CN109813781A - A kind of amine dehydrogenase electrode and its preparation method and application - Google Patents
A kind of amine dehydrogenase electrode and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of amine dehydrogenase electrodes and its preparation method and application.The present invention relates to biosensor technology fields more particularly to a kind of enzyme electrode, enzyme biologic sensor and its preparation method and application.The electrode includes basal electrode, electronic conductive layer and amine dehydrogenase layer, it can be achieved that electronics transfer between effective fixation of enzyme molecule and enzyme and electrode, improves the sensitivity of enzyme molecule catalytic activity and sensor.Grapheme modified, carbon nanotube does electrode material to enzyme electrode of the invention with the conducting polymer polyethyleneimine with good biocompatibility, have the advantages that production method is simple, detection sensitivity is high, quick accurate, stability and reproducible, has a good application prospect.
Description
Technical field
The present invention relates to biosensor technology field, it is related to a kind of enzyme electrode, enzyme biologic sensor and preparation method thereof
And application more particularly to a kind of amine dehydrogenase electrode and its preparation method and application.
Background technique
Biosensor is in biomedical inspection, disease diagnosis and therapy, food analysis, environmental monitoring, industrial process inspection
It surveys and has broad application prospects with fields such as control, poisonous substance detection and war biochemistry early warning.Biosensor technique industrialization
Bottleneck is Primary Component and analysis system manufacturing technology.Attached most importance to enzyme molecule device, in MOLECULE DESIGN, device assembling, analysis
Model construction etc. carries out innovative design and carries out Applied D emonstration in major fields such as medical treatment, environment, industry to be bio-sensing
The research hotspot of device.
Currently, the analysis method of aromatic amine compounds mainly has capillary electrophoresis, high performance liquid chromatography and gas phase
Chromatography.These methods are due to needing complicated sample pretreatment process and huge instrument, it is difficult to meet organic amine substance
Miniaturization, rapid and on-the-spot testing requirements.It is accurate, quick, easy, sensitive and selectively good etc. excellent that enzyme electrode then has
Point provides a kind of effective means for the measurement of aromatic amine substance, such as Munteanu is horseradish peroxidase and grape
Glucocorticoid dehydrogenase, which is embedded in graphite combination electrode, prepares enzyme electrode measurement aromatic amine substance.Yang Shaoming etc. utilizes the bis- enzymes of HRP/GOx
Aromatic amine substance is catalyzed in electrode system to be detected.
Amine dehydrogenase can efficient catalytic amine deamination generate corresponding ketone, while by NAD+It is reduced to NADH (Fig. 1).It reacted
Journey is succinctly efficient, has wide application potential in the enzyme detection of organic amine.However, constructing enzyme electrode currently with amine dehydrogenase
Detection is carried out to have not been reported.To find out its cause, efficient amine dehydrogenase reported at present is less.And organic amine is often insoluble in
Water need to be developed suitable for being detected in organic phase and measure hydrophobicity organic amine suitable for the enzyme electrode of organic phase, especially fragrant
Fragrant amine.And dehydrogenase is often poor to the tolerance of organic solvent, is easy inactivation, the serious exploitation for restricting amine dehydrogenase electrode.
A large amount of amino on branch and straight linear polyethylene imines, not only can be in conjunction with carbon nanomaterial, can also be with
On protease amino acid side chain amino acid (histidine, aspartic acid and arginine etc.) functional group (imidazole radicals, indyl and
Phenolic hydroxyl group etc.) it is reacted.And the microenvironment of polyethyleneimine offer biocompatibility.The multi-level phase interaction of enzyme and carrier
With combining amine dehydrogenase closely on the electrode, and there is also Van der Waals force, hydrogen bond and electrostatic forces therebetween, can use
In highly sensitive and selective biosensor.But does not have also so far and polyethyleneimine is used to prepare amine dehydrogenase electricity
The report of pole.
Summary of the invention
It is an object of the invention in place of overcome the deficiencies in the prior art, provide a kind of amine dehydrogenase electrode and its preparation
Methods and applications, are based on the design of carbon nanomaterial and polyethyleneimine (PEI) composite material, and building is based on hydrogen bond, hydrophobic, quiet
The multi-level interaction force directional assembly enzyme such as electricity and coordination power is conducive to the stability and bio-sensitive film that improve enzyme
Fixation, provide a good microenvironment for the electrochemical reaction of biomolecule, building obtains novel amine dehydrogenase electrode；
Meanwhile the present invention detects the biosensor of amine using the preparation of amine dehydrogenase electrode, and with polyethyleneimine high molecular material work
For electrode auxiliary material, there is good biocompatibility and promotion work can be played in terms of the Direct electron transfer between enzyme and electrode
With realizing the electronics transfer between effective fixation of enzyme molecule and enzyme and electrode, improve enzyme molecule catalytic activity and sensor
Sensitivity, to realize the highly sensitive detection to organic amine.Amine dehydrogenase electrode of the invention is compared with traditional enzyme electrode, tool
There are high detection sensitivity, quick accurate, stability and the advantages such as reproducible.
The technical solution adopted by the present invention to solve the technical problems first is that:
A kind of amine dehydrogenase electrode, including basal electrode, electronic conductive layer and amine dehydrogenase layer；The electronic conductive layer is
Redox graphene-PEI composite material or carbon nanotube；The gene order of the amine dehydrogenase is as shown in SEQ ID NO.1.
The amine dehydrogenase electrode is followed successively by the electronic conductive layer and the amine dehydrogenase centered on basal electrode from inside to outside
Layer.
In one embodiment: the basal electrode is glass-carbon electrode, pyrolytic graphite electrode, carbon paste electrode or metal electrode；It is excellent
It is selected as glass-carbon electrode.
The technical solution adopted by the present invention to solve the technical problems second is that:
A kind of preparation method of amine dehydrogenase electrode, comprising:
(1) graphene oxide is adsorbed into PEI or graphene oxide covalence graft PEI, is restored to obtain oxygen reduction fossil
Black alkene-PEI composite material, the redox graphene-PEI composite material again with amine dehydrogenase mixed immobilization；Or by carbon
(using the space caliber characteristic of carbon nanotube, amine dehydrogenase can be attracted to carbon and receive nanotube with amine dehydrogenase mixed immobilization
On mitron or in caliber)；The gene order of the amine dehydrogenase is as shown in SEQ ID NO.1；
(2) by the product after redox graphene-PEI composite material immobilization amine dehydrogenase or by carbon nanotube-PEI
Product after composite material immobilization amine dehydrogenase is compound in basal electrode surface, obtains amine dehydrogenase electrode.
In one embodiment: the basal electrode is glass-carbon electrode, pyrolytic graphite electrode, carbon paste electrode or metal electrode；It is excellent
It is selected as glass-carbon electrode.The basal electrode first passes through surface preparation when being glass-carbon electrode or pyrolytic graphite electrode in advance, and surface is pre-
Processing method includes: that glass-carbon electrode or pyrolytic graphite electrode are successively used 1.0 μm of diameter, 0.3 μm, 0.05 μm of Al2O3Powder is thrown
Then light is successively cleaned by ultrasonic in dehydrated alcohol and distilled water at mirror surface with distilled water flushing, then by glass-carbon electrode or pyrolysis
Graphite electrode is placed in containing 1~5mM K3Fe(CN)6With carry out electrode activation in the solution of 10~200mM KCl, take out with distillation
Water rinses, and dries to obtain pretreated basal electrode at room temperature.
In one embodiment: the polyethyleneimine is the polyethylene for the linear chain or branched chain that molecular weight ranges are 20~500kDa
Imines, the polyethyleneimine is directly using or using the phosphate buffer of pH 6.0~8.0 or the pH of 1~100mmol/L
8.0~10 ammonium hydroxide-ammonium chloride buffer is configured to the polyethylenimine solution that concentration is 5~200mg/ml and uses.It is described poly-
Aziridine is the straight chain PEI that molecular weight is 22KDa, 87KDa or 217KDa or molecular weight is the branch of 25KDa or 187KDa
PEI。
In one embodiment: the redox graphene-PEI composite material and preparation method thereof includes: by 7~100mL water, 50
~200mg KOH and 150~300mg PEI are stirred evenly, and the finely dispersed GO solution of 4~10mL, 4~6mg/mL is added,
After 75~85 DEG C are heated to reflux 22~26h, 6~10mL, 1~3mol/L sodium borohydride is added and is restored, the reaction was continued 1~
3h；After the reaction was completed, by centrifugation, drying to obtain redox graphene-PEI composite material rGO-PEI.
In one embodiment: the preparation method of the amine dehydrogenase includes: to utilize the amine dehydrogenase as shown in SEQ ID NO.1
Gene prepares the engineering bacteria that can express amine dehydrogenase；Above-mentioned engineering bacteria is inoculated with, inducer, culture gained fermentation liquid centrifugation is added
Obtain cell；Cell ultrasonication, centrifugation, obtains the crude enzyme liquid containing amine dehydrogenase；By the above-mentioned thick enzyme containing amine dehydrogenase
Liquid carries out purification desalination, obtains amine dehydrogenase.
In one embodiment: the preparation side of the redox graphene-PEI composite material and amine dehydrogenase mixed immobilization
Method include: by concentration be 0.05~1mg/ml the redox graphene-PEI composite material and concentration be 0.05~
The amine dehydrogenase solution of 10mg/ml is mixed according to the ratio of volume ratio 1:1~4, and 6000~10000rpm is centrifuged 10~20min,
It rinses, is resuspended in ammonium hydroxide-ammonium chloride buffer of the pH 8.0~10.0 of 0.05~0.1M, preparation contains 0.25~50mg/ml
The mixed solution of amine dehydrogenase, the as product after redox graphene-PEI composite material immobilization amine dehydrogenase；
Or, the carbon nanotube and the preparation method of amine dehydrogenase mixed immobilization include: the load in enrichment carbon nanotube
Amine dehydrogenase solution is added on body, it is dry, it adds amine dehydrogenase solution and drying steps several times repeatedly, obtains amine dehydrogenase-
Carbon nano tube modified object, the as product after carbon nanotube immobilization amine dehydrogenase.
The technical solution adopted by the present invention to solve the technical problems third is that:
A kind of application of amine dehydrogenase electrode in the amine concentration in measurement solution, the measurement of the amine concentration is using three electricity
Polar body system, using the amine dehydrogenase electrode as working electrode, or with the amine dehydrogenase according to prepared by the preparation method
Electrode is working electrode；Reference electrode is saturated calomel electrode, hydrogen electrode, silver | silver chloride electrode or mercury | mercuric oxide electrode, more
Preferably saturated calomel electrode；It is platinum electrode or carbon electrode to electrode.
In one embodiment: measuring the amine concentration in solution in a manner of cyclic voltammetric and/or chrono-amperometric；The circulation volt
The test potential sweep speed of peace mode is 25~200mV/s；It carries out, surveys in the buffer that 1~20mg coenzyme NAD H is added
N is passed through before examination2, the amine is phenyl ethylamine, ortho-methyl aniline, 1,3- dimethyl butylamine, 2- aminoanisole etc.；The amine
Concentration range be 50ppm~5mM；The buffer be phosphate buffer, borate buffer solution, citrate buffer,
At least one of carbonate buffer solution and acetate buffer.
Beneficial effects of the present invention are as follows:
The present invention selects the carbon nanomaterials such as graphene, carbon nanotube and conducting polymer polyethyleneimine to pass as electronics
Pass material and carry out enzyme electrode preparation, prepared composite material because its superior Electronic Performance, can chemical modification performance and good
Biocompatibility etc. realize the stabilization immobilization and efficent electronic transmitting of enzyme.Enzyme electrode of the invention is with good biological
Grapheme modified, carbon nanotube of conducting polymer polyethyleneimine of compatibility etc. does electrode material, simple with production method,
Low in cost, detection sensitivity is high, quick accurate, stability and reproducible advantage.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples.
Fig. 1 is the schematic diagram that enzyme electrode of the invention detects amine.
Fig. 2 is amine dehydrogenase electrode the following under conditions of various concentration 1,3- dimethyl butylamine of the embodiment of the present invention 1
Ring voltammogram.
Fig. 3 is cyclic voltammogram of the amine dehydrogenase electrode of the embodiment of the present invention 5 under conditions of various concentration phenyl ethylamine.
Specific embodiment
It elaborates below by embodiment to the present invention.
Embodiment 1
(1) the thick enzyme preparation of amine dehydrogenase: according to the amine dehydrogenase original series as shown in SEQ ID NO.1, design such as SEQ
Primer shown in ID NO.2 and SEQ ID NO.3 is closed by commercialization company (Shanghai bioengineering Co., Ltd) using PCR method
Plasmid is constructed for connecting with carrier pET28a at the gene order of amine dehydrogenase.
The connection of PCR product (gene order of amine dehydrogenase) and carrier pET28a: use NdeI and XhoI by PCR product
And carrier pET28a double digestion, it is purified after T4DNA ligase effect under 4 DEG C of reaction overnights, obtain Transformation plasmid.It adopts
Competent cell is prepared with Competent Cell Preparation Kit kit completion.It is added into competent cell
The Transformation plasmid of 10ng is placed 30 minutes on ice after mixing gently, and 42 DEG C of water-baths are placed 45 seconds, immediately in placement 1~2 on ice
Minute.Be added the LB culture medium of 37 DEG C of pre-temperatures, 37 DEG C shake culture 1 hour, take appropriate bacterium solution to apply with kalamycin resistance
LB solid plate, 37 DEG C be incubated overnight after choose single colonie, as recombination bacillus coli (E.coli BL21 (DE3)/
pET28a)。
Recombination bacillus coli E.coli BL21 (DE3)/pET28a culture: with 1% inoculum concentration, strain is accessed
In 200mL LB culture medium.The group of LB culture medium becomes 10.0g/L tryptone, 5.0g/L yeast powder, 10g/L NaCl.Culture
Condition are as follows: starting pH 7.0, liquid amount volume fraction be 10%, 37 DEG C of cultivation temperature, shaking speed 200rpm, incubation time 6
Hour.Inducer IPTG is added, makes its final concentration of 10mg/mL, continuation is cultivated 2 hours under the conditions of 30 DEG C, 200rpm.
The preparation of crude enzyme liquid: culture terminates the fermentation liquid obtained, be centrifuged in refrigerated centrifuge (4 DEG C, 8000rpm,
Cell 15min) is obtained, supernatant is abandoned, precipitating is resuspended with Tris-HCl buffer (pH 7.5), is sufficiently centrifuged, is repeated after washing
Operate 3 acquisition cells.Cell liquid is handled using Ultrasonic Cell Disruptor, cell crushing instrument probe is placed in 1cm under liquid level, breaks
Fringe part is ultrasound 2 seconds, is spaced 4 seconds, ultrasound 40 times, power 200W.Then 4 DEG C, 12,000rpm centrifugation 15min removal it is insoluble
Property cell fragment, supernatant is amine dehydrogenase crude enzyme liquid.
(2) the pure enzyme preparation of amine dehydrogenase: amine dehydrogenase crude enzyme liquid is separated using the His Trap nickel column of GE company
Purifying, and ultrafiltration desalination is carried out with the ultra-filtration centrifuge tube of the 10K of PALL company.The purification column that the purification process uses is energy
Enough HisTrap HP columns of protein of the specificity purifying with His-tagged label, step include balance, loading, put down
Weighing apparatus, elution, pillar regeneration；It collects the part of elution and carries out desalination using ultra-filtration centrifuge tube；The liquid obtained after desalination is
The pure enzyme solution body of amine dehydrogenase.And be added thereto with Tris-HCl (0.05M, pH 7.0) buffer, it is prepared into containing amine dehydrogenation
The amine dehydrogenase solution of enzyme, and adjusting amine dehydrogenase concentration therein as needed is that 0.05~50mg/mL is spare.
(3) by glass-carbon electrode (GCE) that diameter is 3mm successively with 1.0 μm of diameter, 0.3 μm, 0.05 μm of Al2O3Powder is thrown
Then light is successively cleaned by ultrasonic 3min in dehydrated alcohol and distilled water with distilled water flushing at mirror surface, with being dried with nitrogen.Again
Glass-carbon electrode is placed in 10mL potassium ferricyanide solution (1mM K3Fe(CN)6+ 10mM KCl) electrode activation is carried out, it takes out with steaming
Distilled water is rinsed, and dries to obtain pretreated glass-carbon electrode at room temperature.
(4) preparation of enzyme electrode decorative material rGO-PEI: Hummer method prepares graphene oxide, after ultrasonic disperse 3h
To finely dispersed GO solution.Under stiring, the straight chain PEI for sequentially adding 45mL water, 50mg KOH and 300mg 22KDa, is stirred
It mixes uniformly, is slowly added to the GO solution of 4.6mL 4.9mg/mL.After (80 DEG C) reflux for 24 hours of heating, 8mL 2mol/L boron hydrogen is added
Change sodium to be restored, the reaction was continued 2h.After the reaction was completed, by centrifugation, drying to obtain rGO-PEI, deionized water weight is added
The outstanding rGO-PEI solution for being prepared into 0.05mg/ml.As control, GO is directly added into 8mL 2mol/L sodium borohydride and is restored
2h obtains rGO.
(5) prepared by rGO-PEI-AmDH: the amine dehydrogenase solution for being 0.05mg/ml by enzyme concentration obtained in step (2)
It is added with the ratio of volume ratio 4:1 into the rGO-PEI solution of 0.05mg/ml obtained in step 4) at 4 DEG C, then will be mixed
Object is closed to be centrifuged 15 minutes under 8000rpm, rinses, is resuspended in ammonium hydroxide-ammonium chloride buffer of the pH8.0 of 0.1M, preparation contains
The rGO-PEI-AmDH mixed solution of 0.25mg/ml amine dehydrogenase.
(6) 5.0 μ L rGO-PEI-AmDH solution are taken, the surface GCE that drop coating is handled well in above-mentioned (3) is dried at 4 DEG C,
Modified electrode is obtained, rGO-PEI-AmDH-GCE is denoted as.As control, rGO modified electrode is prepared with drop-coating, is denoted as rGO-
GCE。
(7) enzyme electrode performance test uses standard three electrode system: obtained amine dehydrogenase electrode rGO-PEI-AmDH-
GCE is working electrode, and platinum electrode is to electrode, and calomel electrode is reference electrode, carries out electrochemistry experiment at room temperature.Modified electrode
The test of rGO-PEI-AmDH-GCE is in the 10ml phosphate buffer (0.02mM, pH 7.0) that 0.4mg coenzyme NAD H is added
It carries out, N is passed through before test2, cyclic voltammetry is used in test process, wherein substrate amine is not added dropwise for blank control.1,3- bis-
The additional amount of methylbutylamine is 0~0.4mM.The sweep speed of cyclic voltammetry be 50mV/s, scanning range -3V~3V, in
It is carried out in the buffer solution that pH is 7.0, the time of timing Ampere currents test is 0~1200s, carries out an amine at interval of 30s
Sample-adding.
It is 1 μM~2mM that concentration, which is added, in 1,3- dimethyl butylamine, and cyclic voltammogram obtained by the performance test of gained enzyme electrode is such as
Shown in Fig. 2.Wherein the 1,3- dimethyl butyrate amine concentration that is corresponding in turn to from low to high of oxidation curve right end be 0mM, 0.05mM,
0.15mM, 0.2mM, it will thus be seen that the enzyme electrode rGO-PEI-AmDH-GCE of the present embodiment can preferably detect the concentration of organic amine,
With the increase of amine concentration, there is the trend increased in oxidation peak, and catalytic current is smaller.Show that enzyme electrode of the present invention can be to solution
In organic amine generate sensitive current-responsive.RGO-GCE modified electrode test as control does not have electric current to ring substrate amine
It answers.
Embodiment 2
(1)~(3) the step of experimental procedure such as embodiment 1 (1)~(3).
(4) preparation of enzyme electrode decorative material rGO-PEI: Hummer method prepares graphene oxide, after ultrasonic disperse 3h
To finely dispersed GO solution.Under stiring, the branch PEI of 100mL water, 200mg KOH and 300mg 187KDa is sequentially added,
It stirs evenly, is slowly added to the GO solution of 4.6mL 4.9mg/mL.After (80 DEG C) reflux for 24 hours of heating, 8mL 2mol/L boron is added
Sodium hydride is restored, the reaction was continued 2h.After the reaction was completed, by centrifugation, drying to obtain rGO-PEI, it is prepared into 0.1mg/
The rGO-PEI solution of mL.
(5) rGO-PEI-AmDH prepare: by enzyme concentration obtained in step (2) be 10mg/ml amine dehydrogenase solution 4
It is added with the ratio of volume ratio 1:1 into the rGO-PEI solution of 0.1mg/mL obtained in step 4) at DEG C, then by mixture
It to be centrifuged 15 minutes under 8000rpm, rinses, is resuspended in ammonium hydroxide-ammonium chloride buffer of the pH10.0 of 0.1M, preparation contains 2mg/
The rGO-PEI-AmDH mixed solution of ml amine dehydrogenase.
(6) 5.0 μ L rGO-PEI-AmDH solution are taken, the surface GCE that drop coating is handled well in above-mentioned (3) is dried at 4 DEG C,
Modified electrode is obtained, rGO-PEI-AmDH-GCE is denoted as.As control, rGO modified electrode is prepared with drop-coating, is denoted as rGO-
GCE。
(7) enzyme electrode performance test uses standard three electrode system: obtained amine dehydrogenase electrode rGO-PEI-AmDH-
GCE is working electrode, and platinum electrode is to electrode, and calomel electrode is reference electrode, carries out electrochemistry experiment at room temperature.Modified electrode
RGO-PEI-AmDH-GCE test be added 20mg coenzyme NAD H 10ml phosphate buffer (0.02mM, pH 7.0) in into
Row is passed through N before test2, cyclic voltammetry is used in test process, wherein substrate amine is not added dropwise for blank control.Cyclic voltammetric
The sweep speed of test is 50mV/s, scanning range -0.8V~0.8V, is carried out in the buffer solution that pH is 7.0, timing ampere
The time of testing current is 0~1200s, carries out amine sample-adding at interval of 30s, ortho-methyl aniline be added concentration be 1 μM~
5mM。
For the present embodiment when amine concentration is 2mM, the oxidation peak current value of measurement is 9 μ A.
Embodiment 3
(1)~(3) the step of experimental procedure such as embodiment 1 (1)~(3).
(4) preparation of enzyme electrode decorative material rGO-PEI: Hummer method prepares graphene oxide, after ultrasonic disperse 3h
To finely dispersed GO solution.Under stiring, 7mL water, 100mg KOH and 200mg PEI are sequentially added, is stirred evenly, slowly
The 4.9mg/mL GO solution of 4.6mL is added.After (80 DEG C) reflux for 24 hours of heating, 8mL 2mol/L sodium borohydride is added and is gone back
Original, the reaction was continued 2h.After the reaction was completed, by centrifugation, drying to obtain rGO-PEI, the rGO-PEI for being prepared into 1mg/ml is molten
Liquid.The polyethyleneimine, the straight chain PEI, concentration 50mg/ml for being 217KDa including molecular weight ranges；
(5) rGO-PEI-AmDH prepare: by enzyme concentration obtained in step (2) be 5mg/mL amine dehydrogenase solution 4
Ratio at DEG C with 2:1 is added into the rGO-PEI solution of 1mg/ml obtained in step 4), then by mixture with
It is centrifuged 15 minutes under 8000rpm, rinses, be resuspended in ammonium hydroxide-ammonium chloride buffer of the pH9.0 of 0.08M, preparation contains 1mg/ml
The rGO-PEI-AmDH mixed solution of amine dehydrogenase.
(6) 5.0 μ L rGO-PEI-AmDH solution are taken, the surface GCE that drop coating is handled well in above-mentioned (3) is dried at 4 DEG C,
Modified electrode is obtained, rGO-PEI-AmDH-GCE is denoted as.As control, rGO modified electrode is prepared with drop-coating, is denoted as rGO-
GCE。
(7) enzyme electrode performance test uses standard three electrode system: obtained amine dehydrogenase electrode rGO-PEI-AmDH-
GCE is working electrode, and platinum electrode is to electrode, and calomel electrode is reference electrode, carries out electrochemistry experiment at room temperature.Modified electrode
RGO-PEI-AmDH-GCE test be added 20mg coenzyme NAD H 10ml phosphate buffer (0.02mM, pH 7.0) in into
Row is passed through N before test2, cyclic voltammetry is used in test process, wherein substrate amine is not added dropwise for blank control.Cyclic voltammetric
The sweep speed of test is 100mV/s, scanning range -1.0V~1.0V, is carried out in the buffer solution that pH is 7.0, timing peace
The time for training testing current is 0~1200s, carries out an amine sample-adding at interval of 30s, it is 1 μM that concentration, which is added, in 2- aminoanisole
~5mM.
For the present embodiment when amine concentration is 1mM, the oxidation peak current value of measurement is 5 μ A.
Embodiment 4
(1)~(3) the step of experimental procedure such as embodiment 1 (1)~(3).
(4) preparation of carbon nanotube electrode material: clip is enriched with the square sheets that the length and width of carbon nanotube are 2mm, is placed on
On clean plate, drop coating 5 μ L NADH is dry at 4 DEG C, repeatedly for three times, obtains the carbon nanotube pieces of NADH modification.5 μ L of drop coating
The amine dehydrogenase solution of 10mg/ml, it is dry at 4 DEG C, repeatedly for three times to get arriving AmDH-NADH-CNT decorative layer；Utilize carbon nanometer
The space caliber characteristic of pipe, NADH and amine dehydrogenase are adsorbed on the carbon nanotubes.AmDH-NADH-CNT decorative layer is sealed
Glue is fixed on the glass-carbon electrode handled well in 1), obtains AmDH-NADH-CNT-GCE enzyme electrode.
(5) enzyme electrode performance test uses standard three electrode system: obtained amine dehydrogenase electrode A mDH-NADH-CNT-
GCE is working electrode, and platinum electrode is to electrode, and calomel electrode is reference electrode, carries out electrochemistry experiment at room temperature.Modified electrode
Test carry out in the 10mL phosphate buffer (0.02mM, pH 7.0), N is passed through before test2, using circulation in test process
Voltammetry, wherein substrate amine is not added dropwise for blank control.The sweep speed of cyclic voltammetry is 50mV/s, scanning range -0.6V
~0.6V, in pH be 7.0 buffer solution in carry out, timing Ampere currents test time be 0~1200s, at interval of 30s into
Amine sample-adding of row, it is 1 μM~5mM that concentration, which is added, in 1,3- dimethyl butylamine.
Embodiment 5
(1)~(3) the step of experimental procedure such as embodiment 1 (1)~(3).
(4) preparation of carbon nanotube electrode material: clip is enriched with the square sheets that the length and width of carbon nanotube are 2mm, is placed on
On clean plate, the amine dehydrogenase solution of the 50mg/mL of 5 μ L of drop coating is dry at 4 DEG C, repeatedly for three times to get arriving AmDH-CNT
Decorative layer；Using the space caliber characteristic of carbon nanotube, amine dehydrogenase is adsorbed on the carbon nanotubes.By AmDH-CNT decorative layer
It is fixed on the glass-carbon electrode handled well in 1) with sealing compound, obtains AmDH-CNT-GCE enzyme electrode.
(5) enzyme electrode performance test uses standard three electrode system: obtained amine dehydrogenase electrode A mDH-CNT-GCE for
Working electrode, platinum electrode are to electrode, and calomel electrode is reference electrode, carries out electrochemistry experiment at room temperature.The survey of modified electrode
It tries to carry out in the 10mL phosphate buffer (0.02mM, pH 7.0) that 20mg coenzyme NAD H is added, N is passed through before test2, test
Cyclic voltammetry is used in the process, and wherein substrate amine is not added dropwise for blank control.The sweep speed of cyclic voltammetry is 50mV/s,
Scanning range -1.5~1.5V, in pH be 7.0 buffer solution in carry out, timing Ampere currents test time be 0~
1200s carries out an amine sample-adding at interval of 30s, and it is 1 μM~5mM that concentration, which is added, in phenyl ethylamine.
Cyclic voltammogram obtained by the performance test of gained enzyme electrode is as shown in Figure 3.Wherein aoxidize curve right end from low to high
The amine concentration being corresponding in turn to is 0mM, 0.5mM, 1.0mM, 1.5mM, it will thus be seen that the enzyme electrode AmDH-CNT-GCE of the present embodiment
The concentration that organic amine can preferably be detected, with the increase of amine concentration, there is the trend increased in oxidation peak, and catalytic current is smaller.Table
Bright enzyme electrode of the present invention can generate sensitive current-responsive to the organic amine in solution.
Embodiment 6
(1)~(3) the step of experimental procedure such as embodiment 1 (1)~(3).
(4) preparation of enzyme electrode decorative material rGO-PEI: Hummer method prepares graphene oxide, after ultrasonic disperse 3h
To finely dispersed GO solution.Under stiring, 80mL water, 100mg KOH and 150mg PEI are sequentially added, is stirred evenly, slowly
The GO solution of 4.6mL 4.9mg/mL is added.After (80 DEG C) reflux for 24 hours of heating, 8mL 2mol/L sodium borohydride is added and is gone back
Original, the reaction was continued 2h.After the reaction was completed, by centrifugation, drying to obtain rGO-PEI, the rGO-PEI for being prepared into 0.5mg/mL is molten
Liquid.The polyethyleneimine, the branch PEI, concentration 40mg/ml for being 25KDa including molecular weight ranges；
(5) rGO-PEI-AmDH prepare: by enzyme concentration obtained in step (2) be 2mg/ml amine dehydrogenase solution 4
Ratio at DEG C with 2:1 is added into the rGO-PEI solution of 0.5mg/mL obtained in step 4), then by mixture with
It is centrifuged 15 minutes under 8000rpm, rinses, be resuspended in ammonium hydroxide-ammonium chloride buffer of the pH8.5 of 0.07M, preparation contains 0.5mg/
The rGO-PEI-AmDH mixed solution of ml amine dehydrogenase.
(6) 5.0 μ L rGO-PEI-AmDH solution are taken, the surface GCE that drop coating is handled well in above-mentioned (3) is dried at 4 DEG C,
Modified electrode is obtained, rGO-PEI-AmDH-GCE is denoted as.As control, rGO modified electrode is prepared with drop-coating, is denoted as rGO-
GCE。
(7) enzyme electrode performance test uses standard three electrode system: obtained amine dehydrogenase electrode rGO-PEI-AmDH-
GCE is working electrode, and platinum electrode is to electrode, mercury | mercury oxide is reference electrode, carries out electrochemistry experiment at room temperature.Modification electricity
The test of pole rGO-PEI-AmDH-GCE is in the 10ml phosphate buffer (0.02mM, pH 7.0) that 20mg coenzyme NAD H is added
It carries out, N is passed through before test2, cyclic voltammetry is used in test process, wherein substrate amine is not added dropwise for blank control.Circulation volt
The sweep speed of peace test is 100mV/s, scanning range -1.2V~1.2V, is carried out in the buffer solution that pH is 7.0, timing
The time of Ampere currents test is 0~1200s, carries out an amine sample-adding at interval of 30s, it is 1 μM that concentration, which is added, in ortho-methyl aniline
~5mM.
For the present embodiment when amine concentration is 3mM, the oxidation peak current value of measurement is 5 μ A.
The above is only the preferred embodiment of the present invention, the range implemented of the present invention that therefore, it cannot be limited according to, i.e., according to
Equivalent changes and modifications made by the invention patent range and description, should still be within the scope of the present invention.
Sequence table
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Claims (10)
1. a kind of amine dehydrogenase electrode, it is characterised in that: including basal electrode, electronic conductive layer and amine dehydrogenase layer；The electricity
Sub- conducting shell is redox graphene-PEI composite material or carbon nanotube；The gene order of the amine dehydrogenase such as SEQ ID
Shown in NO.1.
2. amine dehydrogenase electrode according to claim 1, it is characterised in that: the basal electrode is glass-carbon electrode, pyrolysis
Graphite electrode, carbon paste electrode or metal electrode.
3. a kind of preparation method of amine dehydrogenase electrode, it is characterised in that: include:
(1) graphene oxide is adsorbed into PEI or graphene oxide covalence graft PEI, is restored to obtain redox graphene-
PEI composite material, the redox graphene-PEI composite material again with amine dehydrogenase mixed immobilization；Or by carbon nanotube
With amine dehydrogenase mixed immobilization；The gene order of the amine dehydrogenase is as shown in SEQ ID NO.1；
(2) by the product after redox graphene-PEI composite material immobilization amine dehydrogenase or by carbon nanotube immobilization amine
Product after dehydrogenase is compound in basal electrode surface, obtains amine dehydrogenase electrode.
4. the preparation method of amine dehydrogenase electrode according to claim 3, it is characterised in that: the basal electrode is glass carbon
Electrode, pyrolytic graphite electrode, carbon paste electrode or metal electrode；The basal electrode is pre- when being glass-carbon electrode or pyrolytic graphite electrode
First pass through surface preparation, process for surface preparation include: by glass-carbon electrode or pyrolytic graphite electrode successively use 1.0 μm of diameter,
0.3 μm, 0.05 μm of Al2O3Powder is polished to mirror surface, with distilled water flushing, then successively surpasses in dehydrated alcohol and distilled water
Sound cleaning, then glass-carbon electrode or pyrolytic graphite electrode are placed in containing 1~5mM K3Fe(CN)6With the solution of 10~200mM KCl
Middle carry out electrode activation takes out and uses distilled water flushing, dries to obtain pretreated basal electrode at room temperature.
5. the preparation method of amine dehydrogenase electrode according to claim 3, it is characterised in that: the polyethyleneimine is point
The polyethyleneimine for the linear chain or branched chain that son amount range is 20~500kDa, the polyethyleneimine are directly used or are utilized
Ammonium hydroxide-ammonium chloride buffer of the pH 8.0~10 of the phosphate buffer or 1~100mmol/L of pH6.0~8.0, is configured to dense
The polyethylenimine solution that degree is 5~200mg/ml uses.
6. the preparation method of amine dehydrogenase electrode according to claim 3, it is characterised in that: the reduction-oxidation graphite
Alkene-PEI composite material and preparation method thereof includes: that 7~100mL water, 50~200mg KOH and 150~300mg PEI stirring is equal
It is even, the finely dispersed GO solution of 4~10mL, 4~6mg/mL is added, after 75~85 DEG C are heated to reflux 22~26h, addition 6~
10mL 1~3mol/L sodium borohydride is restored, the reaction was continued 1~3h；After the reaction was completed, also by centrifugation, drying to obtain
Former graphene oxide-PEI composite material rGO-PEI.
7. the preparation method of amine dehydrogenase electrode according to claim 3, it is characterised in that: the preparation of the amine dehydrogenase
Method includes: the engineering bacteria that amine dehydrogenase can be expressed using the preparation of the amine dehydrogenase gene as shown in SEQ ID NO.1；It will be above-mentioned
Engineering bacteria inoculation, is added inducer, and culture gained fermentation liquid centrifugation obtains cell；Cell ultrasonication, centrifugation, obtains containing amine
The crude enzyme liquid of dehydrogenase；The above-mentioned crude enzyme liquid containing amine dehydrogenase is subjected to purification desalination, obtains amine dehydrogenase.
8. the preparation method of amine dehydrogenase electrode according to claim 3, it is characterised in that: the reduction-oxidation graphite
It is to go back described in 0.05~1mg/ml that the preparation method of alkene-PEI composite material and amine dehydrogenase mixed immobilization, which includes: by concentration,
Former graphene oxide-PEI composite material and concentration are the amine dehydrogenase solution of 0.05~10mg/ml according to volume ratio 1:1~4
Ratio mixing, 6000~10000rpm are centrifuged 10~20min, rinse, are resuspended in the ammonia of the pH 8.0~10.0 of 0.05~0.1M
In water-ammonium chloride buffer, the mixed solution of the amine dehydrogenase Han 0.25~50mg/ml, as redox graphene-are prepared
Product after PEI composite material immobilization amine dehydrogenase；
Or, the carbon nanotube and the preparation method of amine dehydrogenase mixed immobilization include: on the carrier of enrichment carbon nanotube
Amine dehydrogenase solution is added, it is dry, it adds amine dehydrogenase solution and drying steps several times repeatedly, obtains amine dehydrogenase-carbon and receive
Mitron modifier, the as product after carbon nanotube immobilization amine dehydrogenase.
9. a kind of application of amine dehydrogenase electrode in the amine concentration in measurement solution, it is characterised in that: the survey of the amine concentration
Surely three-electrode system is used, using amine dehydrogenase electrode of any of claims 1 or 2 as working electrode, or according to claim 3
It is working electrode to amine dehydrogenase electrode prepared by preparation method described in any one of 8；Reference electrode is saturation calomel electricity
Pole, hydrogen electrode, silver | silver chloride electrode or mercury | mercuric oxide electrode；It is platinum electrode or carbon electrode to electrode.
10. application according to claim 9, it is characterised in that: measured in a manner of cyclic voltammetric and/or chrono-amperometric molten
Amine concentration in liquid；The test potential sweep speed of the cyclic voltammetric mode is 25~200mV/s；Being added, 1~20mg is auxiliary
It is carried out in the buffer of enzyme NADH, N is passed through before test2, the amine is phenyl ethylamine, ortho-methyl aniline, 1,3- dimethyl butyrate
Amine, 2- aminoanisole；The concentration range of the amine is 50ppm~5mM；The buffer is phosphate buffer, borate
At least one of buffer, citrate buffer, carbonate buffer solution and acetate buffer.
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