CN102636533B - 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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- 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
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
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Images
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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- Inert Electrodes (AREA)
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 and its preparation method and application, more relate to combination electrode of load dehydrogenasa 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.
Background technology
Nicotinamide adenine dinucleotide (English nicotinamide adenine dinucleotide by name, be abbreviated as NAD) be a kind of coenzyme of dehydrogenasa, the electron carrier in oxidation, no matter be that it all plays core pivotal role in respiration or photosynthesis process.The reduction-state of NAD is NADH, and oxidation state is NAD
+.NAD
+by various dehydrogenasas, from substrate, accept a hydrogen atom and two electronics, become the NADH of reduction-state.This reaction can reversibly be carried out, to realize electronics transmission.NADH participates in exceeding the enzymic catalytic reaction of more than 300 kind of dehydrogenasa, and this makes it at relevant bio-electronic device, as there being very large development prospect in biology sensor and biological fuel cell.But in traditional bare electrode, the Electrocatalytic Oxidation speed of NADH is very slow and need higher overpotential, as be 1.1V in carbon electrode, being 1.3V in platinum electrode, although NADH/NAD
+the thermokinetics electromotive force of electrode is only-0.54V (contrast electrode Ag/AgCl, pH=7.0,25 ℃).More seriously, NADH is oxidized 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.For these problems, first people attempt modifying the eelctro-catalyst with redox active in electrode top, such as oh piperazine dyestuff, the compound that contains quinonyl functional group, metal composite, conducting salt, redox polymers and compound that some are special of azophenlyene, phenothiazine or the fen of monomer or polymeric form, these materials carry out the electrooxidation of catalyzing N ADH as the mediator of electronics transmission at electrode surface.But most these mediator modified electrodes still exist defect, for example mediator is stable not on electrode, and meeting be revealed from electrode, thereby reduces the sensitivity of prepared electrode, thereby limits its application.In recent years, this area researchist is devoted to design and can be used for Electrocatalytic Oxidation and medialess and detect NADH, and can reduce overpotential and reduce the new electrode materials of surface passivation effect.Up to the present, only have little new material, comprise peptide nanotube, poly-1,2-phenylenediamine nanotube and the direct oxidation of the studied NADH of improvement of some carbon-based materials when low superpotential.In these electrode materials, carbon-based material is more attractive.Compared with metal electrode material, carbon-based electrode material more easily obtains, cheaply, there is good biocompatibility, and can be made into multiple different structure.In most electrolytic solution, material with carbon element has better chemical inertness relatively, in aqueous solution, can in a wider potential range, (1.0～1.0V, contrast electrode is saturated calomel electrode) keep higher surfactivity.So far, such as the material with carbon element of the different shapes such as electrode, carbon nano-tube, pyrolytic graphite, carbon nano-fiber, carbon fiber and the carbon cloth of boron doped adamas, preanodize processing, be used to promote the direct oxidation of NADH under lower superpotential.For these carbon-based materials, in the edge carbon of carbon surface or defect and oxy radical, the oxidation of NADH is had to catalytic action.
Summary of the invention
Technical matters to be solved by this invention is that the Electrocatalytic Oxidation speed in order to overcome existing electrode pair NADH is slow, need very high overpotential, need eelctro-catalyst (being mediator), electrode surface easily pollutes, the defects such as sensitivity is low, provide one to be easy to get, inexpensive, there is good biocompatibility and under low superpotential, just can there is combination electrode of good electrical catalytic activity and preparation method thereof, and at the combination electrode of preparing load dehydrogenasa, the preparation oxidasic combination electrode of load or for detection of the application in NADH sensor, provide for detection of NADH sensor and preparation method thereof simultaneously.The present invention also provides combination electrode of a kind of load dehydrogenasa and preparation method thereof, and in the application based in dehydrogenasa biology sensor and biological fuel cell.This biology sensor can detect the corresponding dehydrogenation substrate of dehydrogenasa without mediator catalysis, and has advantages of 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 has good facilitation to oxidasic Direct electron transfer.The characteristic of the combination electrode based on load dehydrogenasa and the oxidasic combination electrode of load, a kind of biological fuel cell without barrier film, direct biological electro catalysis type and preparation method thereof is also 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 by the following technical programs.
The preparation method who the invention provides a kind of combination electrode, it comprises the steps:
(1) by double-layer hollow carbon ball (carbon double-shelled hollow spheres, english abbreviation CS) and solvent, the ultrasonic dispersion liquid that obtains;
(2) described dispersion liquid is evenly dripped and is applied to vitreous carbon (glassy carbon, english abbreviation GC) electrode surface, after being dried, obtain combination electrode.Described combination electrode is the coated prepared combination electrode of double-layer hollow carbon ball on described glassy carbon electrode, for the ease of below setting forth, referred to as CS/GC electrode.
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) be prepared.
Double-layer hollow carbon ball described in the present invention is prepared in the steps below:
(a) at 40 ℃, will be immersed in concentration through cryodesiccated polystyrene hollow ball is sulfonation 1 hour in the 98wt% concentrated sulphuric acid, cleans respectively through water and ethanol, makes the polystyrene hollow ball of sulfonation; At Mg (OH)
2under catalysis, the phenol that mol ratio is 1: 1.3 and formaldehyde react 1～2 hour at 70～100 ℃, are prepared into phenolics, and phenolics and ethanol are mixed to get to the ethanolic solution containing phenolics;
(b) ethanolic solution containing described polystyrene hollow ball is mixed with the described ethanolic solution containing phenolics, under stirring condition, under room temperature, react 4 hours, be heated to 150 ℃ and further react 2 hours, the centrifugal phenolics composite hollow ball that obtains; Wherein, the mass ratio of described polystyrene hollow ball and described phenolics is 1: 4;
(c) by described phenolics composite hollow ball at N
2in atmosphere, calcine 2 hours for 800 ℃.
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 step (1), the content of the ball of double-layer hollow carbon described in described dispersion liquid is preferably 8～10mg/mL.
In step (1), described solvent is the conventional solvent using in this area, is preferably DMF (N, N-dimethylformamide, english abbreviation DMF) or ethanol.
In step (2), the consumption of described dispersion liquid is preferably 4～6 μ L.
In step (2), described glassy carbon electrode is preferably the electrode through polishing and cleaning.The model of described glassy carbon electrode is the conventional model of using in this area, and the diameter of described glassy carbon electrode is preferably 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 the ultrasonic cleaning of water difference 3～5 minutes.
The present invention also provides a kind of combination electrode being 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 preparation oxidasic combination electrode of load or for detection of the application in the sensor of NADH.
The present invention also provides a kind of sensor for detection of 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: dehydrogenasa covering liquid evenly to drip and be applied on described combination electrode, add glutaraldehyde solution to be cross-linked, after being dried, obtain the combination electrode of load dehydrogenasa; Described dehydrogenasa covering liquid comprises dehydrogenasa (dehydrogenase, english abbreviation DH), spreading agent and phosphate buffered solution.The combination electrode of described load dehydrogenasa is the combination electrode of coated dehydrogenasa on described CS/GC electrode, for the ease of below setting forth, referred to as DH-CS/GC electrode.
Wherein, described dehydrogenasa refers to the enzyme of can catalytic specie sloughing hydrogen, it utilizes other chemicals except molecular oxygen as hydrogen acceptor, described dehydrogenasa is the conventional dehydrogenasa using in this area, be preferably lactic dehydrogenase (lactate dehydrogenase, english abbreviation LDH), glucose dehydrogenase or alcohol dehydrogenase.
Preferably, the dehydrogenasa concentration described in described dehydrogenasa covering liquid is 20mg/mL.
Described spreading agent is the conventional spreading agent using in this area, is preferably bovine serum albumin.Preferably, described in described dehydrogenasa covering liquid, the concentration of spreading agent is 6.7mM.
Preferably, the consumption of described dehydrogenasa covering liquid is 8～10 μ L, and the pH value of described dehydrogenasa covering liquid is 7.0.
The consumption of described glutaraldehyde is the conventional amount used of this area, is preferably 4 × 10
-5～8 × 10
-5mmol.
The solvent of described glutaraldehyde solution is water, and preferably, the consumption of described glutaraldehyde solution is 1～2 μ L.
The described conventional drying mode for this area that is dried is generally carried out described dry at the temperature of 0～25 ℃.Preferably, described dry temperature is 0～6 ℃.
The present invention also provides a kind of combination electrode of the load dehydrogenasa being made by above-mentioned preparation method.Preferably, described dehydrogenasa is lactic dehydrogenase.
The combination electrode of load dehydrogenasa of the present invention has the function of molecular recognition and signal conversion simultaneously.
The application of the combination electrode that the present invention also provides described load dehydrogenasa in biology sensor or biological fuel cell.
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 described corresponding dehydrogenation substrate of dehydrogenasa, and dehydrogenation material as corresponding in lactic dehydrogenase is lactic acid.
The present invention also provides a kind of method for detection of dehydrogenation substrate, adopts described biology sensor to detect testing sample, in described testing sample, adds NAD
+, make the described NAD in described sample
+concentration be 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: oxidase covering liquid evenly to drip and be applied on described combination electrode, add glutaraldehyde solution to be cross-linked, after being dried, obtain the oxidasic combination electrode of load; Described oxidase covering liquid comprises oxidase (oxidase, english abbreviation OD), spreading agent and phosphate buffered solution.The oxidasic combination electrode of described load is coated oxidasic combination electrode on described CS/GC electrode, for the ease of below setting forth, referred to as OD-CS/GC electrode.
Wherein, described oxidase refers to directly and as electron accepter, generates water using molecular oxygen, the enzyme of catalytic specie oxidation, described oxidase is the conventional oxidase using in this area, be preferably bilirubin oxidase (bilirubin oxidase, english abbreviation BOD) or laccase (laccase).
Preferably, described in described oxidase covering liquid, oxidasic concentration is 20mg/mL.
Described spreading agent is the conventional spreading agent using in this area, is preferably bovine serum albumin.Preferably, described in described oxidase covering liquid, the concentration of spreading agent is 6.7mM.
Preferably, the consumption of described oxidase covering liquid is 8～10 μ L, and the pH value of described oxidase covering liquid is 7.0.
The consumption of described glutaraldehyde is the conventional amount used of this area, is preferably 4 × 10
-5～8 × 10
-5mmol.
The solvent of described glutaraldehyde solution is water, and preferably, the consumption of described glutaraldehyde solution is 1～2 μ L.
The described conventional drying mode for this area that is dried is generally carried out described dry at the temperature of 0～25 ℃.Preferably, described dry temperature is 0～6 ℃.
The present invention also provides a kind of load being made by above-mentioned preparation method oxidasic combination electrode.Preferably, described oxidase is bilirubin oxidase.
The application of the oxidasic combination electrode of load described in the present invention also provides in biological fuel cell.
The present invention also provides a kind of biological fuel cell, and its Anodic is the combination electrode of described load dehydrogenasa, and negative electrode is the described oxidasic combination electrode of load, and electrolytic solution comprises dehydrogenation substrate, the NAD that described dehydrogenasa is corresponding
+, phosphate buffered solution and oxygen.
Preferably, described anode is the combination electrode of load lactic dehydrogenase, and described electrolytic solution comprises lactic acid, oxygen, NAD
+and phosphate buffered solution.
Preferably, the concentration of described lactic acid is 40～60mM.
Preferably, described NAD
+concentration is 20～40mM.
Preferably, the pH value of described phosphate buffered solution is 7.0, and the described oxygen in described phosphate buffered solution 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 ℃.
Meeting on the basis of this area general knowledge, above-mentioned each optimum condition, can combination in any, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material be commercially available obtaining all.
Positive progressive effect of the present invention is:
(1) adopt carbon-based material as electrode material, it is easy to obtain, cheap.
(2) adopt double-layer hollow carbon ball to modify glassy carbon electrode, the electrode material making has good biocompatibility, and under low superpotential, just has the performance of good catalyzing N ADH electrooxidation.Biology sensor of the present invention carries out electro-catalysis without mediator 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 the combination electrode that covers vitreous carbon with double-deck empty carbon bag application in the biology sensor based on dehydrogenasa and biological fuel cell field at medialess build, the sensitivity of the lactic acid sensor making is up to 4.1 ± 0.2nA/ μ M, and sensing range can reach 0.5～12 μ M.
(5) utilize the combination electrode of process dehydrogenasa and oxidase modification respectively as anode and negative electrode, the battery making can be worked in physiological environment, and does not need mediator to carry out electro-catalysis.
Accompanying drawing explanation
Fig. 1 is the cyclic voltammetry test pattern of the CS/GC electrode of embodiment 1.
Fig. 2 is the graph of a relation between the current peak that records of the CS/GC electrode of different scanning speed and embodiment 1.
Cyclic voltammetry test pattern when Fig. 3 is the CS/GC electrode of embodiment 1 and the NADH content of glassy carbon electrode catalysis 0.5mM; (A) being CS/GC electrode, is (B) glassy carbon electrode.
Fig. 4 is the CS/GC electrode of the embodiment 1 cyclic voltammetry test pattern under different N ADH content.
Fig. 5 is the CS/GC electrode of the embodiment 1 continuous ampere curve map when catalyzing N ADH electrooxidation.
Fig. 6 is the LDH-CS/GC electrode of the embodiment 2 cyclic voltammetry test pattern under different lactic acid contents.
Fig. 7 is the BOD-CS/GC electrode of the embodiment 3 cyclic voltammetry test pattern when catalytic oxidation-reduction.
Fig. 8 is the polarization curve of the biological fuel cell of embodiment 4.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, according to conventional method and condition, or selects according to catalogue.
In following embodiment, raw material sources are:
Polystyrene hollow ball, model is OP-96, purchased from Rhom and Hass (Rohm and Haas);
LDH (891U/mg, extracts from rabbit muscle for LDH, E.C.1.1.1.27) is purchased from the Sigma that strange company in Delhi (Sigma-Aldrich);
Bilirubin oxidase (BOD, E.C.1.3.3.5 extract from myrothecium verrucaria) is purchased from your strange company in Delhi of Sigma (Sigma-Aldrich);
The Pfansteihl sodium of 98wt% is purchased from your strange company in Delhi of Sigma (Sigma-Aldrich);
NAD
+, NADH, bovine serum albumin and 50wt% glutaraldehyde reagent is all purchased from Fluka company;
It is pure that other reagent are analysis.
The pH of 0.1M is that 7.0 phosphate buffered solution is prepared by potassium dihydrogen phosphate and sodium hydrogen phosphate, and its pH value is regulated by NaOH or phosphoric acid.
All aqueous solution are all prepared by the deionized water that distills 3 times.
In following embodiment, the preparation method of double-layer hollow carbon ball is:
(1) at 40 ℃, will be immersed in concentration through cryodesiccated polystyrene hollow ball is sulfonation 1 hour in the 98wt% concentrated sulphuric acid, cleans respectively through water and ethanol, makes the polystyrene hollow ball of sulfonation; At Mg (OH)
2under catalysis, the phenol that mol ratio is 1: 1.3 and formaldehyde react 1.5 hours at 80 ℃, are prepared into phenolics, and phenolics and ethanol are mixed to get to the ethanolic solution containing phenolics;
(2) ethanolic solution containing polystyrene hollow ball is mixed with the ethanolic solution containing phenolics, at room temperature stir 10 minutes, react 4 hours, be heated to 150 ℃ and further react 2 hours, the centrifugal phenolics composite hollow ball that obtains; Wherein, the mass ratio of polystyrene hollow ball and described phenolics is 1: 4;
(3) by phenolics composite hollow ball at N
2in atmosphere, calcine 2 hours for 800 ℃.
The preparation of embodiment 1CS/GC electrode
(1) the double-layer hollow carbon ball of 10mg is mixed to the ultrasonic dispersion liquid that obtains with the DMF of 1mL.
(2) glassy carbon electrode that is 3mm by diameter carries out polishing successively in particle diameter is the oxidation aluminium paste of 0.3 μ m, 0.05 μ m, to remove surperficial booty, with the ultrasonic cleaning 3 minutes respectively of acetone and deionized water.The dispersant liquid drop making in 4 μ L steps (1) is applied to glassy carbon electrode surface, and under light, the dry solvent of removing, makes CS/GC electrode.
The preparation of embodiment 2LDH-CS/GC electrode
The phosphate that is 7.0 by LDH, BSA and pH is mixed to get LDH covering liquid, in LDH covering liquid, the concentration of LDH is that the concentration of 20mg/mL, BSA is 6.7mM, the coated drop of LDH of getting 8 μ L is applied on the CS/GC electrode of embodiment 1, then add the glutaraldehyde solution of 2 μ L concentration 40mM to be cross-linked, in the refrigerator of 4 ℃, be dried, make LDH-CS/GC electrode.
The preparation of embodiment 3BOD-CS/GC electrode
The phosphate that is 7.0 by BOD, BSA and pH is mixed to get BOD covering liquid, in BOD covering liquid, the concentration of BOD is that the concentration of 20mg/mL, BSA is 6.7mM, the coated drop of BOD of getting 8 μ L is applied on the CS/GC electrode of embodiment 1, then add the glutaraldehyde solution that 2 μ L concentration are 40mM to be cross-linked, in the refrigerator of 4 ℃, be dried, make BOD-CS/GC electrode.
The preparation of embodiment 4 biological fuel cells
Using the LDH-CS/GC electrode in embodiment 2 as anode, the BOD-CS/GC electrode in embodiment 3 is as negative electrode, and the phosphate buffered solution that the pH that electrolytic solution is 0.1M is 7.0, wherein contains lactic acid, oxygen and NAD
+, wherein lactic acid concn is 40mM, NAD
+concentration is 20mM, and in phosphate buffered solution, continues to pass into oxygen, and the oxygen content in phosphate buffered solution is reached capacity.
The electrochemical property test of effect embodiment 1～2CS/GC electrode
The chemical property of the CS/GC electrode in glassy carbon electrode and embodiment 1 is tested as follows:
1: glassy carbon electrode and CS/GC electrode, respectively as working electrode, using platinum filament as to electrode, using the Ag/AgCl electrode of saturated KCl as contrast electrode, are made to three-electrode system.All tests are all at room temperature carried out, and the phosphate buffer that employing is 7.0 containing the pH of 0.1M KCl is as electrolytic solution.Each test repeats 3 times and calculates relative standard deviation.
The double-layer capacitance that cyclic voltammetry records the CS/GC electrode in glassy carbon electrode and 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 surface area greatly to the finishing of glassy carbon electrode.
2: adopt Fe (CN)
6 3as redox probe, CS/GC electrode is carried out the test of cyclic voltammetry under different sweep velocitys.CS/GC electrode is containing the Fe of 5mM (CN)
6 3the phosphate buffered solution (pH=7.0) of 0.1M in test result as shown in Figure 1, in figure, curve sweep velocity is from the inside to the outside respectively 10,20,50,100,200,300,400 and 500mV/s, calculates its Δ E
pfor 61mV, I
p ox/ I
p redvalue is 1.0, shows that CS-GC electrode is for Fe (CN)
6 3-/4-present reversible electronics displacement behavior, that is to say that CS/GC electrode is not needing to carry out in pretreated situation, just to have good electrochemical activity.Fig. 2 is the graph of a relation between the current peak that records of the CS/GC electrode of different scanning speed and embodiment 1, when 1/2 power of sweep velocity is at 10-500 (mV/s)
1/2scope time, 1/2 power of current peak and sweep velocity is linear, shows that CS/GC electrode process is diffusion controlled process.
The sensor of the electro catalytic activity of effect embodiment 3CS/GC electrode and detection NADH
Using the CS/GC electrode of embodiment 1 as working electrode, the phosphate buffered solution that the pH value of measuring respectively the NADH that there is no NADH and contain 0.5mM is 7.0, its sweep speed is 20mV/s.Test result as shown in Figure 3, containing the measured cyclic voltammetry curve of NADH, be not wherein dotted line a, containing the measured cyclic voltammetry curve of NADH, be solid line b, at about 0.15V place, there is oxidation peak in the sample containing NADH, show that CS/GC electrode pair NADH oxidation has obvious catalytic action, can directly detect the NADH in sample.But under identical test case, using glassy carbon electrode, when working electrode is tested, the oxidation peak of NADH occurs in the position of 0.60V, proves that CS/GC electrode can make oxidation peak peak decline 450mV.From Fig. 3, can also see that the be about-0.10V of the position that peak of CS/GC electrode goes out the low 500mV in peak position (about 0.40V) than glassy carbon electrode.Visible CS/GC electrode carries out under lower voltage for the energy of oxidation of catalyzing N ADH.
When constantly adding NADH in the phosphate buffered solution that is 7.0 in pH value, electric current in mensuration system under the voltage of 0.15V, its result as shown in Figure 4, adding after NADH, electric current can reach steady state (SS) in 8s, when NADH content is 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) phosphate buffered solution that the pH value of the NADH that employing contains 50 μ M is 7.0, under the voltage of 0.15V, measure response current, after each mensuration, CS/GC electrode is immersed in the phosphate buffered solution that pH value is 7.0, after washing, reuse, replication 11 times, the relative standard deviation (R.S.D.) that calculates its detection is 2.1%.
(2) the CS/GC electrode of 5 embodiment 1 is measured respectively to the phosphate buffered solution that the pH value of the NADH that contains 50 μ M is 7.0, under the voltage of 0.15V, measured response current, the relative standard deviation that calculates its detection is 3.8%.
The contamination resistance test of effect embodiment 4CS/GC electrode detection NADH
Using the CS/GC electrode of embodiment 1 and glassy carbon electrode respectively as working electrode, apply respectively the operating voltage of 0.15V and 0.60V, the continuous ampere that the NADH of 0.1mM is carried out 4000 seconds detects, its result as shown in Figure 5, wherein curve a is the detection schematic diagram on CS/GC electrode, and curve b is the detection schematic diagram on GC electrode.Result shows, after the continuous detecting of 4000 seconds, the electric current of GC electrode declines 47.4%, and CS/GC electrode only loses 16%, shows that CS/GC electrode has stronger contamination resistance.
The performance of effect embodiment 5 lactic acid sensors
Using the LDH-CS/GC electrode in embodiment 2 as working electrode, the phosphate buffered solution containing different content lactic acid that the pH value of measuring 0.1M under the voltage of 0.15V is 7.0, test is carried out under magnetic stirrer state.As shown in Figure 6, current response rate reaches steady state (SS) to result within 10s, lactic acid concn in the scope of 0.5～12 μ M, oxidation current linear growth, its sensitivity is 4.1 ± 0.2nA/ μ M; When the signal to noise ratio (S/N ratio) detecting is 3, detection limit is 3.7 ± 0.2 μ M.
Measure the job stability experiment of lactic acid sensor:
(1) phosphate buffered solution that the pH value of the lactic acid that employing contains 5 μ M is 7.0, under the voltage of 0.15V, measure response current, after each mensuration, LDH-CS/GC electrode is immersed in the phosphate buffered solution that pH value is 7.0, after washing, reuse, replication 11 times, the relative standard deviation (R.S.D.) that calculates its detection is 4.0%.
(2), after storage 2 weeks, when measuring the lactic acid of 5 μ M, response current has declined approximately 9.3% when starting most.
(3) the LDH-CS/GC electrode of 5 embodiment 1 is measured respectively to the phosphate buffered solution that the pH value of the lactic acid that contains 5 μ M is 7.0, under the voltage of 0.15V, measured response current, the relative standard deviation that calculates its detection is 3.1%.
The performance test of effect embodiment 6BOD-CS/GC electrode catalyst hydrogen reduction
Using the BOD-CS/GC electrode in embodiment 3 as working electrode, investigate the electrocatalysis characteristic of this electrode pair oxygen reduction.BOD-CS/GC electrode loop test curve of catalytic oxidation-reduction in 0.10M phosphate buffer solution is shown in Fig. 7.Curve a, b, c are respectively BOD-CS/GC electrode and measure under nitrogen, air and oxygen state of saturation, sweep speed for 10mVs
-1cyclic voltammetry curve under condition.
The electrical property of effect embodiment 7 biological fuel cells
Biological fuel cell in embodiment 4 is carried out to electric performance test, and in Fig. 8, stain represents the power density of biological fuel cell, and white point represents its open-circuit voltage (english abbreviation OCV), and j is current density.As can be seen, its open-circuit voltage is about 0.60V, and power density reaches 3.13 μ W/cm when 0.40V
2.
When the resistance of this battery at additional 1M Ω in normal temperature air continuous firing, its electric power reduced approximately 6% after 24 hours, reduced approximately 41% after 7 days.
NADH is surveyed in the electrode material Electrocatalytic Oxidation that comparative example 1 is different and medialess health check-up
The performance data of different electrode materials during at Electrocatalytic Oxidation and without media detection NADH is as shown in table 1.
NADH is surveyed in the electrode material Electrocatalytic Oxidation that table 1 is different and medialess health check-up
The performance data of the different lactic acid sensors of 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 (16)
1. a preparation method for the combination electrode of load dehydrogenasa, it comprises the steps: dehydrogenasa covering liquid evenly to drip and be applied on a combination electrode, adds glutaraldehyde solution to be cross-linked, and obtains the combination electrode of load dehydrogenasa after being dried; Described dehydrogenasa covering liquid comprises dehydrogenasa, spreading agent and phosphate buffered solution;
Described dehydrogenasa is lactic dehydrogenase, glucose dehydrogenase or alcohol dehydrogenase; Dehydrogenasa concentration described in described dehydrogenasa covering liquid is 20mg/mL; The consumption of described dehydrogenasa covering liquid is 8～10 μ L, and the pH value of described dehydrogenasa covering liquid is 7.0; Described spreading agent is bovine serum albumin; Described in described dehydrogenasa covering liquid, the concentration of spreading agent is 6.7mM;
The consumption of described glutaraldehyde is 4 × 10
-5～8 × 10
-5mmol; The consumption of described glutaraldehyde solution is 1～2 μ L; Described dry temperature is 0～25 ℃;
Described combination electrode is made by following preparation method, and this preparation method comprises the steps:
(1) by double-layer hollow carbon ball and solvent, ultrasonic dispersion liquid;
(2) described dispersion liquid is evenly dripped and is applied to glassy carbon electrode surface, after being dried, obtain combination electrode;
Described double-layer hollow carbon ball is prepared in the steps below:
(a) at 40 ℃, will be immersed in concentration through cryodesiccated polystyrene hollow ball is sulfonation 1 hour in the 98wt% concentrated sulphuric acid, cleans respectively through water and ethanol, makes the polystyrene hollow ball of sulfonation; At Mg (OH)
2under catalysis, the phenol that mol ratio is 1:1.3 and formaldehyde react 1～2 hour at 70～100 ℃, are prepared into phenolics, and phenolics and ethanol are mixed to get to the ethanolic solution containing phenolics;
(b) ethanolic solution containing described polystyrene hollow ball is mixed with the described ethanolic solution containing phenolics, under stirring condition, under room temperature, react 4 hours, be heated to 150 ℃ and further react 2 hours, the centrifugal phenolics composite hollow ball that obtains; Wherein, the mass ratio of described polystyrene hollow ball and described phenolics is 1:4;
(c) by described phenolics composite hollow ball at N
2in atmosphere, calcine 2 hours for 800 ℃;
The content of the ball of double-layer hollow carbon described in described dispersion liquid is 8～10mg/mL, and described solvent is DMF or ethanol, and the consumption of described dispersion liquid is 4～6 μ L.
2. preparation method as claimed in claim 1, is characterized in that, described dry temperature is 0～6 ℃.
3. the combination electrode of the load dehydrogenasa being made by the preparation method described in claim 1 or 2.
4. the combination electrode of load dehydrogenasa as claimed in claim 3, is characterized in that, described dehydrogenasa is lactic dehydrogenase.
5. the application of the combination electrode of the load dehydrogenasa as described in claim 3 or 4 in biology sensor or biological fuel cell.
6. a biology sensor, it comprises the combination electrode of the load dehydrogenasa as described in claim 3 or 4.
7. for detection of a method for dehydrogenation substrate, it is characterized in that, adopt biology sensor as claimed in claim 6 to detect testing sample, in described testing sample, add NAD
+, make the described NAD in described sample
+concentration be greater than 40mM.
8. a preparation method for the oxidasic combination electrode of load, it comprises the steps: oxidase covering liquid evenly to drip and be applied on a combination electrode, adds glutaraldehyde solution to be cross-linked, and after being dried, obtains the oxidasic combination electrode of load; Described oxidase covering liquid comprises oxidase, spreading agent and phosphate buffered solution;
Described oxidase is bilirubin oxidase or laccase; Described in described oxidase covering liquid, oxidasic concentration is 20mg/mL; The consumption of described oxidase covering liquid is 8～10 μ L, and the pH value of described oxidase covering liquid is 7.0; Described spreading agent is bovine serum albumin; Described dispersant concentration is 6.7mM;
The consumption of described glutaraldehyde is 4 × 10
-5～8 × 10
-5mmol; The consumption of described glutaraldehyde solution is 1～2 μ L; Described dry temperature is 0～25 ℃;
Described combination electrode is made by following preparation method, and this preparation method comprises the steps:
(1) by double-layer hollow carbon ball and solvent, ultrasonic dispersion liquid;
(2) described dispersion liquid is evenly dripped and is applied to glassy carbon electrode surface, after being dried, obtain combination electrode;
Described double-layer hollow carbon ball is prepared in the steps below:
(a) at 40 ℃, will be immersed in concentration through cryodesiccated polystyrene hollow ball is sulfonation 1 hour in the 98wt% concentrated sulphuric acid, cleans respectively through water and ethanol, makes the polystyrene hollow ball of sulfonation; At Mg (OH)
2under catalysis, the phenol that mol ratio is 1:1.3 and formaldehyde react 1～2 hour at 70～100 ℃, are prepared into phenolics, and phenolics and ethanol are mixed to get to the ethanolic solution containing phenolics;
(b) ethanolic solution containing described polystyrene hollow ball is mixed with the described ethanolic solution containing phenolics, under stirring condition, under room temperature, react 4 hours, be heated to 150 ℃ and further react 2 hours, the centrifugal phenolics composite hollow ball that obtains; Wherein, the mass ratio of described polystyrene hollow ball and described phenolics is 1:4;
(c) by described phenolics composite hollow ball at N
2in atmosphere, calcine 2 hours for 800 ℃;
The content of the ball of double-layer hollow carbon described in described dispersion liquid is 8～10mg/mL, and described solvent is DMF or ethanol, and the consumption of described dispersion liquid is 4～6 μ L.
9. preparation method as claimed in claim 8, is characterized in that, described dry temperature is 0～6 ℃.
10. the oxidasic combination electrode of the load being made by the preparation method described in claim 8 or 9.
The oxidasic combination electrode of 11. load as claimed in claim 10, is characterized in that, described oxidase is bilirubin oxidase.
The application of 12. oxidasic combination electrodes of load as described in claim 10 or 11 in biological fuel cell.
13. 1 kinds of biological fuel cells, it is characterized in that, its anode is the combination electrode of load dehydrogenasa as claimed in claim 3, and its negative electrode is the oxidasic combination electrode of load as claimed in claim 10, and its electrolytic solution comprises dehydrogenation substrate, the NAD that described dehydrogenasa is corresponding
+, phosphate buffered solution and oxygen.
14. biological fuel cells as claimed in claim 13, is characterized in that, described anode is the combination electrode of load dehydrogenasa as claimed in claim 4, and described electrolytic solution comprises lactic acid, oxygen, NAD
+and phosphate buffered solution; The concentration of described lactic acid is 40～60mM, described NAD
+concentration is 20～40mM, and the pH value of described phosphate buffered solution is 7.0, and the oxygen described in described phosphate buffered solution reaches capacity.
15. the biological fuel cell as described in claim 13 or 14, is characterized in that, described negative electrode is the oxidasic combination electrode of load as claimed in claim 11.
The preparation method of 16. 1 kinds of biological fuel cells as described in claim 13～15 any one, is characterized in that, described anode, described negative electrode and described electrolytic solution are assembled.
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