CN102636533A - Composite electrode, sensor, biological fuel cell, preparation method and application - Google Patents
Composite electrode, sensor, biological fuel cell, preparation method and application Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 49
- 239000000446 fuel Substances 0.000 title claims abstract description 29
- 239000002131 composite material Substances 0.000 title claims abstract description 13
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- 229910052799 carbon Inorganic materials 0.000 claims abstract description 26
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 24
- 102000004316 Oxidoreductases Human genes 0.000 claims abstract description 24
- 229910021397 glassy carbon Inorganic materials 0.000 claims abstract description 23
- 238000001035 drying Methods 0.000 claims abstract description 19
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 claims abstract description 16
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 14
- 101710088194 Dehydrogenase Proteins 0.000 claims abstract description 12
- 239000002904 solvent Substances 0.000 claims abstract description 9
- 238000006356 dehydrogenation reaction Methods 0.000 claims abstract description 8
- 239000000758 substrate Substances 0.000 claims abstract description 8
- 239000002270 dispersing agent Substances 0.000 claims abstract description 4
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 41
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 40
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- 238000012360 testing method Methods 0.000 claims description 18
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- 230000003647 oxidation Effects 0.000 claims description 16
- 238000007254 oxidation reaction Methods 0.000 claims description 16
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- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 claims 1
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- 229950006238 nadide Drugs 0.000 abstract description 15
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- 239000001257 hydrogen Substances 0.000 abstract description 4
- 229910052739 hydrogen Inorganic materials 0.000 abstract description 4
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- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 5
- 239000000463 material Substances 0.000 description 5
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- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 2
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- 229910019142 PO4 Inorganic materials 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
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- 238000004506 ultrasonic cleaning Methods 0.000 description 2
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- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- WJFKNYWRSNBZNX-UHFFFAOYSA-N 10H-phenothiazine Chemical compound C1=CC=C2NC3=CC=CC=C3SC2=C1 WJFKNYWRSNBZNX-UHFFFAOYSA-N 0.000 description 1
- KXGFMDJXCMQABM-UHFFFAOYSA-N 2-methoxy-6-methylphenol Chemical compound [CH]OC1=CC=CC([CH])=C1O KXGFMDJXCMQABM-UHFFFAOYSA-N 0.000 description 1
- 241001103808 Albifimbria verrucaria Species 0.000 description 1
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- 108091006149 Electron carriers Proteins 0.000 description 1
- 241000662429 Fenerbahce Species 0.000 description 1
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- 101100412856 Mus musculus Rhod gene Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108010038006 Peptide Nanotubes Proteins 0.000 description 1
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- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical class Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
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- 229910052751 metal Inorganic materials 0.000 description 1
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- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 1
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- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
- Y02E60/30—Hydrogen technology
- Y02E60/50—Fuel cells
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Abstract
The invention discloses a composite electrode and a preparation method thereof. The preparation method includes (1) uniformly mixing carbon double-shell hollow spheres with solvent to form dispersing agent; and (2) uniformly dripping the dispersing agent on the surface of a glassy carbon electrode and drying. Application of the composite electrode in terms of preparation of a composite electrode loaded with dehydrogenase or oxidase and preparation of a sensor for detecting NADH (nicotinamide adenine diuncleotide hydrogen) is realized. The invention further discloses the sensor for detecting NADH, a preparation method of the sensor, the composite electrode loaded with dehydrogenase or oxidase, a preparation method of the composite electrode loaded with dehydrogenase or oxidase, application of the composite electrode in fields of biosensors and biological fuel cells, a biosensor, a biological fuel cell and a preparation method of the biosensor and the biological fuel cell. The biosensor can detect dehydrogenation substrates corresponding to the dehydrogenase without catalysis of media, and has the advantages of fast response, fine selectivity and high sensitivity and accuracy. The biological fuel cell can work in a physiological environment and can realize electro-catalysis without media.
Description
Technical field
The present invention relates to electrode material; Relate in particular to combination electrode; More relate to combination electrode and preparation method thereof and the application in biology sensor and biological fuel cell and the oxidasic combination electrode of load and preparation method thereof and the application in biological fuel cell of load dehydrogenasa.
Background technology
NADH (English nicotinamide adenine dinucleotide by name; Be abbreviated as NAD) be a kind of coenzyme of dehydrogenasa; Be the electron carrier in the oxidation, no matter be that it all plays the core pivotal role in respiration or photosynthesis process.The ortho states of going back of NAD is NADH, and oxidation state is NAD
+NAD
+Through various dehydrogenasas, from substrate, accept a hydrogen atom and two electronics, become the NADH that goes back ortho states.This reaction can be carried out reversiblely, to realize electron transport.NADH participates in surpassing the enzymic catalytic reaction of more than 300 kind of dehydrogenasa, and this makes it at relevant bio-electronic device, as very big development prospect is arranged in biology sensor and the biological fuel cell.Yet the very slow and higher overpotential of needs of the direct electrooxidation speed of NADH in traditional bare electrode is as being 1.1V in carbon electrode, in platinum electrode, being 1.3V, although NADH/NAD
+The thermokinetics electromotive force of electrode is merely-0.54V (contrast electrode Ag/AgCl, pH=7.0,25 ℃).Even more serious ground, NADH is oxidation meeting contaminated electrode surface under high overpotential, follows the accumulation of reaction product simultaneously, greatly reduces catalysis sensitivity, stability and the mission life of electrode.To these problems; People at first attempt modifying the eelctro-catalyst with redox active in electrode top; Such as azophenlyene, phenothiazine or the fen of monomer or polymeric form oh piperazine dyestuff, the compound that contains quinonyl functional group, metal composite, conducting salt, redox polymers and some particular compound, these materials come the electrooxidation of catalyzing N ADH as the mediator of electron transport at electrode surface.Yet still there is defective in most these mediator modified electrodes, and for example mediator is stable inadequately on electrode, can reveal from electrode, thereby reduce the sensitivity of prepared electrode, thereby limit its application.In recent years, this area researchist is devoted to design and can be used for direct electrooxidation and medialess detection NADH, and can reduce overpotential and reduce the novel electrode material of surface passivation effect.Up to the present, have only new material seldom, comprise the peptide nanotube, gather 1,2-phenylenediamine nanotube is studied with some carbon-based materials and improves the direct oxidation of NADH when hanging down superpotential.In these electrode materials, carbon-based material is more attractive.Compare with metal electrode material, the carbon-based electrode material more is prone to obtain, cheaply, have excellent biological compatibility, and can be made into multiple various structure.In most electrolytic solution, material with carbon element has better chemical inertness relatively, in the WS, can in the potential range of a broad, (1.0～1.0V, contrast electrode are saturated calomel electrode) keep higher surface activity.The material with carbon element of the different shapes of handling such as boron doped adamas, preanodize so far, such as electrode, CNT, pyrolytic graphite, carbon nano-fiber, carbon fibre and carbon cloth is used to promote the direct oxidation of NADH under lower superpotential.For these carbon-based materials, the oxidation of NADH had catalytic action at the edge carbon of carbon surface or defective and oxy radical.
Summary of the invention
Technical matters to be solved by this invention is slow in order to overcome the direct electrooxidation speed that has electrode pair NADH now; Need very high overpotential; Need eelctro-catalyst (promptly being mediator), defective such as electrode surface be prone to pollute, and sensitivity is low; Provide a kind of and be easy to get, inexpensive; Have good biocompatibility and under low superpotential, just can have combination electrode of good electrical catalytic activity and preparation method thereof, and at the combination electrode of preparation load dehydrogenasa, prepare the oxidasic combination electrode of load or be used for detecting the application of NADH sensor, provide simultaneously to be used to detect NADH sensor and preparation method thereof.The present invention also provides combination electrode of a kind of load dehydrogenasa and preparation method thereof, and based on the application in dehydrogenasa biology sensor and the biological fuel cell.This biology sensor need not mediator catalysis and can detect the pairing dehydrogenation substrate of dehydrogenasa, and has the advantage that response is quick, selectivity is good, highly sensitive and accuracy is high.The present invention also provides oxidasic combination electrode of a kind of load and preparation method thereof, its application in biological fuel cell.The oxidasic combination electrode of this load shifts oxidasic direct electron has good facilitation.Characteristic based on the combination electrode and the oxidasic combination electrode of load of load dehydrogenasa; A kind of biological fuel cell that does not have barrier film, direct biological electro catalysis type and preparation method thereof also is provided; This biological fuel cell can be worked in physiological environment, and does not need mediator to carry out electro-catalysis.
The present invention solves the problems of the technologies described above through following technical scheme.
The invention provides a kind of preparation method of combination electrode, it comprises the steps:
(1) with double-layer hollow carbon ball (carbon double-shelled hollow spheres, english abbreviation CS) and solvent, the ultrasonic dispersion liquid that gets;
(2) even the dripping of described dispersion liquid is applied to vitreous carbon (glassy carbon, english abbreviation GC) electrode surface, promptly gets combination electrode after the drying.Described combination electrode is on described glassy carbon electrode, to coat the prepared combination electrode of double-layer hollow carbon ball, sets forth for the ease of hereinafter, abbreviates the CS/GC electrode as.
Wherein, described double-layer hollow carbon ball can be by document General Synthetic Route toward Functional Hollow Spheres with Double-Shelled Structures (M.Yang, J.Ma, C.Zhang, Z.Yang; Y.Lu, Angew.Chem.Int.Ed.2005,44,6727-6730) or Phenolic Resin and Derived Carbon Hollow Spheres (M.Yang, J.Ma; S.Ding, Z.Meng, J.Liu, T.Zhao, L.Mao; Y.Shi, X.Jin, Y.Lu, Z.Yang; Macromol.Chem.Phys., 2006,207,1633-1639) prepare.
Double-layer hollow carbon ball described in the present invention prepares by following step:
(a) be sulfonation 1 hour in the 98wt% concentrated sulphuric acid being immersed in concentration through cryodesiccated polystyrene hollow ball under 40 ℃, clean through water and ethanol respectively, make the polystyrene hollow ball of sulfonation; At Mg (OH)
2Under the catalysis, mol ratio be 1: 1.3 phenol and formaldehyde 70～100 ℃ of reactions 1～2 hour down, be prepared into phenolics, mix phenolics to such an extent that contain the ethanolic solution of phenolics with ethanol;
The ethanolic solution that (b) will contain said polystyrene hollow ball mixes with the described ethanolic solution that contains phenolics, and under stirring condition, reaction is 4 hours under room temperature, is heated to 150 ℃ and further reacts 2 hours, the centrifugal phenolics composite hollow ball that gets; Wherein, the mass ratio of said polystyrene hollow ball and said phenolics is 1: 4;
(c) with described phenolics composite hollow ball at N
2Calcined 2 hours for 800 ℃ in the atmosphere, get final product.
The particle diameter of the double-layer hollow carbon ball that the present invention makes is generally 450～540nm, and the specific surface area of described double-layer hollow carbon ball is generally 194～200m
2/ g.
In the step (1), the content of the ball of double-layer hollow carbon described in the described dispersion liquid preferably is 8～10mg/mL.
In the step (1), described solvent is the conventional solvent that uses in this area, preferably is N, dinethylformamide (N, N-dimethylformamide, english abbreviation DMF) or ethanol.
In the step (2), the consumption of described dispersion liquid preferably is 4～6 μ L.
In the step (2), described glassy carbon electrode is preferably the electrode through polishing and cleaning.The model of said glassy carbon electrode is the conventional model of using in this area, and the diameter of said glassy carbon electrode preferably is 3mm.
Wherein, described polishing is undertaken by the conventional method of this area, and it is that the oxidation aluminium paste of 0.3 and 0.05 μ m carries out that particle diameter is used in described polishing successively.Described cleaning is the conventional cleaning way of this area, generally uses acetone and water difference ultrasonic cleaning 3～5 minutes.
The present invention also provides a kind of combination electrode that is made by above-mentioned preparation method.
The present invention also provides the combination electrode of a kind of described combination electrode at preparation load dehydrogenasa, the oxidasic combination electrode of preparation load or has been used for detecting the application of the sensor of NADH.
The present invention also provides a kind of sensor that is used to detect NADH, and it comprises described combination electrode.
The present invention also provides a kind of preparation method of combination electrode of load dehydrogenasa; It comprises the steps: even the dripping of dehydrogenasa covering liquid is applied on the described combination electrode; The adding glutaraldehyde solution carries out crosslinked, promptly gets the combination electrode of load dehydrogenasa after the drying; Described dehydrogenasa covering liquid comprises dehydrogenasa (dehydrogenase, english abbreviation DH), spreading agent and PBS.The combination electrode of described load dehydrogenasa is the combination electrode that on described CS/GC electrode, coats dehydrogenasa, sets forth for the ease of hereinafter, abbreviates the DH-CS/GC electrode as.
Wherein, Described dehydrogenasa is meant the enzyme of can catalytic specie sloughing hydrogen; Its utilization other chemicals except that molecular oxygen are as hydrogen acceptor; Described dehydrogenasa is the conventional dehydrogenasa that uses in this area, preferably is lactic dehydrogenase (lactate dehydrogenase, english abbreviation LDH), GDH or alcohol dehydrogenase.
Preferably, the dehydrogenasa concentration described in the said dehydrogenasa covering liquid is 20mg/mL.
Described spreading agent is the conventional spreading agent that uses in this area, preferably is bovine serum albumin.Preferably, the concentration of spreading agent is 6.7mM described in the said dehydrogenasa covering liquid.
Preferably, the consumption of said dehydrogenasa covering liquid is 8～10 μ L, and the pH value of said dehydrogenasa covering liquid is 7.0.
The consumption of described glutaraldehyde is the conventional amount used of this area, preferably is 4 * 10
-5～8 * 10
-5Mmol.
The solvent of said glutaraldehyde solution is a water, and preferably, the consumption of described glutaraldehyde solution is 1～2 μ L.
Described drying is the conventional drying mode of this area, generally under 0～25 ℃ temperature, carries out said drying.Preferably, the temperature of said drying is 0～6 ℃.
The present invention also provides a kind of combination electrode of the load dehydrogenasa that is made by above-mentioned preparation method.Preferably, described dehydrogenasa is a lactic dehydrogenase.
The combination electrode of load dehydrogenasa of the present invention has the function of molecular recognition and signal conversion simultaneously.
The present invention also provides the application of combination electrode in biology sensor or biological fuel cell of said load dehydrogenasa.
The present invention also provides a kind of biology sensor, and it comprises the combination electrode of described load dehydrogenasa.
Described biology sensor can detect the corresponding dehydrogenation substrate of described dehydrogenasa, is lactic acid like the corresponding dehydrogenation material of lactic dehydrogenase.
The present invention also provides a kind of method that is used to detect the dehydrogenation substrate, adopts described biology sensor that testing sample is detected, and in described testing sample, adds NAD
+, make the said NAD in the said sample
+Concentration greater than 40mM.
The present invention also provides the preparation method of the oxidasic combination electrode of a kind of load; It comprises the steps: even the dripping of oxidase covering liquid is applied on the described combination electrode; The adding glutaraldehyde solution carries out crosslinked, promptly gets the oxidasic combination electrode of load after the drying; Described oxidase covering liquid comprises oxidase (oxidase, english abbreviation OD), spreading agent and PBS.The oxidasic combination electrode of described load is on described CS/GC electrode, to coat oxidasic combination electrode, sets forth for the ease of hereinafter, abbreviates the OD-CS/GC electrode as.
Wherein, Described oxidase is meant directly and generates water with molecular oxygen as electron accepter, and the enzyme of catalytic specie oxidation, described oxidase are the conventional oxidase that uses in this area; Preferably be bilirubin oxidase (bilirubin oxidase, english abbreviation BOD) or laccase (laccase).
Preferably, oxidasic concentration is 20mg/mL described in the said oxidase covering liquid.
Described spreading agent is the conventional spreading agent that uses in this area, preferably is bovine serum albumin.Preferably, the concentration of spreading agent is 6.7mM described in the said oxidase covering liquid.
Preferably, the consumption of said oxidase covering liquid is 8～10 μ L, and the pH value of said oxidase covering liquid is 7.0.
The consumption of described glutaraldehyde is the conventional amount used of this area, preferably is 4 * 10
-5～8 * 10
-5Mmol.
The solvent of said glutaraldehyde solution is a water, and preferably, the consumption of described glutaraldehyde solution is 1～2 μ L.
Described drying is the conventional drying mode of this area, generally under 0～25 ℃ temperature, carries out said drying.Preferably, the temperature of said drying is 0～6 ℃.
The present invention also provides a kind of oxidasic combination electrode of load that is made by above-mentioned preparation method.Preferably, described oxidase is a bilirubin oxidase.
The present invention also provides the application of the oxidasic combination electrode of described load in biological fuel cell.
The present invention also provides a kind of biological fuel cell, and wherein anode is the combination electrode of described load dehydrogenasa, and negative electrode is the oxidasic combination electrode of described load, and electrolytic solution comprises the corresponding dehydrogenation substrate of said dehydrogenasa, NAD
+, PBS and oxygen.
Preferably, described anode is the combination electrode of load lactic dehydrogenase, and described electrolytic solution comprises lactic acid, oxygen, NAD
+And PBS.
Preferably, said concentration of lactic acid is 40～60mM.
Preferably, described NAD
+Concentration is 20～40mM.
Preferably, the pH value of described PBS is 7.0, and the described oxygen in the said PBS reaches capacity.
Preferably, described negative electrode is the combination electrode of load bilirubin oxidase.
The present invention also provides a kind of preparation method of biological fuel cell, and described anode, described negative electrode and described electrolytic solution are assembled by this area conventional method.
Room temperature described in the present invention is this area conventional sense, is generally 5～40 ℃.
On the basis that meets this area general knowledge, above-mentioned each optimum condition, but combination in any promptly get each preferred embodiments of the present invention.
Agents useful for same of the present invention and raw material are all commercially available to be got.
Positive progressive effect of the present invention is:
(1) adopt carbon-based material as electrode material, it is easy to obtain, and is cheap.
(2) adopt double-layer hollow carbon ball that glassy carbon electrode is modified, the electrode material that makes has excellent biological compatibility, and under low superpotential, just has the performance of good catalyzing N ADH electrooxidation.Biology sensor of the present invention need not mediator to carry out electro-catalysis and can detect NADH, and it detects, and response is fast, selectivity is high, sensing range can reach 0.20～100 μ M, and sensitivity is up to 7.3 ± 0.2nA/ μ M, and accuracy is high.
(3) CS/GC electrode provided by the present invention has good contamination resistance.
(4) opened up cover vitreous carbon with double-deck empty carbon bag combination electrode in biology sensor and the biological fuel cell Application for Field of medialess build based on dehydrogenasa; The sensitivity of the lactic acid sensor that makes is up to 4.1 ± 0.2nA/ μ M, and sensing range can reach 0.5～12 μ M.
(5) combination electrode that utilizes the modification of process dehydrogenasa and oxidase is respectively as anode and negative electrode, and the battery that makes can be worked in physiological environment, and does not need mediator to carry out electro-catalysis.
Description of drawings
Fig. 1 is the cyclic voltammetry test pattern of the CS/GC electrode of embodiment 1.
Graph of a relation between the current peak that Fig. 2 records for the CS/GC electrode of different scanning speed and embodiment 1.
Cyclic voltammetry test pattern when Fig. 3 is the NADH content of CS/GC electrode and glassy carbon electrode catalysis 0.5mM of embodiment 1; (A) being the CS/GC electrode, (B) is glassy carbon electrode.
Fig. 4 is the cyclic voltammetry test pattern of CS/GC electrode under different N ADH content of embodiment 1.
Fig. 5 is the continuous ampere curve map of CS/GC electrode when catalyzing N ADH electrooxidation of embodiment 1.
Fig. 6 is the cyclic voltammetry test pattern of LDH-CS/GC electrode under different lactic acid contents of embodiment 2.
Fig. 7 is the cyclic voltammetry test pattern of BOD-CS/GC electrode when catalytic oxidation-reduction of embodiment 3.
Fig. 8 is the polarization curve of the biological fuel cell of embodiment 4.
Embodiment
Mode through embodiment further specifies the present invention below, but does not therefore limit the present invention among the described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example according to conventional method and condition, or is selected according to catalogue.
Among the following embodiment, raw material sources are:
Polystyrene hollow ball, model are OP-96, available from Rhom and Hass (Rohm and Haas);
L-lactic dehydrogenase (891U/mg extracts from rabbit muscle for LDH, E.C.1.1.1.27) is purchased that strange company in Delhi (Sigma-Aldrich) in Sigma;
Bilirubin oxidase (BOD, E.C.1.3.3.5 extract from myrothecium verrucaria) is available from your strange company in Delhi (Sigma-Aldrich) of Sigma;
The L-sodium lactate of 98wt% is available from your strange company in Delhi (Sigma-Aldrich) of Sigma;
NAD
+, NADH, bovine serum albumin and 50wt% glutaraldehyde reagent is all available from Fluka company;
It is pure that other reagent are analysis.
0.1M pH be 7.0 PBS by potassium dihydrogen phosphate and sodium hydrogen phosphate preparation, its pH value is by NaOH or phosphoric acid adjusting.
All WS are all by the deionized water preparation of distilling 3 times.
Among the following embodiment, the preparation method of double-layer hollow carbon ball is:
(1) is sulfonation 1 hour in the 98wt% concentrated sulphuric acid being immersed in concentration through cryodesiccated polystyrene hollow ball under 40 ℃, cleans through water and ethanol respectively, make the polystyrene hollow ball of sulfonation; At Mg (OH)
2Under the catalysis, mol ratio be 1: 1.3 phenol and formaldehyde 80 ℃ of reactions 1.5 hours down, be prepared into phenolics, mix phenolics to such an extent that contain the ethanolic solution of phenolics with ethanol;
The ethanolic solution that (2) will contain the polystyrene hollow ball mixes with the ethanolic solution that contains phenolics, at room temperature stirs 10 minutes, reacts 4 hours, is heated to 150 ℃ and further reacts 2 hours, the centrifugal phenolics composite hollow ball that gets; Wherein, the mass ratio of polystyrene hollow ball and said phenolics is 1: 4;
(3) with the phenolics composite hollow ball at N
2Calcined 2 hours for 800 ℃ in the atmosphere, get final product.
The preparation of embodiment 1CS/GC electrode
(1) with the double-layer hollow carbon ball of 10mg and the N of 1mL, dinethylformamide mixes, the ultrasonic dispersion liquid that gets.
(2) be that the glassy carbon electrode of 3mm polishes in particle diameter is the oxidation aluminium paste of 0.3 μ m, 0.05 μ m successively with diameter, removing surperficial booty, with the ultrasonic cleaning 3 minutes respectively of acetone and deionized water.The dispersant liquid drop that makes in the 4 μ L steps (1) is applied to the glassy carbon electrode surface, and the dry solvent of removing makes the CS/GC electrode under light.
The preparation of embodiment 2LDH-CS/GC electrode
With LDH, BSA and pH be 7.0 phosphate mix the LDH covering liquid; The concentration of LDH is that the concentration of 20mg/mL, BSA is 6.7mM in the LDH covering liquid; The LDH that gets 8 μ L coats drop and is applied on the CS/GC electrode of embodiment 1; The glutaraldehyde solution that adds 2 μ L concentration 40mM then carries out crosslinked, in 4 ℃ refrigerator, carries out drying, makes the LDH-CS/GC electrode.
The preparation of embodiment 3BOD-CS/GC electrode
With BOD, BSA and pH be 7.0 phosphate mix the BOD covering liquid; The concentration of BOD is that the concentration of 20mg/mL, BSA is 6.7mM in the BOD covering liquid; The BOD that gets 8 μ L coats drop and is applied on the CS/GC electrode of embodiment 1; The glutaraldehyde solution that adds 2 μ L concentration then and be 40mM carries out crosslinked, in 4 ℃ refrigerator, carries out drying, makes the BOD-CS/GC electrode.
The preparation of embodiment 4 biological fuel cells
As anode, the BOD-CS/GC electrode among the embodiment 3 is as negative electrode with the LDH-CS/GC electrode among the embodiment 2, and electrolytic solution is that the pH of 0.1M is 7.0 PBS, wherein contains lactic acid, oxygen and NAD
+, wherein lactic acid concn is 40mM, NAD
+Concentration is 20mM, and continues aerating oxygen in the PBS, and the oxygen content in the PBS is reached capacity.
The electrochemical property test of effect embodiment 1～2CS/GC electrode
The chemical property of the CS/GC electrode among glassy carbon electrode and the embodiment 1 is tested as follows:
1: glassy carbon electrode and CS/GC electrode respectively as working electrode, as to electrode, as contrast electrode, are processed three-electrode system with the Ag/AgCl electrode of saturated KCl with platinum filament.All tests are all at room temperature carried out, and adopting the pH contain 0.1M KCl is that 7.0 phosphate buffer is as electrolytic solution.Each test repetition 3 times is also calculated relative standard deviation.
The double-layer capacitance that cyclic voltammetry records the CS/GC electrode among glassy carbon electrode and the embodiment 1 is respectively 12.7 μ F/cm
2And 2.1mF/cm
2, their background charge electric current is respectively 2.31 μ A/cm
2With 420 μ A/cm
2, visible double-layer hollow carbon ball increases its electrochemical activity area greatly to the finishing of glassy carbon electrode.
2: adopt Fe (CN)
6 3As the redox probe, under different sweep velocitys, the CS/GC electrode carried out the test of cyclic voltammetry.The CS/GC electrode is containing the Fe of 5mM (CN)
6 3The PBS (pH=7.0) of 0.1M in test result as shown in Figure 1, curve sweep velocity from the inside to the outside is respectively 10,20,50,100,200,300,400 and 500mV/s among the figure, calculates its Δ E
pBe 61mV, I
p Ox/ I
p RedValue is 1.0, shows that the CS-GC electrode is for Fe (CN)
6 3-/4-Demonstrate reversible electron transfer behavior, that is to say that the CS/GC electrode need not carry out just having good electrochemical activity under the pretreated situation.Graph of a relation between the current peak that Fig. 2 records for the CS/GC electrode of different scanning speed and embodiment 1, when 1/2 power of sweep velocity at 10-500 (mV/s)
1/2Scope the time, 1/2 power of current peak and sweep velocity is linear, shows that the CS/GC electrode process is the diffusion control process.
The electro catalytic activity of effect embodiment 3CS/GC electrode and the sensor that detects NADH
The CS/GC electrode of embodiment 1 as working electrode, is measured respectively that not have NADH and the pH value that contains the NADH of 0.5mM be 7.0 PBS, and its sweep speed is 20mV/s.Test result is as shown in Figure 3; Wherein not containing the measured cyclic voltammetry curve of NADH is dotted line a; Containing the measured cyclic voltammetry curve of NADH is solid line b; The sample that contains NADH oxidation peak occurs at about 0.15V place, shows that CS/GC electrode pair NADH oxidation has obvious catalysis, can directly detect the NADH in the sample.Yet under identical test case, when glassy carbon electrode was tested as working electrode, the oxidation peak of NADH occurred in the position of 0.60V, proves that the CS/GC electrode can make oxidation peak peak decline 450mV.Can also see that from Fig. 3 the position that the peak of CS/GC electrode is about-0.10V, hang down 500mV than the peak position (about 0.40V) that goes out of glassy carbon electrode.It is thus clear that the CS/GC electrode carries out under lower voltage for the energy of oxidation of catalyzing N ADH.
When being when constantly adding NADH in 7.0 the PBS in the pH value, electric current in the mensuration system under the voltage of 0.15V, its result is as shown in Figure 4; After adding NADH, electric current can reach steady state (SS) in 8s, when NADH content in the concentration range of 0.20～100 μ M; It demonstrates linear relationship; Its related coefficient is 0.991, and its detection sensitivity is 7.3 ± 0.2nA/ μ M, and its detection limit is 0.08 ± 0.03 μ M.
The job stability experiment of determination sensor:
(1) adopting the pH value of the NADH contain 50 μ M is 7.0 PBS; Under the voltage of 0.15V, measure response current; After each the mensuration CS/GC electrode being immersed the pH value is in 7.0 the PBS; Reuse after the washing, replication 11 times, the relative standard deviation (R.S.D.) that calculates its detection is 2.1%.
(2) the pH value of the CS/GC electrode of 5 embodiment 1 being measured the NADH that contains 50 μ M respectively is 7.0 PBS, under the voltage of 0.15V, measures response current, and the relative standard deviation that calculates its detection is 3.8%.
The contamination resistance test of effect embodiment 4CS/GC electrode detection NADH
With the CS/GC electrode of embodiment 1 and glassy carbon electrode respectively as working electrode; Apply the WV of 0.15V and 0.60V respectively; The NADH of 0.1mM carried out 4000 seconds continuous Amperometric Detection Coupled; Its result is as shown in Figure 5, and wherein curve a is the detection synoptic diagram on the CS/GC electrode, and curve b is the detection synoptic diagram on the GC electrode.The result shows that after 4000 seconds continuous detecting, the electric current of GC electrode descends 47.4%, and the CS/GC electrode only loses 16%, shows that the CS/GC electrode has stronger contamination resistance.
The performance of effect embodiment 5 lactic acid sensors
As working electrode, the pH value of under the voltage of 0.15V, measuring 0.1M is 7.0 the PBS that contains different content lactic acid with the LDH-CS/GC electrode among the embodiment 2, and test is carried out under the magnetic stirrer state.The result is as shown in Figure 6, and current-responsive speed reaches steady state (SS) within 10s, lactic acid concn in the scope of 0.5～12 μ M, the oxidation current linear growth, its sensitivity is 4.1 ± 0.2nA/ μ M; When the signal to noise ratio (S/N ratio) that detects was 3, detection limit was 3.7 ± 0.2 μ M.
Measure the job stability experiment of lactic acid sensor:
(1) adopting the pH value of the lactic acid contain 5 μ M is 7.0 PBS; Under the voltage of 0.15V, measure response current; After each the mensuration LDH-CS/GC electrode being immersed the pH value is in 7.0 the PBS; Reuse after the washing, replication 11 times, the relative standard deviation (R.S.D.) that calculates its detection is 4.0%.
(2) after 2 weeks of storage, when measuring the lactic acid of 5 μ M, response current has descended about 9.3% when beginning most.
(3) the pH value of the LDH-CS/GC electrode of 5 embodiment 1 being measured the lactic acid that contains 5 μ M respectively is 7.0 PBS, under the voltage of 0.15V, measures response current, and the relative standard deviation that calculates its detection is 3.1%.
The performance test of effect embodiment 6BOD-CS/GC electrode catalyst hydrogen reduction
BOD-CS/GC electrode among the embodiment 3 as working electrode, is investigated the electrocatalysis characteristic of this electrode pair oxygen reduction.BOD-CS/GC electrode loop test curve of catalytic oxidation-reduction in the 0.10M phosphate buffer solution is seen Fig. 7.Curve a, b, c are respectively the BOD-CS/GC electrode and under nitrogen, air and oxygen state of saturation, measure, and sweep speed and are 10mVs
-1Cyclic voltammetry curve under the condition.
The electrical property of effect embodiment 7 biological fuel cells
Biological fuel cell among the embodiment 4 is carried out electric performance test, and stain is represented the power density of biological fuel cell among Fig. 8, and white point is represented its open-circuit voltage (english abbreviation OCV), and j is a current density.As scheme visiblely, and its open-circuit voltage is about 0.60V, and power density reaches 3.13 μ W/cm when 0.40V
2
When this battery is adding the resistance of 1M Ω in normal temperature air continuous firing, it is about 6% that its electric power reduced after 24 hours, after 7 days, reduces about 41%.
NADH is surveyed in comparative example's 1 different direct electrooxidation of electrode material and medialess health check-ups
The performance data of different electrode materials in direct electrooxidation and when not having media detection NADH is as shown in table 1.
NADH is surveyed in direct electrooxidation of electrode material that table 1 is different and medialess health check-up
The performance data of the different lactic acid sensors of the performance data of comparative example's 2 different lactic acid sensors is as shown in table 2.
The performance data of the different lactic acid sensors of table 2
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Claims (25)
1. the preparation method of a combination electrode, it comprises the steps:
(1) with double-layer hollow carbon ball and solvent, ultrasonic dispersion liquid;
(2) even the dripping of described dispersion liquid is applied to the glassy carbon electrode surface, promptly gets combination electrode after the drying.
2. preparation method as claimed in claim 1 is characterized in that, described double-layer hollow carbon ball prepares by following step:
(a) be sulfonation 1 hour in the 98wt% concentrated sulphuric acid being immersed in concentration through cryodesiccated polystyrene hollow ball under 40 ℃, clean through water and ethanol respectively, make the polystyrene hollow ball of sulfonation; At Mg (OH)
2Under the catalysis, mol ratio be 1: 1.3 phenol and formaldehyde 70～100 ℃ of reactions 1～2 hour down, be prepared into phenolics, mix phenolics to such an extent that contain the ethanolic solution of phenolics with ethanol;
The ethanolic solution that (b) will contain said polystyrene hollow ball mixes with the described ethanolic solution that contains phenolics, and under stirring condition, reaction is 4 hours under room temperature, is heated to 150 ℃ and further reacts 2 hours, the centrifugal phenolics composite hollow ball that gets; Wherein, the mass ratio of said polystyrene hollow ball and said phenolics is 1: 4;
(c) with described phenolics composite hollow ball at N
2Calcined 2 hours for 800 ℃ in the atmosphere, get final product.
3. preparation method as claimed in claim 1 is characterized in that, the content of the ball of double-layer hollow carbon described in the described dispersion liquid is 8～10mg/mL, and described solvent is N, and dinethylformamide or ethanol, the consumption of described dispersion liquid are 4～6 μ L.
4. want 1 described preparation method like right; It is characterized in that; Described glassy carbon electrode is through polishing and the electrode that cleans, and it is that the oxidation aluminium paste of 0.3 and 0.05 μ m carries out that particle diameter is adopted in described polishing successively, and described cleaning is for using acetone and water difference ultrasonic 3～5 minutes; The diameter of said glassy carbon electrode is 3mm.
5. combination electrode that makes by each described preparation method of claim 1～4.
6. combination electrode as claimed in claim 5 is at combination electrode, the oxidasic combination electrode of preparation load of preparation load dehydrogenasa or be used for detecting the application of the sensor of NADH.
7. a sensor that is used to detect NADH is characterized in that, it comprises combination electrode as claimed in claim 5.
8. the preparation method of the combination electrode of a load dehydrogenasa; It comprises the steps: even the dripping of dehydrogenasa covering liquid is applied on the combination electrode as claimed in claim 5; The adding glutaraldehyde solution carries out crosslinked, promptly gets the combination electrode of load dehydrogenasa after the drying; Described dehydrogenasa covering liquid comprises dehydrogenasa, spreading agent and PBS.
9. preparation method as claimed in claim 8 is characterized in that, described dehydrogenasa is lactic dehydrogenase, GDH or alcohol dehydrogenase; Dehydrogenasa concentration described in the said dehydrogenasa covering liquid is 20mg/mL; The consumption of said dehydrogenasa covering liquid is 8～10 μ L, and the pH value of said dehydrogenasa covering liquid is 7.0; Described spreading agent is a bovine serum albumin; The concentration of spreading agent is 6.7mM described in the said dehydrogenasa covering liquid.
10. like claim 8 or 9 described preparation methods, it is characterized in that the consumption of said glutaraldehyde is 4 * 10
-5～8 * 10
-5Mmol; The consumption of described glutaraldehyde solution is 1～2 μ L; The temperature of said drying is 0～25 ℃, preferably is 0～6 ℃.
11. the combination electrode of a load dehydrogenasa that makes by each described preparation method of claim 8～10.
12. the combination electrode of load dehydrogenasa as claimed in claim 11 is characterized in that, described dehydrogenasa is a lactic dehydrogenase.
13. the application of combination electrode in biology sensor or biological fuel cell like claim 11 or 12 described load dehydrogenasas.
14. a biology sensor, it comprises the combination electrode like claim 11 or 12 described load dehydrogenasas.
15. a method that is used to detect the dehydrogenation substrate is characterized in that, adopts biology sensor as claimed in claim 14 that testing sample is detected, and in described testing sample, adds NAD
+, make the said NAD in the said sample
+Concentration greater than 40mM.
16. the preparation method of the oxidasic combination electrode of load; It comprises the steps: even the dripping of oxidase covering liquid is applied on the combination electrode as claimed in claim 5; The adding glutaraldehyde solution carries out crosslinked, promptly gets the oxidasic combination electrode of load after the drying; Described oxidase covering liquid comprises oxidase, spreading agent and PBS.
17. preparation method as claimed in claim 16 is characterized in that, described oxidase is bilirubin oxidase or laccase; Oxidasic concentration is 20mg/mL described in the said oxidase covering liquid; The consumption of said oxidase covering liquid is 8～10 μ L, and the pH value of said oxidase covering liquid is 7.0; Described spreading agent is a bovine serum albumin; Said dispersant concentration is 6.7mM.
18. preparation method as claimed in claim 16 is characterized in that, the consumption of described glutaraldehyde is 4 * 10
-5～8 * 10
-5Mmol; The consumption of described glutaraldehyde solution is 1～2 μ L; The temperature of said drying is 0～25 ℃, preferably is 0～6 ℃.
19. oxidasic combination electrode of load that makes by each described preparation method of claim 16～18.
20. the oxidasic combination electrode of load as claimed in claim 19 is characterized in that, described oxidase is a bilirubin oxidase.
21. like claim 19 or the application of the oxidasic combination electrode of 20 described loads in biological fuel cell.
22. biological fuel cell; It is characterized in that; Its anode is the combination electrode of load dehydrogenasa as claimed in claim 11, and its negative electrode is the oxidasic combination electrode of load as claimed in claim 16, and its electrolytic solution comprises the corresponding dehydrogenation substrate of said dehydrogenasa, NAD
+, PBS and oxygen.
23. biological fuel cell as claimed in claim 22 is characterized in that, described anode is the combination electrode of load dehydrogenasa as claimed in claim 12, and described electrolytic solution comprises lactic acid, oxygen, NAD
+And PBS; Preferably, said concentration of lactic acid is 40～60mM, described NAD
+Concentration is 20～40mM, and the pH value of described PBS is 7.0, and the oxygen described in the said PBS reaches capacity.
24., it is characterized in that described negative electrode is the oxidasic combination electrode of load as claimed in claim 20 like claim 22 or 23 described biological fuel cells.
25. the preparation method like each described biological fuel cell of claim 22～24 is characterized in that, described anode, described negative electrode and described electrolytic solution are assembled.
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| CN104730129A (en) * | 2015-04-10 | 2015-06-24 | 江南大学 | Bi-enzyme modified electrode-based electrochemical method for quickly detecting ethyl carbamate content of solution |
| CN108978189A (en) * | 2018-07-13 | 2018-12-11 | 武汉纺织大学 | Carbon nano-tube/poly pyrroles composite fibre and preparation method and its application in transistor sensor |
| CN111307900A (en) * | 2020-02-07 | 2020-06-19 | 山东省科学院生物研究所 | Coenzyme factor compound, enzyme electrode, enzyme sensor, preparation method and application thereof |
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