Be hydrogen, butyl or hexyl independently.

[0049] chitosan of micella hydrophobically modified can be with the hydrophobic grouping modification extremely in various degree.The hydrophobically modified degree is recently measured with respect to the percentage of the number of free amino group in the unmodified chitosan by the free amino group that adopts the hydrophobic grouping modification.Can be from acid base titration and/or nulcear magnetic resonance (NMR) (NMR), particularly ¹Calculate the hydrophobically modified degree in the H NMR data.The hydrophobically modified degree can vary widely and be at least about 1,2,4,6,8,10,12,14,16,18,20,25,30,32,24,26,28,40,42,44,46,48% or bigger.Preferably, the hydrophobically modified degree is about 10%～about 45%; About 10%～about 35%; About 20%～about 35%; Perhaps about 30%～about 35%.

[0050] hydrophobic grouping that is used for the modification chitosan play (1) domination material hole size and (2) modification chitosan electronic environment to keep the double action in acceptable orifice ring border, the two all makes enzyme stable.About first kind of effect of hydrophobic grouping, the chitosan of hydrophobically modified produces enzyme immobilization material, and its intermediate pore size depends on the size of hydrophobic grouping.Thus, size, shape and the degree of the chitosan modification of employing hydrophobic grouping influence the size and the shape in hole.This effect of dewatering cationic allow hole dimension make greater or lesser or difformity meeting concrete enzyme, by changing the size and the branching of hydrophobic grouping.

[0051] about second kind of effect of hydrophobic grouping, changes the performance of the chitosan films of hydrophobically modified by using hydrophobic grouping modification chitosan.This hydrophobically modified of chitosan influences the orifice ring border by the number that increases the obtainable exchange of proton site.Except the pH that influences material, the hydrophobically modified of chitosan provides the film that intercepts for machinery, and it further protects stable enzyme.

[0052] table 1 has shown the number for the obtainable proton exchange of the chitosan films of hydrophobically modified site.

Table 1: every gram chitosan polymer, the number in obtainable proton exchange site

Film	Exchange site/gram (* 10 ^-4molSO ₃/g)
Film	Exchange site/gram (* 10 ^-4molSO ₃/g)	Chitosan	10.5±0.8
The butyl modification	226±21	Chitosan	10.5±0.8
The butyl modification	226±21	The hexyl modification	167±45
The octyl group modification	529±127	The hexyl modification	167±45
The octyl group modification	529±127	The decyl modification	483±110

In addition, this polycationic polymer can stabilized enzyme and is increased the activity that is stabilized in enzyme wherein, with respect to the activity of the same enzyme in cushioning liquid.In the various execution modes, polycationic polymer is the polysaccharide of hydrophobically modified, especially, and the chitosan of hydrophobically modified.For example,, adopt the operation among the embodiment 6, measure the enzymatic activity of glucose oxidase for described hydrophobically modified.In the chitosan of the hexyl modification that is suspended in tertiary pentyl alcohol, observe the enzymatic activity high of glucose oxidase.These fixedly film show with respect to the enzyme in the cushioning liquid and increasing by 2.53 times aspect the glucose oxidase enzymatic activity.Table 2 describes the glucose oxidase activity of the chitosan of various hydrophobically modifieds in detail.

Table 2: the glucose oxidase activity of modification chitosan.

Film/solvent enzymatic activity (unit/gm)

Buffer solution 103.61 ± 3.15

Unmodified chitosan 214.86 ± 10.23

The hexyl chitosan

Chloroform 248.05 ± 12.62

Tert-pentyl alcohol 263.05 ± 7.54

50% acetate 118.98 ± 6.28

The decyl chitosan

Chloroform 237.05 ± 12.31

Tert-pentyl alcohol 238.05 ± 10.02

50% acetate 3.26 ± 2.82

The octyl group chitosan

Chloroform 232.93 ± 7.22

Tert-pentyl alcohol 245.75 ± 9.77

50% acetate 127.55 ± 11.98

The butyl chitosan

Chloroform 219.15 ± 9.58

Tert-pentyl alcohol 217.10 ± 6.55

50% acetate 127.65 ± 3.02

[0053] in order to prepare the hydrophobically modified chitosan that has as the alkyl of modifier of the present invention, chitosan gel is suspended in the acetate, adds alcoholic solvent subsequently.In this chitosan gel, add aldehyde (for example butyraldehyde, hexanal, octanal or capraldehyde), add sodium cyanoborohydride subsequently.The product that obtains is washed by isolated by vacuum filtration and with alcoholic solvent.Subsequently that the chitosan of modification is dry under 40 ℃ in vacuum drying oven, obtain the sheet white solid.

[0054] in order to prepare the hydrophobically modified chitosan that has as the redox mediators of modifier of the present invention, by with 4,4 '-dimethyl-2,2 '-bipyridine contacts with diisopropylamine lithium, add alkylene dihalide subsequently to make 4-methyl-4 '-(6-haloalkyl)-2,2 '-bipyridine derives redox mediators.Subsequently with this part and Ru (bipyridine) ₂Cl ₂Hydrate contacts in the presence of inorganic base and refluxes in water-alcohol mixture, up to Ru (bipyridine) ₂Cl ₂Depleted.Subsequently with product with ammonium hexafluorophosphate or randomly sodium perchlorate or sylvite precipitation, recrystallization subsequently.The redox mediators that to derive (Ru (bipyridine) subsequently ₂(4-methyl-4 '-(6-bromo hexyl)-2,2 '-bipyridine) ^{+ 2}) contact with deacetylated chitosan and heat.Chitosan with the redox mediators modification precipitates and recrystallization subsequently.

[0055] the hydrophobically modified chitosan films has favourable insoluble in ethanol.For example, above-mentioned chitosanase immobilization material can be fixed and stabilized enzyme in having at most greater than the solution of about 99 weight % or 99 volume % ethanol usually.In the various execution modes, the chitosan immobilization material have 15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or the solution of bigger weight % or volume % ethanol in effective.

[0056] in other execution mode, micella or reversed micelle enzyme immobilization material are multi-anion copolymer, as the polysaccharide of hydrophobically modified, especially, the alginates of hydrophobically modified.Alginates are the linearity of L-guluronic acid (guluronic acid) residue of the D-mannuronic acid (mannuronic acid) that contains β-(1-4)-connect and α-(1-4)-be connected branched polymer not.In unprotonated form, the D-mannuronic acid of β-(1-4)-connect is corresponding to the structure of formula 3A

And in unprotonated form, the L-guluronic acid of α-(1-4)-connect is corresponding to the structure of formula 3B

The heteropolymerization thing that alginates are made up of the polymer blocks of the polymer blocks of mannuronic acid residue and guluronic acid residues.

[0057] alginate polymer modification in many ways.One type is to adopt greater than ammonium ion (NH ₄ ⁺) the alginates of dewatering cationic modification.This dewatering cationic plays (1) domination polymer hole dimension and (2) double action as the chemical buffer of the pH level that helps retaining hole, and the two all makes enzyme stable.About first kind of effect of dewatering cationic, adopt dewatering cationic modification alginates to produce enzyme immobilization material, its intermediate pore size depends on the size of hydrophobic grouping.Thus, size, shape and the degree of employing dewatering cationic modification alginates influence the size and the shape in hole.This effect of dewatering cationic allow hole dimension make greater or lesser or difformity meeting concrete enzyme, by changing the size and the branching of dewatering cationic.

[0058] about second kind of effect of dewatering cationic, by with the dewatering cationic exchange as on the alginates-CO ₂ ^-The proton of the counter ion of group, the performance of change alginates.The change of this counter ion has improved the cushioning effect to pH because dewatering cationic with respect to proton right-CO ₂ ^-The site affinity is bigger.The sort buffer effect of alginates film makes that the pH in hole keeps constant in fact when changing pH value of solution; In other words, the variation of the pH in hole opposing pH value of solution.In addition, this alginates film provides mechanical obstruct, and it is the fixing enzyme of protection further.

[0059] in order to prepare the alginates film of modification, the first step be casting with the suspension of the alginate polymer of the solution of dewatering cationic to form film.From this film, extract excessive dewatering cationic and their salt subsequently, and then cast membrane.When casting once more, this film contain with the alginates film-CO ₂ ^-The dewatering cationic that the site links mutually.The salt of removing dewatering cationic from this film causes more stable and reproducible film, because excessive salt can become trapped in the hole or cause hole in the film.

[0060] in a kind of execution mode, the alginates film of modification is to prepare by the suspension of casting with the polymer of the alginates of the solution of dewatering cationic salt such as bromination quaternary ammonium.It being cast once more, from this film, remove excessive bromination quaternary ammonium or hydrogen bromide with before forming the film that extracts salt.The salt of film extracts the existence that has kept quaternary ammonium cation on carboxylic acid exchange site, but has eliminated the problem that may be trapped in the hole or may cause the excess salt of the hole in the balance film.Exemplary dewatering cationic is cation, quaternary ammonium cation, alkyl trimethyl ammonium cation, alkyl triethyl ammonium cation, organic cation, phosphonium cation, triphenyl phosphonium, pyridylium, glyoxaline cation, cetyl pyridinium, second ingot, purpurine, methyl viologen, benzyl viologen, two (triphenylphosphine) imines, metal complex, Bipyridine metal complexes, the metal complex based on the phenanthroline, [Ru (bipyridine) based on ammonium ₃] ²⁺[Fe (phenanthroline) ₃] ³⁺

[0061] in a kind of preferred implementation, this dewatering cationic is the cation based on ammonium.Especially, this dewatering cationic is a quaternary ammonium cation.In another execution mode, this quaternary ammonium cation is by shown in the formula 4:

[0062] studied the result of the alginates film that has shown a kind of hydrophobically modified among pore property and Figure 11.The pore structure of this this film fixedly is desirable for enzyme, because the hole is hydrophobic, structure is a micella, cushions outside pH and changes, and have high hole interconnectivity.

[0063] in another experiment, ultra-low molecular amount alginates and lauryl amine are placed 25% ethanol and backflow, make the alginates of dodecyl modification thus by the amidatioon of hydroxy-acid group.Can replace lauryl amine to make alkyl-modified alginates with various alkylamines, it has the C of the reactive hydroxy-acid group of the varying number that is connected in the alginates structure ₄-C ₁₆Alkyl.In the various execution modes, at least about 1,2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48% or more hydroxy-acid group and alkylamine.

[0064] the alginates film of hydrophobically modified has favourable insolublely in ethanol, and for example, the effect of above-mentioned alginates enzyme immobilization material is fixing and stabilized enzyme in having at least about the solution of 25 weight % or 25 volume % ethanol normally.In the various execution modes, the alginates immobilization material have 25,30,35,40,45,50,55,60,65,70,75,80,85,90 or the solution of bigger weight % or volume % ethanol in effective.

4. biological-cathode execution mode

[0065] various biological-cathodes can be attached in the biological fuel cell of the present invention.For example, this biological-cathode is described in U.S. Patent application 10/,931 147 (open with U.S. Patent Application Publication 2005/0095466), during it all is incorporated herein as a reference.

B. biological anode

[0066] in a kind of execution mode, this biology anode comprises electronic conductor and the enzyme that is fixed in the enzyme immobilization material.The biological anode component of Que Dinging is adjacent one another are above; Show that they connect by suitable mode physics or chemistry.Because this component is identical with the biological-cathode component usually, the difference of each element composition and the difference of function are paid close attention in discussion below, when appropriate the time.

1. electronic conductor

[0067] 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 a kind of execution mode, this biology anode electronic conductor is a carbon paper.

2. enzyme

[0068] enzyme is in the oxidation reaction of biological anode place catalytic fuel fluid.Particularly, the exemplary enzyme that is used for biological anode is the enzyme with oxidized fuel fluid of reacting, and comprises more than one redox center.For example, Shi Yi anode enzyme comprises PQQ-dependence (PQQ-dependent) dehydrogenase, lipoxygenase (lipoxygenase) or its combination.PQQ-dependence alcohol dehydrogenase extracts from glucose bacillus (gluconobacter).

3. enzyme immobilization material

[0069] as mentioned above, biological anode and/or biological-cathode place utilize enzyme immobilization material in this biological fuel cell.Find among the I.A.3 in the above about the composition of this enzyme immobilization material and the further detailed content of fixed mechanism.In a kind of execution mode, this biology anode enzyme immobilization material fuel permeable fluid and stable and fix this enzyme.This immobilization material fuel permeable fluid thus can be by the oxidation of fixing enzymatic fuel fluid at biological anode place.In some execution modes, this enzyme immobilization material is the chitosan of the polysaccharide of hydrophobically modified, particularly hydrophobically modified.

4. biological anode execution mode

[0070] preferred biological anode is described in U.S. Patent application 10/,617 452 (open with U.S. Patent Application Publication 2004/0101741), during its full content is incorporated herein as a reference.Other potential suitable biological anode is described in US 6 531 239 and 6 294 281, also is introduced into herein as a reference.

C. fuel fluid and oxidant

[0071] can oxidized fuel fluid be the component of biological fuel cell of the present invention at biological anode place with the oxidant that can be reduced at the biological-cathode place to produce water with the generation electronics.

[0072] oxidation reaction of fuel fluid at the redox center of immobilized enzyme that is used for biological anode is consumed.The molecular dimension of fuel fluid is enough little, makes by the diffusion coefficient of this enzyme immobilization material big.Exemplary fuel fluid 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; the G-6-P ester; lactate; lactate-6-phosphate; the D-lactate; the L-lactate; fructose; galactolipin-1; galactolipin; aldose; sorbose and mannose); monoglyceride; coacetylase; acetyl group Co-A; malate; the isocitric acid ester; formaldehyde; acetaldehyde; acetic acid esters; citrate; the L-gluconate; beta-hydroxysteroid; alpha-hydroxysteroid; lactic aldehyde (lactaldehyde); testosterone; gluconate; aliphatic acid; lipid; the phosphoglycerol acid esters; retinene; estradiol; cyclopentanol; hexadecanol; long-chain alcohol; coniferyl-alcohol; cinnamyl-alcohol; formic acid esters; long-chain aldehyde; pyruvate; butyraldehyde; acyl group-CoA; steroids; amino acid; flavine; NADH; NADH ₂, NADPH, NADPH ₂, hydrocarbon and amine.In the various preferred implementations, this fuel fluid is alcohol, more preferably methyl alcohol and/or ethanol; And most preferred ethanol.

[0073] electronics that adopts biological anode to provide is provided the reduction reaction of oxidant at the redox center of immobilized enzyme that is used for biological-cathode.The molecular dimension of oxidant is enough little, makes by the diffusion coefficient of this enzyme immobilization material big.Can utilize the variety of way that oxidant source is provided as known in the art.

[0074] in the preferred implementation, this oxidant is a gaseous oxygen, by diffusion it is transferred to biological-cathode.In other preferred implementation, this oxidant is a peroxide.

[0075] biological fuel cell of execution mode can comprise (i) aforesaid biological anode; (ii) aforesaid biological-cathode; (iii) aforesaid biological anode and biological-cathode; The biological-cathode of describing in (iv) aforesaid biological anode and the U.S. Patent application 10/,931 147 (open) with U.S. Patent Application Publication 2005/0095466; (v) biological anode and the aforesaid biological-cathode of describing in the U.S. Patent application 10/,617 452 (open) with U.S. Patent Application Publication 2004/0101741.

[0076] biological fuel cell of the present invention can comprise polymer dielectric film (" PEM " or salt bridge, for example,

117) so that the anode chamber is separated with cathode chamber.But, for execution mode with biological anode and biological-cathode, PEM and nonessential and biological fuel cell that make no film.It is unnecessary that the enzyme that uses in biological anode and the biological-cathode makes that to oxidant or the preferential selectivity of the catalytic action of fuel fluid reaction the physical separation of anode chamber and cathode chamber becomes.

II. microfluidic biofuel cell

[0077] among the various aspects of the present invention, comprises the microfluidic biofuel cell that utilizes fuel fluid to produce electric power, the redox reaction that its enzyme that takes place by means of the little molded microelectrode place that has immobilized enzyme therein mediates.As in the standard biological fuel cell, biological anode is to be used for the oxidation reaction of fuel fluid, to discharge the place of electronics simultaneously.Electronics by electric connector from biological anode some energy consumption equipments that lead.To another electric connector, it will be transmitted electronically to the biological-cathode of biological fuel cell to electronics by this equipment moving, and wherein electronics is used to make the oxidant reduction to produce water.In this way, biological fuel cell of the present invention is as the energy source (electric power) of the external electrical load that is used to be connected to this.In order to promote the redox reaction of fuel fluid, this microelectrode comprises electronic conductor, enzyme and enzyme immobilization material.

[0078] still, be different from the standard biological fuel cell, biological fuel cell of the present invention utilizes at least one little molded electrode.In a kind of execution mode, this little molded electrode has by allow flowing of structure that fuel flows in this microelectrode.With respect to the traditional biological fuel cell electrode, this structure produces bigger current density, because the amount of the microelectrode surface area that contacts with fuel is bigger.In another execution mode, this little molded electrode has irregularly shaped.In addition, the current density of microelectrode is because the more substantial surface area that contacts with fuel and bigger with respect to the traditional biological fuel cell electrode.These features and further feature disclosed herein combination have formed the biological fuel cell that increases with respect to traditional biological fuel cell current density by raw material littler on the size thus.At last, can be used for valuably producing economically can disposable fuel cell for method of the present invention.

A. microfluid passage

[0079] except that biological anode and/or biological-cathode, this microfluidic biofuel cell is characterised in that at least one microfluid passage, and it holds this biology anode and/or biological-cathode, fuel fluid, and oxidant when using.This microfluid passage configuration can be according to application change.In a kind of execution mode, this microfluid passage can be a rectangular chamber simply, and the biological anode of this biological fuel cell and/or biological-cathode are with which.Referring to Fig. 2.In other execution mode, the configuration of this microfluid passage can be more elaborate, is used for any desired purpose, so guarantee biological anodic dissolution and biological-cathode solution not each other physics contact.Referring to Fig. 3.

[0080] with reference to Fig. 2 and 3, fuel fluid and/or oxidant flow on microelectrode or wherein by microfluid passage (34), from the end (inlet) (33) of microfluid passage to end opposite (outlet) (35).Among Fig. 3, (41) biological anode of expression and (40) expression biological-cathode.Microfluid passage should promote fuel fluid and/or the convection current of oxidant on this microelectrode, prevents its seepage outside microfluid passage (34) simultaneously.

B. electric connector

[0081] electric connector provides electrically contacting to microfluidic biofuel cell from the microelectrode to the external electrical load.In the most general implication, electric connector can be any material and structure, and it promotes electronics from biological anode to electric loading with get back to the transfer of biological-cathode.In a kind of preferred implementation, the electric connector of this microfluidic biofuel cell provides can be with another equipment and its physics and the welding lead that electrically contacts.This miscellaneous equipment, for example, copper cash transmits electronics subsequently, is sent to external electrical load and therefrom transmission.

[0082] in a kind of preferred implementation, this electric connector is the thin layer connector, before other is handled it is formed on the substrate of microfluidic biofuel cell.In this execution mode, arrange the microelectrode that forms subsequently, make them that their each electric connector is intersected mutually.In the execution mode of replacing, this electric connector is cylindrical electric conducting material, and it is connected in microelectrode after their processing.

III. microfluidic biofuel cell is made

[0083] when making, uses the substrate that constitutes other biological fuel cell assembly thereon according to microfluidic biofuel cell of the present invention.In the preferred implementation, the first step is to form electric connector, makes microelectrode subsequently and randomly defines the bio-fuel chamber.In the execution mode of replacing, after further feature, form this electric connector.

A. the making of electric connector

[0084] microfluidic biofuel cell of the present invention is by providing the substrate that forms the residue assembly on it to form.This substrate can be made by nonconducting any material, its will be not can the passivation microelectrode electric conducting material, by processing thereon, and mould reversibly can be sealed on it the electric conducting material adhesion.In a kind of execution mode, this substrate is a glass.In the preferred implementation, this substrate is for gathering (dimethyl siloxane) (PDMS).In another preferred implementation, this substrate is a Merlon.In a kind of execution mode, this substrate is flat.In the execution mode of replacing, this substrate can present the geometry that advantageously is fit to application-specific.

[0085] in the preferred implementation, first biological fuel cell that forms on this substrate is characterized as electric connector, and it will be electrically connected with microelectrode in the biological fuel cell of finishing, and be provided for external electrical load is connected in the approach of microelectrode thus.This connector can be made by any electric conducting material.Exemplary materials comprises alloy, carbon, nickel, copper and the stainless steel of platinum, palladium, gold, these noble metals.In the preferred implementation, this connector is made by platinum.

[0086] can adopt traditional photography lithographic printing known in the silicon wafer industry that this connector is formed on the substrate.For example, in order to form thin layer platinum electric connector, sputtered titanium adhesive layer on substrate at first.Sputter platinum layer on titanium layer subsequently.These two sputter procedure can, for example, in argon gas-ion sputtering system, carry out.By photo imprint lithography definition connector, wherein photoresist is administered to the link position of expecting with protection on the platinum layer subsequently.Adopt these two layers of etchant chemistry etching of commercially available acquisition, peel off photoresist subsequently, will produce final platinum electric connector.In the execution mode of replacing, this electric connector is the last feature that forms.Describe this execution mode below in detail.

B. the making of microelectrode

[0087] form on the substrate of biological fuel cell after the electric connector, next step is to make biological anode and biological-cathode.These can form successively or simultaneously.

1. biological anode is made

[0088] in a kind of execution mode, on substrate, forms biological anode and biological-cathode successively, wherein formation order and non-key.Only, at first describe biological anode in detail and make for the purpose of statement.The first step of making the biological anode of minute yardstick is the pattern that forms the microchannel in the surface of mold.Usually, mold can be made by nonconducting any material, its will be not can passivation conducting material and can reversibly be sealed on the substrate, wherein exemplary materials comprises silicon, glass and polymer.Mold preferably polymer is made, even more preferably make by PDMS.Most preferably, mold is made by Merlon.

[0089] in the preferred implementation, wherein mold is a polymer, forms pattern by adopting known flexible offset printing art technology, to make the microchannel to define the shape and size of biological anode in mold.Flexible offset printing art technology requires to make the process of prepolymer with respect to the mother matrix molding of the raised image with desired design of imprint lithography definition usually.The flexible offset printing art technology that adopts should be able to produce about 1 μ m～about 1mm, about 1 μ m～about 200 μ m, preferably about 10 μ m～about 200 μ m, more preferably from about 10 μ m～about 100 μ m and most preferably little about 10 μ m or the littler microchannel arrived in mold.Exemplary flexible offset printing art technology comprises near field phase shift imprint lithography, duplicating molded, micrometastasis molding (μ TM), little contact molding (SAMIM) that solvent is auxiliary, and micro-contact printing (μ CP).Preferably, this microchannel adopts duplicating molded to make.

[0090] in mold, forms after the microchannel, the pattern of mold is formed side be adhered to substrate to finish the molded of this microelectrode.Referring to Fig. 4 (a).Being pre-formed on substrate in the execution mode of electric connector (31), should align on electric connector in this microchannel, makes the microelectrode of finishing to be electrically connected with connector.In addition, tubular connector (30) is adhered to substrate to keep to become subsequently the position of inlet container.

[0091] next, with reference to Fig. 4 (b), make electronic conductor solution by the inlet container (32) that in mold, formed in the microchannel of a side inflow mold of microchannel.This inlet container (32) is similar to the casting groove in the conventional art of casting of metals.Excess solution will leave this microchannel in the outlet that is positioned at the microchannel end relative with the inlet container.

[0092] electronic conductor solution can be to comprise the electronic conductor source and can remove any solution with the liquid-carrier that produces the solid microelectrode by curing.A large amount of potential electron conductor material are listed among the I.A.1 in the above.In a kind of preferred implementation, this electronic conductor source is a carbon source.In the preferred execution mode, this electronic conductor source is the printing ink based on carbon.In this execution mode, liquid-carrier is the printing ink reducer based on carbon, for example Ercon N160 solvent diluent.According to the person's character of liquid-carrier in the solution, can form microelectrode structure-solid microelectrode of two types or flow through microelectrode according to the present invention.Adopt more low viscous liquid-carrier, make the solid microelectrode.These microelectrodes come down to continuous and solid, and fuel fluid flows on this microelectrode between the operating period.Adopt more full-bodied liquid-carrier, make and flow through microelectrode, its structure can make fuel fluid therefrom flow during use, increases the surface area of the microelectrode that contacts with fuel fluid effectively.

[0093] irrelevant with ad hoc structure, the microelectrode that forms according to the present invention has several advantages with respect to the microelectrode (it must have flat shape) that adopts traditional handicraft to form.So, any fluid that flows on traditional microelectrode has the flow pattern of common rule and contacts with the microelectrode surface area of common limited amount.This flat geometrical surface calculates by the rectangular tables area of the top of flat microelectrode and side is added up.Because the electric current of microelectrode produces major part and decided by the surface area that contacts with fuel fluid,, the electric current of flat microelectrode only can increase by increasing its size so producing ability.On the contrary, the microelectrode of foundation formation of the present invention has highly irregular, three dimensional shapes, and it produces two tangible advantages at least.At first, fully increased the effective surface area of microelectrode of the present invention with respect to the microelectrode of flat silk screen printing.The effective surface area of described microelectrode is each peak of this microelectrode shape of sign and the surface area sum of paddy herein.A kind of exact method that calculates this effective surface area is, the electric current output of the microelectrode that will form according to the present invention is compared with the flat microelectrode of equal length, width and height dimension.For example, the analysis of this microelectrode shows that the electric current for microelectrode of the present invention is output as 9.85 * 10 ^-4A/cm ², relatively traditional glass matter carbon electrode is 2.06 * 10 ^-4A/cm ²In addition, the irregularly shaped turbulent flow that can form fluid of microelectrode.This flow pattern is favourable, because it causes the mixing of fluid on microelectrode, it has increased the transmission rate of fluid to microelectrode again.Increase the reaction that the transmission rate of fluid helps taking place in microelectrode, increased the current capacity ability of microelectrode thus.

[0094] in a kind of execution mode of replacement, before introducing electronic conductor solution, priming paint is flowed in the microchannel of mold and rapid draing.This priming paint can be to prevent that with helping electronic conductor from becoming partly-for good and all being adhered to any material of mold.For example, in printing ink execution mode, can use printing ink reducer based on carbon as priming paint, if desired based on carbon.

[0095] solution is filled after the microchannel of mold, applies heat so that electronic conductor solution solidifies.Usually, should heat being enough to remove under the temperature of liquid-carrier from solution, but enough low so that the microelectrode of acquisition can not be damaged.In a kind of preferred implementation, under about 75 ℃, heat.In addition, should apply heat is enough to fully remove whole liquid-carriers from solution time.In a kind of preferred implementation, apply heat at least about 1 hour.In another preferred implementation, under about 75 ℃, applied heat about 1 hour.With reference to Fig. 4 (c), because the evaporation of carrier, solidification process produces the curing microchannel (36) with respect to the original size little about 20% of mold microchannel.

[0096] according in the method for the present invention, microelectrode is handled so that enzyme and enzyme immobilization material are imparted to wherein to form biological anode.In some execution modes, the enzyme immobilization material that will contain enzyme is administered to the microelectrode of curing.In order to form biological anode, after solidifying microelectrode, remove mold from substrate.Referring to Fig. 4 (c).With reference to Fig. 4 (d), in the position of mold, the mould that will have the gas-permeable of the microchannel that width is about twice of mold microchannel (34) reversibly is sealed on the microelectrode.The mould of this gas-permeable can be by nonconducting, can not the passivation electronic conductor and any material of helping the evaporation of solvent make.Preferably, silicon polymer is as PDMS, as the mold materials of this gas-permeable.More preferably, thermoplastic resin as Merlon, is the mold materials of this gas-permeable.After the mould of gas-permeable was in place, the enzyme immobilization material that will contain biological anode enzyme was administered to the microelectrode of curing.This point is finished to outlet (35) by the cast-solution syringe pump being delivered in the inlet container (33) and by the mould of gas-permeable.For the biological anode of finishing, referring to Fig. 4 (d).

[0097] in all execution modes, the concrete composition and the enzyme of enzyme immobilization material were described in detail among I.B.2～I.B.3 in the above.The preferred enzyme immobilization material of biological anode is the perfluorinated sulfonic acid-PTFE copolymer of tetra-allkylammonium modification or the polysaccharide of hydrophobically modified, especially, and the chitosan of hydrophobically modified.The preferred enzyme in anode place is the PQQ-dependent dehydrogenase.In addition, mold can comprise microchannel more than one in all execution modes.

2. biological-cathode is made

[0098] in order to form according to biological-cathode of the present invention, the identical general operation that can adopt the biological anode of making to take is made biological-cathode.The execution mode of handling biological-cathode with enzyme immobilization material and enzyme is with to be used for those of biological anode identical.The concrete composition and the enzyme of enzyme immobilization material were described in detail among I.A.2～I.A.3 in the above.The preferred enzyme immobilization material of biological-cathode is the perfluorinated sulfonic acid-PTFE copolymer of tetra-allkylammonium modification or the polysaccharide of hydrophobically modified, especially, and the chitosan of hydrophobically modified.For biological-cathode, preferred enzyme is a bilirubin oxidase in addition.

3. form exercisable biological fuel cell

[0099] after foundation the present invention has formed biological anode and biological-cathode, randomly removes the mould of casting or gas-permeable.In this optional execution mode, biological anode and biological-cathode are retained on the substrate.After the mould of removing casting or gas-permeable, the microfluid passage form is alignd on biological anode and biological-cathode.The little pattern of this form is formed, form the microfluid passage that the fuel fluid of at least one biological fuel cell can therefrom flow thus.This form can be by nonconducting, can not passivation conducting material and any material that will be adhered to substrate make.Preferably, this form is PDMS.More preferably, this cover layer is a Merlon.Little pattern of the microfluid passage of this form can form by using any known flexible offset printing art technology.In a kind of execution mode, about 2～4 times of this microfluid passage than microelectrode.In another execution mode, this microfluid passage and microelectrode are approximate same size.The microfluid passage of this form defines wherein basically, and fuel fluid will form the electrochemical cell at interface with microelectrode.When only using a microfluid passage to hold biological anode, biological-cathode, fuel fluid and oxidant, the mixture of fuel fluid and oxidant can not damage the function of microelectrode of the present invention in the identical microfluidic chamber, because their redox reaction is optionally.In other words, biological anode will be only and the fuel fluid reaction, and biological anode will be only and oxidant reaction, and cross reaction does not take place.

[0100] in the execution mode of Ti Huaning, the mould of casting or gas-permeable keeps contacting and playing with substrate the effect of the microfluid passage of definition biological fuel cell, as above-mentioned microfluid passage form.In this execution mode, fuel fluid is by the microchannel of mould and the spatial movement between biological anode or the biological-cathode.In this execution mode, the processing that must carry out subsequently is connected to form before each biological anode and biological-cathode microfluid passage.In order to form connection, in molding, form the passage that connects each microfluidic chamber by any suitable way, as apply normal force to the top of mould or from mould, remove enough materials.Then, by will seal this connection with restrain operating period fuel fluid or the material of the seepage of oxidant cover this passage.This material must be able to be connected to mold materials to form suitable sealing.In a kind of execution mode, this cladding material only is a flat mold materials, as PDMS or Merlon.

4. Ren Xuan formation execution mode

[0101] the microelectrode manufacturing technology of describing among the III.B.1. above refers to the execution mode that wherein forms biological anode and biological-cathode successively, by means of the microchannel biological anode is connected with biological-cathode to form the method for biological fuel cell subsequently.In the execution mode of replacing, can form this biology anode and biological-cathode simultaneously.In this execution mode, single mold is formed pattern to form biological anode and biological-cathode simultaneously.Replaceability ground can use the combination of mold to form single biological anode and biological-cathode.No matter in the sort of situation, form at the same time after biological anode and the biological-cathode, by or use the microfluid passage form or the modification mold forms exercisable biological fuel cell, as describing in detail among the top III.B.3.

[0102] execution mode of describing among the III.A. above has been described the formation of electric connector on substrate before other procedure of processing.In the execution mode of replacing, the procedure of processing as last is added to electric connector in the microfluidic biofuel cell.At this moment, in the mold of microfluid passage form or modification, produce the part exposure of hole with each biological anode and biological-cathode.Then, with the expose portion of electric connector physical connection to each biological anode and biological-cathode.In this execution mode, this electric connector can be to make external electrical load keep any material of any structure be electrically connected with biological anode and biological-cathode.In a kind of preferred implementation, electric connector is the cylindrical, copper body.In addition, can adopt any interconnection technique that electrically contacts that can keep between electric connector and biological anode and the biological-cathode.In a kind of preferred implementation, can use silver-colored epoxy creme incoming call to connect electric connector and biological anode and biological-cathode.This execution mode has the advantage that increases the conductivity between these assemblies.

[0103] above-mentioned execution mode has been described wherein biological anode and biological-cathode the two has been contained in biological fuel cell in the microchannel of biological fuel cell.Though this is preferred embodiment, replacement execution mode of the present invention comprises the male or female of the outside, microchannel that is positioned at biological fuel cell.At this moment, by with the biological anode of microfluid or biological-cathode and suitable external anode or the incompatible formation fuel cell of cathode sets.

C. the purposes of microfluidic biofuel cell

[0104] after the making of finishing exercisable microfluidic biofuel cell of the present invention, can use it for multiple application, wherein can obtain fluid fuel source and oxidant for biological anode and biological-cathode respectively.During use, fuel fluid flows to contact biological anode and biological-cathode by microfluid passage with oxidant.At this, the redox reaction described in the I. is to produce current source above taking place.Microfluidic biofuel cell of the present invention need can be used to any application of supply of electric power, as electronic equipment, commercial toy, inner Medical Devices and electrically driven vehicles.In addition, microfluidic biofuel cell of the present invention can be transplanted in the live body, wherein fuel fluid obtains from organism and electric current is used for providing power to the equipment of implanting live body.

[0105] in addition, a plurality of microfluidic biofuel cells of the present invention can be connected to form the biological fuel cell heap with series circuit.Referring to Fig. 5, be electrically connected with the biological-cathode (40) of another biological fuel cell by biological anode (41) a biological fuel cell, it is connected with another biological anode (41) again up to the heap that obtains expectation, forms series stack.Fuel fluid and/or oxidant flow into the microfluidic chamber in the inlet container (33).By forming heap, total output voltage of microfluidic biofuel cell circuit is the output voltage sum in each microfluidic biofuel cell of series connection in theory.The bigger whole output voltage of this heap is applicable to that the electronic equipment, toy, Medical Devices and the vehicle that are higher than the power that single microfluidic biofuel cell can provide to power requirement provide electric power.

IV. electricity-generating method

[0106] the present invention includes a kind of electricity-generating method, it comprises makes fuel fluid in anodic oxidation with make oxidant in cathodic reduction, wherein adopts to comprise that the biological fuel cell of aforesaid biological anode and/or biological-cathode generates electricity.

Definition

[0107] term used herein " hydrocarbon " and " alkyl " have been described compound or the group only be made up of elemental carbon and hydrogen.These parts comprise alkyl, alkenyl, alkynyl and aryl moiety.These parts also comprise alkyl, alkenyl, alkynyl and the aryl moiety that is replaced by other aliphat or cyclic hydrocarbon group, as alkaryl, and alkene aryl and alkynes aryl.Unless point out on the contrary, otherwise these parts preferably include 1～20 carbon atom.

[0108] part of describing herein " alkyl of replacement " is by the hydrocarbyl portion of the atom of at least one non-carbon replacement, comprises that wherein the carbochain atom is by the part of hetero-atom such as nitrogen, oxygen, silicon, phosphorus, boron, sulphur or halogen atom replacement.These substituting groups comprise hydroxyl, ketone, acyl group, acyloxy, nitro, amino, amide groups, nitro, cyano group, mercaptan, ketal, acetal, ester and the ether of halogen, heterocycle, alkoxyl, alkenyloxy, alkynyloxy group, aryloxy group, hydroxyl, protection.

[0109] unless points out that on the contrary otherwise described alkyl is preferably the low alkyl group that contains 1～8 carbon atom and maximum 20 carbon atoms in the main chain herein.They can be straight chain or branched chain or ring-type, and comprise methyl, ethyl, propyl group, isopropyl, butyl, hexyl etc.

[0110] unless points out that on the contrary otherwise described alkenyl is preferably the low-grade alkenyl that contains 2～8 carbon atoms and maximum 20 carbon atoms in the main chain herein.They can be straight chain or branched chain or ring-type, and comprise vinyl, acrylic, isopropenyl, cyclobutenyl, isobutenyl, hexenyl etc.

[0111] unless points out that on the contrary otherwise described alkynyl is preferably the low-grade alkynyl that contains 2～8 carbon atoms and maximum 20 carbon atoms in the main chain herein.They can be straight chain or branched chain or ring-type, and comprise acetenyl, propinyl, butynyl, isobutyl alkynyl, hexin base etc.

[0112] chooses the isocyclic aryl that replaces wantonly individually or as term " aryl " or " virtue " expression that the part of another group is used herein, preferred monocycle or bicyclic radicals, in loop section, contain 6～12 carbon atoms, 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.

[0113] represents chlorine, bromine, fluorine and iodine individually or as term " halogen " or " halogen " that the part of another group is used herein.

[0114] from the group-COOH of organic carboxyl acid, remove the part that hydroxyl forms individually or as term " acyl group " expression that the part of another group is used herein, for example RC (O)-, wherein R is R ₁

[0115] R ₁O-, R ₁R ₂N-or R ₁S-, R ₁Be alkyl, assorted alkyl or the heterocycle that replaces, and R ₂Alkyl for hydrogen, alkyl or replacement.

[0116] herein individually or term " acyloxy " expression of using as the part of another group by oxygen connect (--O--) the aforesaid acyl group of bonding, RC (O) O-for example, wherein R as with the relevant definition of term " acyl group ".

[0117] to answer implication be atom outside de-carbon and the hydrogen to term " hetero-atom ".Herein individually or group optional that replace, fully saturated or undersaturated, monocycle of the term " heterocycle " that uses as the part of another group or " heterocycle " expression or dicyclo, aromatics or non-aromatics, have at least one hetero-atom in its at least one ring, and 5 or 6 atoms during preferably each encircles.During preferably encircling, heterocyclic group has 1 or 2 oxygen atom, 1 or 2 sulphur atom, and/or 1～4 nitrogen-atoms, and can be by carbon or hetero-atom bonding in the remainder of molecule.Exemplary heterocycle comprises heteroaryl such as furyl, thienyl, pyridine radicals, oxazolyl, pyrrole radicals, indyl, quinolyl or isoquinolyl etc.Exemplary substituting group comprises one or more following groups: the hydroxyl of the alkyl of alkyl, replacement, ketone, hydroxyl, protection, acyl group, acyloxy, alkoxyl, alkenyloxy, alkynyloxy group, aryloxy group, halogen, amide groups, amino, nitro, cyano group, mercaptan, ketal, acetal, ester and ether.

The following example has been set forth the present invention.

Embodiment

Embodiment 1: use the direct electron of bilirubin oxidase to shift on different carbon surfaces

[0118] carbon paste electrode modification: adopt new carbon paste electrode of filling to carry out each experiment.After filling carbon paste, adopt a kind of material with carbon element to come 4 electrodes of modification: the carbon nano-tube of carbon black, carbon worm, diameter 20nm and long 5-20 micron and Pt (on Vulcan XC-72).Use unmodified carbon paste electrode in contrast.Modified electrode is immersed in 4 ℃ of bilirubin oxidase solution in the following pH 7.15pH cushioning liquid 15 minutes.The bilirubin oxidase solution that will contain the 1.0mg bilirubin oxidase is dissolved in the 10mL 0.1M pH7.15 phosphate buffer.In case electrode reaches balance in enzyme solutions, they were placed vacuum desiccator dry about 15 minutes.In case dry, electrode is carried out the volt-ampere test in the contrast solution of degassing pH 7.15 PBSs.Modification carbon paste electrode is connected with the SCE reference electrode electrode with platinum guaze then as work electrode.With the sweep speed of 0.01V/s with each electrode from 0.8V～-0.1V scanning.Each modification carbon paste electrode after the test, with the testing liquid oxidation and adopt previous described identical parameters to scan each electrode, is determined whether to have taken place the direct electron transfer thus in degassing phosphate buffer.

[0119] result of these researchs provides in following table.These data show, adopt the electrode modification of carbon nano-tube to obtain maximum flux raising.

[0120] table. the flux that is adsorbed on the bilirubin oxide on the modification carbon paste electrode in the buffer solution improves.

The electrode modification agent	Flux improves
The electrode modification agent	Flux improves	Carbon black	4.88±1.88
Expansion carbon worm	1.81±0.67	Carbon black	4.88±1.88
Expansion carbon worm	1.81±0.67	Pt on the Vulcan XC-72	4.03±0.58
Carbon nano-tube	14.12±3.17	Pt on the Vulcan XC-72	4.03±0.58
Carbon nano-tube	14.12±3.17	Carbon paste	2.46±0.82

[0121] three kinds of independent methods of employing prepare the TBAB modification

Carbon nano tube compound material.First method (method 1) comprises uses 0.1 micrometer alumina that nature of glass carbon electrode is polished on Buehler cloth, washes to guarantee not having previous electrode dirt with the first alcohol and water subsequently.Use 0.05g nanotube and the bilirubin oxidase solution combination that makes as previously mentioned, preparation carbon nano-tube slurry.Make this creme drying form moist creme up to it.Subsequently this carbon paste is placed vacuum desiccator and makes its finish-drying.Secondly, with the TBAB modification in dry creme of 2.0mg and the 100 microlitre solution

Polymer mixed.With suspension in Scientific Industries Vortex Genie 2 eddy current to guarantee abundant mixing.This suspension of 20 microlitres is moved on the end of polished glass matter carbon electrode (GCE) with pipette.Modified electrode is carried out spin coating with the speed that does not wait from 10～50rpm, with being placed in the vacuum desiccator and making its dry about 15 minutes.In identical as previously mentioned mode to having the suspension TBAB modification of enzyme in the suspension/nanotube creme

Test, in pH 7.15 cushioning liquid of the degassing, use the conduct of SCE reference electrode and platinum guaze electrode.Adopt cyclic voltammetry with the sweep speed of 0.01V/s with each electrode from 0.8V～-0.1V scanning.After the control experiment, with the oxidation at least 15 minutes before carrying out sample experiment of pH 7.15 PBSs.Make the total data connection in these experiments and adopt the CH Instruments pressurizer that is connected in PC to carry out record.

[0122] second method (method 2) comprises polished glass matter carbon electrode as described above.Be suspended in nanotube in the described bilirubin oxidase solution that makes of 1.0mL by containing 0.05g, make nanotube and bilirubin oxidase creme.With enzyme/nanotube suspension in Scientific IndustriesVortex Genie 2 eddy current to guarantee abundant mixing.This suspension of 20 microlitres is moved on the end of polished glass matter carbon electrode with pipette.With electrode in vacuum desiccator dry about 15 minutes.After the drying, with 5 microlitre TBAB modifications Spin coating on the electrode of nanotube/bilirubin oxidase creme modification.Subsequently with the electrode of spin coating in vacuum desiccator dry about 15 minutes.In described identical mode to adopting the TBAB modification

The electrode of the enzyme of spin coating/nanotube creme is tested.

[0123] the third method (method 3) also is to adopt polished glass matter carbon electrode to begin.Subsequently, be suspended in nanotube in 1.0mLpH 7.15 phosphate buffers, make the nanotube creme by containing 0.05g.With nanotube suspension in Scientific Industries Vortex Genie 2 eddy current to guarantee abundant mixing.This nanotube suspension of 20 microlitres is moved on the end of the electrode on the drying nano pipe creme with pipette, make its early in the vacuum desiccator dry about 15 minutes subsequently.The bilirubin oxidase solution that 20 microlitres are made as mentioned above moves on the end of the electrode on the drying nano pipe creme with pipette.In case dry, with these electrodes with 5 microlitre TBAB modifications

Polymer is with the 50rpm spin coating.Make electrode in vacuum desiccator dry about 15 minutes.In identical as previously mentioned mode to adopting the TBAB modification

The electrode of the nanotube creme/enzyme solutions spin coating that applies is tested.

[0124] these result of experiment are tabulated below.These data show that method 1 is for forming carbon nano-tube/enzyme/TBAB modification

The method for optimizing of composite material is because it provides the remarkable flux with littler variation to improve with respect to method 2.

[0125] table 2. is for forming carbon nano-tube/enzyme/TBAB modification

The contrast that the flux of the distinct methods of composite material improves

Embodiment 2: the bilirubin oxidase negative electrode in the biological fuel cell

[0126] anode and the negative electrode in employing biocatalyst (enzyme) the preparation biological fuel cell.The TBuA modification

NAD ⁺The biological anode of-dependence alcohol dehydrogenase is used for these experiments.The biological anode of development is by 1cm ²Carbon cloth is formed.(from Myrotheciumverrucaria, units activity=10 .mg of unit Sigma) is added in 100 microlitre DE, the 520 Nafion film suspension and eddy current 20 minutes with the 0.5mg bilirubin oxidase.Move to carbon electrode on pipette 2 microlitres enzyme/film cast-solution and make its dry 12 hours.Under the room temperature of from 20 to 25 ℃ of variations, carry out all electrochemistry experiments.Electrode is introduced with in oxygen-saturated pH 7.15,7.5 of dissolving and 8.0 phosphate buffers.Measure being connected on the CH Instruments pressurizer model 900 of PC computer.By 0.09672g TBAB (TBAB) is prepared DE 520 in 1mL DE 520 Nafion

Film suspension.Subsequently this mixture solution is cast in the weighing ship and make its dried overnight.In case after the drying, mixture-cast membrane is immersed in the 18M Ω water 24 hours to remove all excessive bromide salt.Remove desalt after, with film with 18M Ω water cleaning down three times and make its drying.This film is suspended in the 1mL ethanol once more.

[0127] used two types electrochemical cell.Test conventional fuel cell in the U-shaped glass guide channel, wherein anode and cathode chamber pass through

117PEM film (Alfa Aesar) separately.For second type of fuel cell (no membrane cell), biological anode and biological-cathode are incorporated in the 50mL beaker that contains fuel solution.This fuel solution is by 1.0mM ethanol in the phosphate buffer of pH 7.15,7.5 and 8.0 and 1.0mM NAD ⁺Form.Make this solution in air, reach the oxygen of balance to dissolve in the buffer solution before guaranteeing to test.Positioning of electrode is separated about 1cm can not contact with each other to guarantee them.

[0128] test conventional fuel cell in the U-shaped glass guide channel, wherein anode and cathode chamber pass through

117PEM film (Alfa Aesar) separately.Anolyte is the 1.0mM fuel solution in the phosphate buffer of different pH, and catholyte is the cushioning liquid with different pH that is exposed to air simultaneously.Experimental session, unique oxygen source are the oxygen that dissolves in the buffer solution.With the NAD that finishes ⁺The biological anode of-dependence is introduced anode fuel cell chamber separately, and it is connected in its oneself the cathode chamber that contains the bilirubin oxidase biological-cathode.

[0129] by biological-cathode and biological anode are placed the 1.0mM NAD that contains at pH 8.0 cushioning liquid that are exposed to air ⁺In the beaker of 1.0mM ethanol, form the ethanol/oxygen biological fuel cell of no film.The normal duration of test of Yan Zhi biological anode formerly is not with NAD ⁺Be added in the cushioning liquid, because NAD ⁺Static is fixed in biological anode.But, with NAD ⁺Be added to any NAD of solution that is used for testing this system to guarantee from biological anode, to leach ⁺Can not influence biological-cathode reaction and biological-cathode life-span.The initial open circuit voltage of no film biological fuel cell is that 1.20V and maximum power density are 0.64mW/cm ²Can point out that the two is higher for no film system for open circuit voltage and power density.For the biological fuel cell of the bilirubin oxidase oxygen-free reduction amboceptor wherein biological fuel cell with respect to the bilirubin oxidase that contains redox mediators, open circuit voltage increases to 0.30V and power density increases to 0.25mW/cm ²

[0130] table 3 contrasted at room temperature with different pH of buffer under the data that obtain of different biological fuel cells.As can be seen, adopt the traditional biological fuel cell of the pH value of solution with increase, open circuit voltage, current density and power density also increase.The maximum open circuit voltage that obtains for 8.0 times at pH is that 1.16V and current density are 7.65mA/cm ²With power density be 0.45mW/cm ²For no film biological fuel cell, under identical condition of work, the maximum open circuit voltage that obtains for 8.0 times at pH is that 1.10V and current density are 11.7mA/cm ²With power density be 0.64mW/cm ²Obtain more high open circuit voltage 1.20V for 7.15 times at pH, but be lower than value under the pH 8 in electric current and power density under this pH.Can sum up for no film biological fuel cell, the increase of fuel solution pH causes the small increase of open circuit voltage and the increase of electric current and power density.

Table 3

The 1.0mM NAD in pH 8.0 phosphate buffers at room temperature ⁺In the solution, collect other experimental data of not having the film biological fuel cell as mentioned above.Fig. 6 has shown the schematic power curve of this system.Fig. 7 has shown the variation of the power output of this system with the time in making.Fig. 8 has shown that power output is with variation of temperature under 50% humidity.Following table is described maximum open circuit voltage, maximum current density and the maximum power density under the different ethanol concentration in detail.

Concentration of alcohol (mM)	Maximum open circuit voltage (V)	Maximum current density (mA/cm ²)	Maximum power density (mW/cm ²)
Concentration of alcohol (mM)	Maximum open circuit voltage (V)	Maximum current density (mA/cm ²)	Maximum power density (mW/cm ²)	1	1.10	11.70	0.67
10	1.03	10.78	0.31	1	1.10	11.70	0.67
10	1.03	10.78	0.31	100	1.05	11.60	0.20
500	1.08	6.53	0.17	100	1.05	11.60	0.20
500	1.08	6.53	0.17	1000	0.99	6.78	0.08

Embodiment 3: the preparation of the biological anode of lipoxygenase

[0131] prepares as described above that various ammonium salts handle

The suspension of enzyme immobilization material.The stock solution of preparation lipoxygenase.Lipoxygenase and modification with equivalent Suspension mixes and this solution is moved to 1cm with pipette ²On the surface of carbon paper carrier and finish-drying.

[0132] utilization has Nafion ^TM117 films are separated the U-shaped glass guide channel of anode and cathode chamber.The cathode side of fuel cell is filled buffer solution (pH～7.15) and platinum cathode partly is suspended in the solution.The anode-side of fuel cell is filled in the fuel solution after the sonicated that contains 10 microlitre soybean oils in the 100mL buffer solution.Anode is suspended in the solution fully.

With TBAB (TBAB), triethyl group hexyl ammonium bromide (TEHA), trimethyl hexyl ammonium bromide (TMHA), trimethyl octyl ammonium bromide (TMOA), trimethyl dcyl ammonium chloride (TMDA), trimethyldodecane base ammonium bromide (TMDDA) or trimethyl Tetra-n-decylammonium bromide (TMTDA) modification.Following table is described in detail and is contained modification

The result of the various biological anodes of film and lipoxygenase.

	TBAB	TEHA	TMHA	TMOA	TMDA	TMDDA	TMTDA
	TBAB	TEHA	TMHA	TMOA	TMDA	TMDDA	TMTDA	Best open circuit voltage (V)	0.96	0.91	0.90	0.91	0.91	0.97	0.96
Maximum current (mA/cm ²)	7.67	9.18	9.53	8.23	9.23	10.6	8.83	Best open circuit voltage (V)	0.96	0.91	0.90	0.91	0.91	0.97	0.96
Maximum current (mA/cm ²)	7.67	9.18	9.53	8.23	9.23	10.6	8.83	Maximum power (mW/cm ²)	3.78	3.85	3.89	3.54	3.95	4.39	4.14
MaLS	More than 1 year	More than 1 year	More than 1 year	More than 1 year	More than 1 year	More than 1 year	More than 1 year	Maximum power (mW/cm ²)	3.78	3.85	3.89	3.54	3.95	4.39	4.14

Embodiment 4: the preparation of alkyl-modified chitosan

[0133] by stirring fast middle molecular weight chitosan (obtaining from Aldrich) (0.500g) is dissolved in 15mL 1% acetate.Obtain the gelatinous solution of thickness like this and add 15mL methyl alcohol subsequently.Make the chitosan gel stir about 15 minutes, and subsequently 20mL aldehyde (butyraldehyde, hexanal, octanal or capraldehyde) was added in the chitosan gel, add the 1.25g sodium cyanoborohydride subsequently.With the gel continuous stirring up to the suspension cool to room temperature.With the product that obtains by isolated by vacuum filtration and with the methanol wash of 150mL increment three times.Subsequently with the modification chitosan in vacuum drying oven 40 ℃ dry 2 hours down, obtain the sheet white solid.In 50% acetate, chloroform and tert-pentyl alcohol, form 1 weight % suspension of every kind of polymer.

Embodiment 5: the fluorescence imaging of hydrophobically modified chitosan

[0134] every kind of polymer suspension with 2 microlitres casts on the micro-slide of glass (Fisher) and drying in drier.0.01mM Ru (bpy) with 20 microlitre volumes ₃ ²⁺Or 0.01mM FITC moves on the polymer of casting and makes its submergence 2 minutes with pipette.After the submergence, slide is washed and makes its drying in drier with 18M Ω water.(Melville NY) makes the polymer imaging to utilize Olympus BX60M surface fluorescence (epifluorescence) microscope.Has observation polymer under the 40X overlength focal length camera lens of video camera (SonySSC-DC50A).Adopt mercury lamp to realize fluorescence excitation.(Indianapolis IN) collects image for Integral Technologies, Inc., and utilizes SPOT software (Diagnostic Instruments, Inc.) analysis image on Dell PC to use the frame grabber card.Carry out at Ru (bpy) ₃ ²⁺With the fluorescence imaging of each hydrophobically modified polyelectrolyte in the fluorescein, to determine the form influence of hydrophobically modified.Fluorescence studies show that formation accumulation body and form change with alkyl chain length in the hydrophobically modified chitosan.The chitosan of butyl modification show have little, fibrous interconnected, but the chitosan of hexyl modification has the big territory that contains littler micella territory.Along with alkyl chain length increases, the number in micella territory reduces, but the size in territory increases.The fluorescence micrograph of unmodified chitosan does not show distinct territory, does not observe micellar structure for unmodified chitosan thus.

Embodiment 6: the electrochemical measurement of hydrophobically modified chitosan

[0135] (diameter 3mm CHInstruments) is polishing on the Buehler polishing cloth and is washing in 18M Ω water with nature of glass carbon work electrode to use 0.05 micrometer alumina.Be cast to every kind of polymer suspension of 2 microlitres on the nature of glass carbon electrodes and make its in the vacuum desiccator drying up to use.Adopt cyclic voltammetry to measure at the flux of electrode surface redox material by polymer film.Make work electrode reach balance as supporting in electrolytical, as the to contain 0.1M sodium sulphate 1.0mM redox substance solution, the platinum guaze that also has is together measured to electrode and with respect to saturated calomel reference electrode.The redox material of research is caffeine, the potassium ferricyanide and Ru (bpy) ₃ ²⁺Collection and analysis data on the Dell computer that is connected in CH Instruments pressurizer model 810.Carry out cyclic voltammetric with the sweep speed of scope from 0.05V/s to 0.20V/s.All experiments repeat three times and the error reported corresponding to a standard deviation.

[0136] having carried out two kinds of hydrophobically modified polyelectrolyte studies with the cyclic voltammetric of the variation of the alkyl chain length of hydrophobically modified.All cyclic voltammetry experiments show linear i _pTo v ^1/2Figure, the chemical property of expression transmission restriction.Because the electrochemistry flux changes with concentration, as shown in equation 2, KD ^1/2Value is reported as the concentration independent solution of comparison flux (flux) in this article

Flux = \frac{i}{nFA} = \frac{2.69 x 10^{5} n^{3 / 2} AC {* v}^{1 / 2} {KD}^{1 / 2}}{nFA}

Equation 2

Wherein, i is a peak current, the electron number of n for shifting, and F is a Faraday constant, A is the area of electrode, C ^*Be the concentration of redox material, v is a sweep speed, and K is extraction coefficient (extrationcoeifficient), and D is a diffusion coefficient.The degree of swelling of polymer during solvent has been determined to cast again.Most of literature research about chitosan and chitosan derivative adopt acetate as the solvent that suspends again, still, and importantly from KD ^1/2Point out in the value that chloroform provides higher average flux.Unmodified chitosan only is dissolved in acetic acid solution.The KD of unmodified chitosan in the caffeine ^1/2 values are 5.52 (± 0.14) * 10 ^-3Obviously, the hydrophobically modified of chitosan can reduce the flux of caffeine, improves but can not obtain tangible flux.

[0137] on the other hand, the little variation of pore structure/size aspect can appreciable impact big, hydrophobicity ion such as Ru (bpy) ₃ ²⁺Transmission.Ru (bpy) by unmodified chitosan transmission ₃ ²⁺KD ^1/2 values are 2.17 (± 0.33) * 10 ^-4Obviously, for the octyl group modification chitosan films in being suspended in tert-pentyl alcohol, the hydrophobically modified of chitosan has improved Ru (bpy) in all scenario ₃ ²⁺Nearly 11.1 times of transmission.

Embodiment 7: the preparation of electrode

[0138] is suspended in the solution of 2 weight % hydrophobically modified chitosan polymer in the tert-pentyl alcohol and adds glucose oxidase solution.This solution is moved on the electrode material with pipette.This electrode material is generally carbon cloth or other material with carbon element.

Embodiment 8: be used for the glucose oxidase activity test of hydrophobically modified chitosan

[0139] glucose oxidase (GOx) catalysis β-D-glucose discharges hydrogen peroxide simultaneously to the oxidation reaction of D-glucopyrone.This is high specific and do not act on alpha-D-glucose for β-D-glucose.When having peroxidase, hydrogen peroxide enters into second reaction of this test, and it comprises p-hydroxybenzoic acid and 4-amino-antipyrine, quantitatively forms quinone imines (quinoneimine) dye complexes simultaneously, and it is measured under 510nm.In every kind of hydrophobically modified Nafion and chitosan films, measure the activity of GOx enzyme.After being fixed on the GOx enzyme within the hydrophobically modified chitosan films and casting in it in plastic bottle, absorb with respect to water gaging at 510nm.All experiments repeat three times and the error reported corresponding to a standard deviation.

[0140] as mentioned above with table 2 in tabulation show like that, in being suspended in the hexyl modification chitosan of tert-pentyl alcohol, observe the enzymatic activity high of glucose oxidase.These fixedly film show that the GOx enzymatic activity increases by 2.53 times with respect to the enzyme in the buffer solution.

Embodiment 9: chitosan-butyl biological-cathode

[0141] bilirubin oxidase.By with 0.01g hydrophobically modified chitosan (butyl, hexyl, octyl group or decyl) and 1mL

DE 520 is mixed that chitosan mixes and adopts and mixes the pearl eddy current and made the chitosan mixture in 1 hour.Subsequently with chitosan

40 mul aliquots samples of mixture mixed 1 minute with 20 mul aliquots samples of bilirubin oxidase (1mg enzyme in 10mL pH 7.15 phosphate buffers).Chitosan/enzyme mixing is moved to 1cm with pipette ²On the carbon paper sheet to make negative electrode and to make its finish-drying in vacuum desiccator.For with (1) TBA modification

NAD ⁺-dependence alcohol dehydrogenase anode (Fig. 9) or (2) butyl chitosan NAD ⁺The butyl chitosan bilirubin oxidase negative electrode of-dependence alcohol dehydrogenase anode (Figure 10) combination is collected power curve data.

[0142] in addition, carried out determining the research of the optimum temperature that various biological fuel cells are operated.Under different temperatures, measure about (1) TBA modification NAD ⁺-dependence alcohol dehydrogenase anode and butyl chitosan bilirubin oxidase negative electrode, (2) butyl chitosan NAD ⁺-dependence alcohol dehydrogenase anode and TBA modification

Bilirubin oxidase negative electrode and (3) butyl chitosan NAD ⁺Maximum open circuit voltage (V), the maximum current density (mA/cm of-dependence alcohol dehydrogenase anode and butyl chitosan bilirubin oxidase negative electrode ²) and maximum power density (mW/cm ²).These temperature datas have been provided in the following table.

Table. the biological anode of mediation (comprises the TBA modification

And NAD ⁺-dependence alcohol dehydrogenase) and direct electron shift biological-cathode (comprising butyl chitosan and bilirubin oxidase)

Table. the biological anode of mediation (comprises butyl chitosan and NAD ⁺-dependence alcohol dehydrogenase) and direct electron shift biological-cathode and (comprise the TBA modification

And bilirubin oxidase)

Table. the biological anode of mediation (comprises butyl chitosan and NAD ⁺-dependence alcohol dehydrogenase) and direct electron shift biological-cathode (comprising butyl chitosan and bilirubin oxidase)

Embodiment 10: the preparation of alkyl-modified shell alginates

[0143] by using the 3 weight % alginates suspension bromination quaternary ammonium of casting altogether, forms the alginates film that combines the bromination quaternary ammonium.The polymer that uses is extremely low, low or middle molecular weight alginates.Make mixture-cast-solution by the bromination quaternary ammonium being added in the 3 weight % suspension.Make all mixture-cast-solution, make the concentration excess in carboxylic acid site in the concentration ratio alginates suspension of bromination quaternary ammonium.After the optimization, the bromination quaternary ammonium concentration that definite the most stable and reproducible film has is three times of exchange site concentration.

[0144] 1 milliliter of cast-solution is placed the weighing ship and makes its drying.Be added to 7.0mL 18M Ω water in the weighing ship and its submergence is spent the night.Remove anhydrate and with film with 18M Ω water cleaning down and drying.Subsequently, film is resuspended in the 1.0mL methyl alcohol.Use tetrapropyl ammonium (T3A), four pentyl ammonium (T5A), tetrahexyl ammonium (T6A), four heptyl ammoniums (T7A), trimethyl eicosyl ammonium (TMICA), trimethyl octadecyl ammonium (TMODA), trimethyl cetyltrimethyl ammonium (TMHDA), trimethyl myristyl ammonium (TMTDA), trimethyl octyl group ammonium (TMOA), trimethyldodecane base ammonium (TMDDA), trimethyl decyl ammonium (TMDA), trimethyl hexyl ammonium (TMHA), the bromination ammonium salt of TBuA (TBA) produces best micellar structure as alginates modifier so that whom to understand.Micellar structure effectively fixedly is important for enzyme.

[0145], places three every kind of polymer on the slide subsequently and make its drying in order to determine pore property.After the finish-drying, they are immersed in 1mM Ru (bpy) in the ethanol ₃ ²⁺In at least 3 hours.After falling with alcohol flushing,, make the polymer drying adopting before the fluorescence microscope imaging observes micellar structure.The example that has shown structure among Figure 11.

[0146] in another experiment, very low molecular weight alginates and lauryl amine are placed 25% ethanol and backflow, make the alginates of dodecyl modification with amidatioon by hydroxy-acid group.

Embodiment 11: the preparation of alginates electrode

[0147] will adopt 3 weight % solution of the alginate polymer of hydrophobic ammonium cation modification described in the embodiment 10 to be suspended in the alcohol (alcohol), and add enzyme solutions (for example, bilirubin oxidase).This solution is moved on the electrode material with pipette.This electrode material is generally carbon cloth or other material with carbon element.

Embodiment 12: the alginates biological fuel cell

[0148] by moving on the carbon cloth with pipette with the solution mixture casting hydrophobically modified alginates of enzyme and buffer solution and with mixture, thus, form similar above those the biological anode described in the embodiment 10, make biological fuel cell with the anode enzyme that is fixed in the hydrophobically modified alginates.Can use the hydrophobically modified that comprises described in top and U.S. Patent application 10/931,147 (open) with U.S. Patent Application Publication 2,005,/00 95466

The biological-cathode of film forms the biological fuel cell with biological anode and biological-cathode.Replaceability ground by moving on the carbon cloth with pipette with the solution mixture casting hydrophobically modified alginates of enzyme and buffer solution and with mixture, thus, forms biological-cathode, makes to have the biological fuel cell that is fixed on the cathode enzyme in the hydrophobically modified alginates.Can use the hydrophobically modified that comprises described in top and U.S. Patent application 10/617,45 (open) with U.S. Patent Application Publication 2004/0101741

The biological anode of film forms the biological fuel cell with biological anode and biological-cathode.In another execution mode, can make the biological fuel cell that has the cathode enzyme that is fixed in the hydrophobically modified alginates that make as mentioned above and have the biological anode that is fixed on the anode enzyme in the hydrophobically modified alginates that make as mentioned above.

Embodiment 13: biological fuel cell

[0149] by moving on the carbon cloth with pipette with the solution mixture casting hydrophobically modified chitosan of enzyme and buffer solution and with mixture, thus, form biological anode, make biological fuel cell with the anode enzyme that is fixed in the hydrophobically modified chitosan.Can use the hydrophobically modified that comprises described in U.S. Patent application 10/931,147 (open) with U.S. Patent Application Publication 2005/0095466

The biological-cathode of film forms the biological fuel cell with biological anode and biological-cathode.Replaceability ground, by moving on the carbon cloth with pipette with the solution mixture casting hydrophobically modified chitosan of enzyme and buffer solution and with mixture, thus, form biological-cathode, make and have the biological fuel cell that is fixed on the cathode enzyme in the hydrophobically modified chitosan.Can use the hydrophobically modified that comprises described in U.S. Patent application 10/617,452 (open) with U.S. Patent Application Publication 2004/0101741

The biological anode of film forms the biological fuel cell with biological anode and biological-cathode.By moving on the carbon cloth with pipette with the solution mixture casting hydrophobically modified chitosan of enzyme and buffer solution and with mixture, thus, be formed for the biological anode of biological fuel cell, make biological anode with the anode enzyme that is fixed in the hydrophobically modified chitosan.

Embodiment 14: microfluidic biofuel cell

[0150] (Brewer Science, Rolla MO) apply 4 inches silicon chips with the negative photoresist of SU-810 and make the motherboard that is used to produce the little molded passage of PDMS, adopt the rotation program of 1000rpm to operate 30 seconds with little molded passage to pass through to adopt spin coater.For flow channel, the rotation program of employing 1750rpm 30 seconds uses the negative photoresist of SU-850.With photoresist 90 ℃ of following prebake conditions 5 minutes, adopt subsequently near-(Autoflood 1000, Optical Associates in UV floodlight source, Milpitas, CA) by containing little molded passage or flow channel design structure (Jostens, Topeka, negativity film UV exposure KS) 4 minutes.(SanFrancisco obtains slide in the computer pattern of describing in CA) for PC Version 8.0, Macromedia Inc. from Freehand.(KS) adopting respectively, the picture regulator of the little 2400dpi of rate is a slide with this pattern transformation for Jostens, Topeka by printing equipment.After this exposure, wafer was cured 5 minutes and video picture in Nano SU-8 developer after under 90 ℃.The wafer that will contain the pattern of expectation washes to remove any excessive, the unexposed photoresist that may be retained on the silicon chip with acetone and isopropyl alcohol.The employing profilograph (Alpha Step-200, Tencor Instruments, Mountain View, the CA) thickness of measurement photoresist, it is corresponding to the channel depth of PDMS structure.

[0151] 10: 1 mixtures of the degassing of Sylgard 184 elastomers and curing agent is poured on the silicon chip and at 75 ℃ solidified about 2 hours down subsequently.By from wafer, peeling off, PDMS is removed from motherboard along the edge and with PDMS.Motherboard can reuse to produce the duplicate of many PDMS passages.The PDMS flow channel that obtains is that 200mm is wide, 100mm is dark and 3.0cm long.

[0152] buys soda-lime glass (soda-lime glass) plate from local glass store.This plate is that 7cm is wide, 10cm is long and 1.54mm is thick.By glass plate being immersed in the piranha solution (70% concentrated sulfuric acid/30% hydrogen peroxide) 15 minutes, they are cleaned up to remove organic impurities.(18M Ω-cm) the water cleaning down is also used the nitrogen drying with Nanopure with glass subsequently.Adopt traditional lithographic printing and sputtering technology, at the platinum electrode of this making specific pattern on glass.Each plate can hold the several flow channels with electrode.This point is more particularly finished by the argon ion sputtering of titanium layer (being used for bond properties) and palladium layer.In order to realize this point, glass is placed in the sedimentary system (Thin FilmDeposition System, Kurt J.Lesker Co.) with plated metal.Utilize the thickness of quartz crystal deposition monitor (InficonXTM/2, Leybold Inficon) monitoring metal.With～ Speed from the Ti-target titanium deposition to

The degree of depth.With～

Speed from the Pd-target depositing Pd to

The degree of depth.AZ 1518 positive photoetching rubbers dynamically are scattered in the on glass of coating palladium.After 95 ℃ of following prebake 1 minute, by 9 seconds of positivity film uv-exposure.Remove this film and glass is placed 45 seconds of developer (AZ 300MIF developer) of commercially available acquisition.Water washes and with after the nitrogen drying, this glass was cured 1 minute after under 95 ℃.Adopt wet etching, utilized chloroazotic acid (8: 7: 1H ₂O: HCl: HNO ₃) remove unwanted palladium and utilize the titanium etchant from glass, to remove unwanted titanium.In case after finishing, this glass is also used the nitrogen drying with acetone and isopropyl alcohol flushing to remove residual photoresist.

[0153] adopts 1-mm diamond bit and Dremel throw (Dremel),, be immersed under water simultaneously by the mobile access hole of each glass twist drill.Adopt the Dremel throw and together with cutting disc remove the injection connector part of leur adapter.Adopt after the emery disc polishing, adopt J.B.Weld that the leur adapter is fixed on the glass plate.Before using epoxy resin (75 ℃) in baking oven was solidified 2 hours.Obtain and being connected of platinum electrode by copper cash and collargol.

[0154], at first the little molded channel seal of PDMS is arrived on the glass plate that contacts with the platinum lead that thoroughly cleans (adopting the leur assembling to be connected) in order to make the carbon printing ink microelectrode.At first use solvent diluent (N-160) with PDMS passage prime (prime).Remove diluent by in a container, applying vacuum.One removes diluent, just the mixture of the carbon printing ink of commercially available acquisition and solvent diluent is added in the passage and by apply vacuum (by means of water aspirator) in the opposite end to make it pass passage.Preparation printing ink/diluent mixture, the volume of the feasible diluent that adds is 0.2% (v/w) of initial ink by weight.After the carbon printing ink filling channel, place 75 ℃ in baking oven following 1 hour with printing ink/diluent solution filling and with entire chip in the container that has wherein applied vacuum.During this period of time, can from glass, remove PDMS, stay the carbon microelectrode that is connected in glass surface.By chip being placed 12 ℃ in independent baking oven following 1 hour, finish last curing/regulating step.Adopt the height of talysurf measurement carbon microelectrode and measure width by microscope.

[0155] in order further to characterize the carbon printing ink electrode, (Austin TX) with 3 electrode modes, adopts and carries out cyclic voltammetry to utilize CH Instruments 810 bistable depressors.The carbon microelectrode is a work electrode, and silver/silver chlorate is that reference electrode and platinum line are as auxiliary electrode.By (cutting out small pieces (1cm * 2cm), in a PDMS, be formed for the static cell of cyclic voltammetry experiment among the 2cm * 3cm) at the PDMS of bulk more; Subsequently this sheet PDMS being sealed in makes the whole length of electrode be exposed to solution on the carbon electrode.For flowing experiment, with the PDMS microchannel (～200mm is wide, 100mm dark and～2cm is long) be sealed on the carbon electrode, make entire electrode is sealed in inside microchannels.By using electrochemical cell fixture (CH Instruments) to make auxiliary and reference electrode is contained in the exit vessel.

[0156] pore volume that flows into that gets out in glass plate is permitted from syringe pump (pump 11, HarvardApparatus, Holliston, mobile entering MA).Syringe is filled the solution of selecting and placed syringe pump.Adopt high pressure assembling, leur adapter and Teflon PEEK pipeline, syringe is connected in the glass microchip.Flow velocity by 200 microns wide PDMS flow channels changes from 0 mul/min～15 mul/min, adopts at an end that flows into the hole its alignment.Passage directly is sealed on the electrode.At the other end of passage, form container and be placed in one with negative electrode or reference with to electrode by punching.

[0157] the carbon printing ink electrode is generally the 2.5cm long electrode, and its width is 55 microns and highly is 87 microns.Solution as electrolytical 1mM three (2,2 '-bipyridyl) ruthenous chloride (II) hexahydrate and 0.1M sodium sulphate is used to adopt cyclic voltammetry to characterize electrode response.When flow velocity increases, reckon with that current density increases owing to reach electrode surface quickly along with flow velocity increases analyte.At first, by in 0.05M phosphate buffer (pH 7.4), applying 1.5V 3 minutes, utilize electrochemical pre-treatment to clean electrode.

[0158] to above-mentioned operation slight modification, to simplify the process that forms the electrode that comprises electronic conductor and enzyme immobilization material.For this reason, with the electronic conductor solution modification to comprise enzyme immobilization material.By the hydrophobically modified alginate soln in the alcohol of hydrophobically modified chitosan soln in the tert-pentyl alcohol that adds 2 weight % or 3 weight %, be suspended in Ercon N160 solvent diluent and thorough eddy current, make another material.At last, this modification diluent of 1mL is added in 0.5g Ercon E-978 (I) the carbon back printing ink.The die cavity that described modification electronic conductor flow of solution is crossed form and make its curing according to the above method in the present embodiment by mold and substrate.

[0159] in order forming, to adopt the above-mentioned general step among this embodiment, wherein to finish this anode by another material that after curing and activation stage, on electronic conductor, flows according to biological anode of the present invention.By hydrophobically modified alginate soln in the alcohol of hydrophobically modified chitosan soln in the tert-pentyl alcohol of 2 weight % or 3 weight % and the enzyme solutions in the lower aliphatic alcohols are mixed, form the cast-solution of remaining anode assemblies.Subsequently with this solution thorough eddy current and pass through about 100mm microchannel pumping together with the flow velocity of about 1mL/min.Make electronic conductor and cast-solution dried overnight subsequently.

[0160], adopt lithoprinting as above to make microchip and passage motherboard described in this embodiment for biological-cathode.Can be with the carbon printing ink microelectrode that makes by little molding process with aforesaid hydrophobically modified chitosan or the further modification of hydrophobically modified alginates film mixture.

[0161] with the modification of carbon microelectrode with as biological anode.Punching is to form the bulk container and be placed on around the microelectrode and to comprise the Ag/AgCl reference electrode and as the platinum line of auxiliary electrode in PDMS.Especially, it is a static cell.

[0162] adopts the microchannel and the hydraulic pressure that reversibly are sealed on the microelectrode to flow, enzyme/hydrophobically modified chitosan mixture or enzyme/hydrophobically modified modification alginates mixture are fixed on the carbon microelectrode.The size of this flow channel makes that alignment is possible on the microelectrode, but wideer unlike electrode.In order to realize this point, with PDMS microchannel (130mm is wide, 100mm is dark and～2cm is long) be sealed in carbon electrode (～40mm is wide ,～2cm long and～the 100mm height) on, make entire electrode be sealed in inside microchannels.Prepare the enzyme of 2: 1 ratios and hydrophobically modified chitosan mixture or hydrophobically modified alginates mixture and eddy current up to abundant mixing.(Harvard Apparatus, Brookfield OH) introduces mixture in the passage by syringe with 1.0mL/min to use syringe pump.In case mixture moves to the whole length (visual monitoring) of passage, and solvent is at room temperature evaporated.This point is possible, because the PMDS permeable gas.After the evaporation fully, remove PDMS, stay the anode of coating.

[0163] in order forming, to adopt the general step of describing among this embodiment, wherein to finish this biological-cathode on electronic conductor by after curing and activation stage, another material being flowed according to biological-cathode of the present invention.

[0164] for the modification electronic conductor, with about 20 minutes of the cast-solution of bilirubin oxidase and hydrophobically modified chitosan or hydrophobically modified alginates eddy current together.Next, with this solution with the flow velocity of about 1mL/min by about 100mm microchannel pumping.Make electronic conductor and cast-solution dried overnight subsequently.

[0165] forms biological-cathode with the pattern that is similar to above-mentioned biological anode.The PDMS microchannel is sealed on the carbon printing ink microelectrode.The hydrophobically modified chitosan is mixed with cathode enzyme.Subsequently mixture is passed through the passage pumping with 1.0mL/min,, make solvent evaporation afterwards up to its end that arrives passage.Remove the PDMS flow channel then, stay electrode coated as biological-cathode.

[0166] in view of foregoing, what will find out is to have realized several purpose of the present invention and obtained other useful result.

[0167] can in said method, carry out multiple change, and not deviate from scope of the present invention, mean contain in the above-mentioned specification or accompanying drawing in all the elements of showing should be interpreted as illustrative and and the infinite meaning.

[0168] other execution mode within the scope of claim herein is to those skilled in the art in the explanation of considering invention disclosed herein be obvious when implementing.Mean this specification should think it only is exemplary with embodiment, scope and spirit wherein of the present invention are shown by the claim after the embodiment.

Claims

1, a kind of biological anode comprises:

(a) electronic conductor;

(b) at least a can with fuel fluid reaction anode enzyme with the fuel fluid that generates oxidised form, this anode endonuclease capable discharges electronics to electronic conductor; With

(c) can fix and stablize the enzyme immobilization material of this enzyme, this material fuel permeable fluid.

2, a kind of biological-cathode comprises:

(a) electronic conductor;

(b) at least a can with oxidant reaction to generate the cathode enzyme of water, this cathode enzyme can obtain electronics from electronic conductor; With

(c) can fix and stablize the enzyme immobilization material of this enzyme, the permeable oxidation agent of this material.

3, a kind of biological fuel cell that is used to generate electricity comprises:

Fuel fluid;

The biological anode of claim 1; With

The biological-cathode of claim 2.

4, a kind of biological fuel cell that is used to generate electricity comprises:

Fuel fluid;

The biological anode of claim 1; With

Negative electrode.

5, a kind of biological fuel cell that is used to generate electricity comprises:

Fuel fluid;

Anode; With

The biological-cathode of claim 2.

6, each biological anode, biological-cathode or biological fuel cell in the claim 1～5, wherein this enzyme immobilization material comprises micella or reversed micelle structure.

7, each biological anode, biological-cathode or biological fuel cell in the claim 1～6, wherein this enzyme immobilization material comprises the perfluorinated sulfonic acid-PTFE copolymer of modification.

8, each biological anode, biological-cathode or biological fuel cell in the claim 1～6, wherein this enzyme immobilization material comprises the alginates of hydrophobically modified.

9, claim 7 or 8 biological anode, biological-cathode or biological fuel cell wherein adopt greater than NH ₄ ⁺Dewatering cationic to this enzyme immobilization material modification.

10, biological anode, biological-cathode or the biological fuel cell of claim 9, wherein this dewatering cationic comprises cation, quaternary ammonium cation, alkyl trimethoxy ammonium cation, organic cation, phosphonium cation, triphenyl phosphonium, pyridylium, glyoxaline cation, cetyl pyridinium, second ingot, purpurine, methyl viologen, benzyl viologen, two (triphenylphosphine) imines, metal complex, Bipyridine metal complexes, the metal complex based on the phenanthroline, [Ru (bipyridine) based on ammonium ₃] ²⁺Or [Fe (phenanthroline) ₃] ³⁺

11, biological anode, biological-cathode or the biological fuel cell of claim 9, wherein this dewatering cationic comprises the quaternary ammonium ion shown in the formula 4:

Wherein, R 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 ₄In at least one be not hydrogen.

12, biological anode, biological-cathode or the biological fuel cell of claim 11, wherein R ₁, R ₂, R ₃And R ₄Be hydrogen, methyl, ethyl, propyl group, butyl, amyl group, hexyl, heptyl, octyl group, nonyl or decyl, wherein R independently ₁, R ₂, R ₃And R ₄In at least one be not hydrogen.

13, biological anode, biological-cathode or the biological fuel cell of claim 11, wherein R ₁, R ₂, R ₃And R ₄Identical and be methyl, ethyl, propyl group, butyl, amyl group or hexyl.

14, biological anode, biological-cathode or the biological fuel cell of claim 11, wherein R ₁, R ₂, R ₃And R ₄Be butyl.

15, biological anode, biological-cathode or the biological fuel cell of claim 11, wherein R ₁, R ₂, R ₃And R ₄In one be hexyl, octyl group, decyl, dodecyl or myristyl, and other is methyl, ethyl or propyl group independently.

16, each biological anode, biological-cathode or biological fuel cell in the claim 1～6, wherein this enzyme immobilization material is the polysaccharide of hydrophobically modified.

17, biological anode, biological-cathode or the biological fuel cell of claim 16, wherein this polysaccharide comprises chitosan, cellulose, chitin, starch, amylose, alginates and combination thereof.

18, biological anode, biological-cathode or the biological fuel cell of claim 17, the polysaccharide of wherein said micella hydrophobically modified is corresponding to formula 1

Wherein, n is an integer;

R ₁₀Be the alkyl of hydrogen, alkyl or replacement independently; With

R ₁₁Be the alkyl of hydrogen, alkyl or replacement independently.

19, biological anode, biological-cathode or the biological fuel cell of claim 18, wherein the molecular weight of the polysaccharide of this hydrophobically modified is about 90000～about 500000.

20, biological anode, biological-cathode or the biological fuel cell of claim 18, wherein the molecular weight of the polysaccharide of this hydrophobically modified is about 225000～about 275000.

21, biological anode, biological-cathode or the biological fuel cell of claim 18, wherein R ₁₀Be hydrogen or alkyl independently, and R ₁₁Be hydrogen or alkyl independently.

22, biological anode, biological-cathode or the biological fuel cell of claim 18, wherein R ₁₀Be hydrogen or hexyl independently, and R ₁₁Be hydrogen or hexyl independently.

23, biological anode, biological-cathode or the biological fuel cell of claim 18, wherein R ₁₀Be hydrogen or octyl group independently, and R ₁₁Be hydrogen or octyl group independently.

24, each biological anode, biological-cathode or biological fuel cell in the claim 1～23, wherein this electronic conductor comprises the conductor of material, metallic conductor, semiconductor, metal oxide or modification based on carbon.

25, each biological anode, biological-cathode or biological fuel cell in the claim 1～23, wherein this electronic conductor comprises the conductor based on carbon.

26, biological anode, biological-cathode or the biological fuel cell of claim 25, wherein this electronic conductor comprises purifying flake graphite, the high-performance graphite of graphite worm, the layering of the electrode, carbon black, carbon dust, carbon fiber, Single Walled Carbon Nanotube, double-walled carbon nano-tube, multi-walled carbon nano-tubes, carbon nano pipe array of carbon cloth, carbon paper, carbon filament reticulated printing, diamond coated conductor, vitreous carbon, mesoporous carbon, graphite, not compacting, pyrolytic graphite, pyrolytic graphite or the polycrystalline graphite of high rule.

27, biological anode, biological-cathode or the biological fuel cell of claim 25, wherein this electronic conductor comprises carbon nano-tube.

28, each biological anode, biological-cathode or biological fuel cell in the claim 1～27, wherein this cathode enzyme or anode enzyme comprise more than one redox center.

29, biological anode, biological-cathode or the biological fuel cell of claim 28, wherein this cathode enzyme or anode enzyme comprise bilirubin oxidase, laccase, PQQ-dependence hydrogenase, lipoxygenase, superoxide dismutase, peroxidase or its combination.

30, each biological-cathode or biological fuel cell in the claim 2,3 and 5～29, wherein this cathode enzyme comprises bilirubin oxidase or superoxide dismutase.

31, each biological anode or biological fuel cell in the claim 1,3,4 and 6～30, wherein this anode enzyme comprises PQQ-dependence hydrogenase or lipoxygenase.

32, each biological fuel cell in the claim 3～31, wherein this oxidant comprises oxygen or peroxide.

33, the biological fuel cell of claim 32, wherein this oxidant comprises oxygen.

34; each biological fuel cell in the claim 3～33, wherein this fuel fluid comprises ammonia; methyl alcohol; ethanol; propyl alcohol; isobutanol; butanols; isopropyl alcohol; allyl alcohol; aryl alcohol; glycerine; propylene glycol; sweet mellow wine; glucuronic acid; aldehyde; carbohydrate; glucose; glucose-1; D-glucose; L-glucose; the G-6-P ester; lactate; lactate-6-phosphate; the D-lactate; the L-lactate; fructose; galactolipin-1; galactolipin; aldose; sorbose; mannose; monoglyceride; coacetylase; acetyl group Co-A; malate; the isocitric acid ester; formaldehyde; acetaldehyde; acetic acid esters; citrate; the L-gluconate; beta-hydroxysteroid; alpha-hydroxysteroid; lactic aldehyde; testosterone; gluconate; aliphatic acid; lipid; the phosphoglycerol acid esters; retinene; estradiol; cyclopentanol; hexadecanol; long-chain alcohol; coniferyl-alcohol; cinnamyl-alcohol; formic acid esters; long-chain aldehyde; pyruvate; butyraldehyde; acyl group-CoA; steroids; amino acid; flavine; NADH; NADH ₂, NADPH, NADPH ₂Or hydrogen.

35, the biological fuel cell of claim 34, wherein this fuel fluid comprises methyl alcohol, ethanol or propyl alcohol.

36, the biological fuel cell of claim 34, wherein this fuel fluid comprises ethanol.

37, each biological fuel cell in the claim 3 and 6～36, wherein should the biology anode or biological-cathode not separately by salt bridge or polymer dielectric film.

38, a kind of method that adopts each biological fuel cell generating in the claim 3～37 is included in oxidized fuel fluid and reduction-oxidation agent on negative electrode or biological-cathode on anode or the biological anode.