CN103954666B - Preparation method for enzyme sensor and method for determining hydrogen peroxide - Google Patents
Preparation method for enzyme sensor and method for determining hydrogen peroxide Download PDFInfo
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- CN103954666B CN103954666B CN201410212146.2A CN201410212146A CN103954666B CN 103954666 B CN103954666 B CN 103954666B CN 201410212146 A CN201410212146 A CN 201410212146A CN 103954666 B CN103954666 B CN 103954666B
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- horseradish peroxidase
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Abstract
The invention relates to a preparation method for an enzyme sensor and a method for determining hydrogen peroxide. According to the method, a high catalytic performance of horse radish peroxidase to hydrogen peroxide is used and a signal enhancement effect of a carbon nano tube is utilized. According to the method, the enzyme sensor is prepared by loading hydrotalcite-enzyme on the surface of a carbon nano tube modified basic electrode through electrolytic deposition; a simple electrolytic deposition method is used for preparing hydrotalcite and fixing the horse radish peroxidase on the surface of an electrode so that the enzyme is tightly loaded on the surface of the electrode; the fixation of the enzyme and direct electron transmission of the enzyme are easy to realize.
Description
Technical field
The present invention relates to enzyme sensor and technical field of electroanalytical chemistry detection, more particularly, to one kind is with brucite as carrier
Measure the preparation method of enzyme sensor and its mensure of hydrogen peroxide.
Background technology
Brucite is the tradable anion institute by positively charged metal hydroxidess and layer and interlayer filling for the class
The lamellar compound constituting.Because brucite has, strong adsorptivity, specific surface area be big, biocompatibility is good and ion exchangeable
Can be good the features such as, it is constantly subjected to the extensive concern of people in recent years, and is applied to the load as immobilized enzyme in enzyme sensor
As dressing agent in body and chemically modified electrode.But because the weak inductive of brucite is unfavorable for the electronics of brucite modified electrode
Transmission, reduces the chemical property of electrode material, limits its greatly developing in electrochemical field.Recently, golden nanometer particle,
The nano material that CNT and Graphene etc. have satisfactory electrical conductivity is incorporated in brucite modified electrode and improves its electrochemistry
Performance.Build repairing of mensure methanol as wang etc. prepares golden nanometer particle-brucite composite nano materials by coprecipitation method
Decorations electrode, due to the synergism of golden nanometer particle and brucite, the peak current of this electrode catalyst Oxidation of Methanol is much larger than neatly
Peak current (wang y., zhang d., tang m., xu s., the li m. of stone modified electrodeelectrochimica acta, 2010,55:4045-4049.).
Since CNT self-discovery, received much concern with its unique structure, electrochemistry and mechanical performance, due to
Its diameter is little, and high surface energy and Atomic coordinate are not enough, very high to the Surface atomic mobility making CNT, and electronics easily occurs
Transferance, due to its unique structure so as to have big specific surface area and preferable biocompatibility, is good electrification
Learn nano material.
Hydrogen peroxide is a kind of important chemical products, also assists in the enzymatic reaction of peroxidase simultaneously, it biological,
Environmental protection, food, the quantitative determination in the field such as medical and industrial have great importance.With respect to traditional hydrogen peroxide determination side
The method such as method such as ultraviolet visible spectrophotometry, chemiluminescence and fluorescence, based on the electrochemical sensor method of enzyme electrode, because of it
Sensitivity is high, and the good feature of fast response time, selectivity, by the pro-gaze of vast researcher.As chen etc. will by drop-coating
Horseradish peroxidase and nickel aluminum hydrotalcite are fixed on glassy carbon electrode surface, the enzyme sensor (chen of formation determination hydrogen peroxide
x., fu c., wang y., yang w., evans d.g., biosensors and bioelectronics, 2008,
24:356-361), but due to the shortcoming in the method for immobilized enzyme, enzyme is made to be easy to come off, protected from environmental big, limit
Make its application.
At present, the carrier with brucite as immobilized enzyme is prepared hydrogen peroxide sersor and is used for detecting peroxidating
The report of hydrogen, is all to first pass through coprecipitation or hydro-thermal method synthetic hydrotalcite, then adopts drop-coating or the preparation of layer assembly method
Hydrogen peroxide sersor.Said method is loaded down with trivial details, complexity is time-consuming, and patent of the present invention passes through simple electrodeposition process by Radix Cochleariae officinalises
Peroxidase is fixed in hydrotalcite supports prepares hydrogen peroxide sersor, prepares water by simple electrodeposition process
Horseradish peroxidase is fixed on electrode surface by Talcum makes enzyme closely be supported on electrode surface, be conducive to enzyme immobilizatio and
The direct electron transfer of enzyme；In addition, CNT is incorporated into basic electrode surface by patent of the present invention, improves brucite and modify
The electric conductivity of electrode and improve the performance of enzyme sensor.
Content of the invention
The purpose of the present invention is to answer for the method preparing hydrogen peroxide sersor with brucite for carrier at present
The shortcoming of the poorly conductive of miscellaneous, loaded down with trivial details and brucite modified electrode, by a simple step electrodeposition process carbon nano tube modified
Basic electrode surface is prepared for the hydrogen peroxide sersor with brucite as carrier and is used for detecting hydrogen peroxide.
Realization the technical scheme is that, the present invention by the method for drop coating first by carbon nano tube modified in electrode table
Then brucite and horseradish peroxidase are fixed on carbon nano tube modified electrode surface by a step electrodeposition process by face,
Using horseradish peroxidase to the catalysis reduction of hydrogen peroxide and the direct electron transfer of enzyme, establish a kind of new mensure
The method of hydrogen oxide.
The present invention prepares the preparation method of enzyme sensor with brucite for carrier, and methods described step is as follows:
(1) 0.3～2.0 mg/ml carbon nano tube suspension taking 2～15 μ l scattered with microsyringe is uniform
Deca, in clean glassy carbon electrode surface, is dried at room temperature naturally, and deionized water is rinsed well, obtains carbon nano tube modified
Electrode.
(2) first prepare and contain cobalt nitrate 15～25 mmol/l, aluminum nitrate 5～10 mmol/l, potassium nitrate 0.1～0.5 mol/
Then carbon nano tube modified electrode is placed in electrodeposit liquid by l and the electrodeposit liquid of horseradish peroxidase 0.3～1.5 mg/ml
In carry out potentiostatic electrodeposition under -0.6～-1.2 v, the time is 10～80s, deposition terminate after deionized water rinse dry
Only, obtain brucite-horseradish peroxidase/carbon nano tube modified electrode, this horseradish peroxidase modified electrode is to be surveyed
Determine the enzyme sensor of hydrogen peroxide.
The present invention prepares enzyme sensor with brucite for carrier and detects that the method for hydrogen peroxide is as follows:
The present invention utilizes horseradish peroxidase that the catalysis of hydrogen peroxide is reduced with the direct electron and enzyme and electrode between
Transmission, using aforementioned horseradish peroxidase modified electrode as working electrode, reference electrode is ag/agcl electrode, auxiliary electrode
For platinum electrode, form three-electrode system, you can realize the detection to hydrogen peroxide.
The range of linearity that this sensor measures hydrogen peroxide is 2.5 × 10-6～7.85 × 10-4Mol/l, detection is limited to
1.6×10-6Mol/l, sensitivity is 0.00881 a l/mol.With 5 modified electrodes with a collection of preparation to 0.25mmol/l
h2o2It is measured, its relative standard deviation is 3.2%.5 detections are carried out using same modified electrode to hydrogen peroxide, it is right
The relative standard deviation of hydrogen peroxide response is 2.1%.Enzyme modified electrode is in 0.25mmol/l h2o2Examine in the environment being placed in 4 DEG C
Examine its stability, after surrounding, still retain the 83% of response current value.Show that this electrode has good repeatability and stability.
The invention has the beneficial effects as follows, the present invention utilizes bigger serface, good electric conductivity and the biology of CNT
Horseradish peroxidase is supported on neatly by a step electrodeposition process on carbon nano tube modified basic electrode surface by the compatibility
The Direct Electrochemistry of enzyme is achieved on stone and is used for measuring hydrogen peroxide.
Brief description
Fig. 1 is the cyclic voltammogram in 0.2 mol/l pbs (ph 7.0) for the enzyme modified electrode in the present invention, (a) not
Plus hydrogen peroxide, (b) adds 125 μm of ol/l hydrogen peroxide；
Fig. 2 is the cyclic voltammetry curve of different modifying electrode in the present invention, (a) brucite modified electrode, (b) brucite/
Carbon nano tube modified electrode, (c) horseradish peroxidase/carbon nano tube modified electrode, (d) brucite-horseradish peroxidase/
Carbon nano tube modified electrode；
Fig. 3 is the chronoa mperometric plot to hydrogen peroxide for the enzyme modified electrode；
The calibration curve to concentration of hydrogen peroxide for the current-responsive of Fig. 4 enzyme modified electrode.
Specific embodiment
Below by specific embodiment, the present invention is described in detail, and following examples contribute to those skilled in the art
Member further understands the present invention, but is never limited in protection scope of the present invention.
Embodiment 1
Enzyme sensor is prepared for carrier with brucite.
(1) take the scattered uniform Deca of 0.5 mg/ml carbon nano tube suspension of 3 μ l clean with microsyringe
Glassy carbon electrode surface, naturally dry at room temperature, deionized water is rinsed well, obtain carbon nano tube modified electrode.
(2) first prepare containing cobalt nitrate 15 mmol/l, aluminum nitrate 5 mmol/l, potassium nitrate 0.1 mol/l and Radix Cochleariae officinalises peroxidating
The electrodeposit liquid of thing enzyme 0.3 mg/ml, then carbon nano tube modified electrode is placed in electrodeposit liquid and carries out perseverance under -0.6 v
Potential deposition, the time be 80s, deposition terminate after deionized water rinse well, obtain brucite-horseradish peroxidase/
Carbon nano tube modified electrode, this horseradish peroxidase modified electrode is the enzyme sensor measuring hydrogen peroxide.
Embodiment 2
Enzyme sensor is prepared for carrier with brucite.
(1) take the scattered uniform Deca of 0.5 mg/ml carbon nano tube suspension of 5 μ l clean with microsyringe
Glassy carbon electrode surface, naturally dry at room temperature, deionized water is rinsed well, obtain carbon nano tube modified electrode.
(2) first prepare containing cobalt nitrate 21 mmol/l, aluminum nitrate 7 mmol/l, potassium nitrate 0.2 mol/l and Radix Cochleariae officinalises peroxidating
The electrodeposit liquid of thing enzyme 0.5 mg/ml, then carbon nano tube modified electrode is placed in electrodeposit liquid and carries out perseverance under -0.8 v
Potential deposition, the time be 70s, deposition terminate after deionized water rinse well, obtain brucite-horseradish peroxidase/
Carbon nano tube modified electrode, this horseradish peroxidase modified electrode is the enzyme sensor measuring hydrogen peroxide.
Embodiment 3
Enzyme sensor is prepared for carrier with brucite.
1) take the scattered uniform Deca of 1.0 mg/ml carbon nano tube suspension of 5 μ l clean with microsyringe
Glassy carbon electrode surface, dries at room temperature naturally, and deionized water is rinsed well, obtains carbon nano tube modified electrode.
(2) first prepare containing cobalt nitrate 22.5 mmol/l, aluminum nitrate 7.5 mmol/l, potassium nitrate 0.3 mol/l and Radix Cochleariae officinalises mistake
Then carbon nano tube modified electrode is placed in electrodeposit liquid to enter under -0.9 v by the electrodeposit liquid of oxide enzyme 1.0 mg/ml
Row potentiostatic electrodeposition, the time is 30s, and after deposition terminates, deionized water is rinsed well, obtains brucite-horseradish peroxidase
Enzyme/carbon nano tube modified electrode, this horseradish peroxidase modified electrode is the enzyme sensor measuring hydrogen peroxide.
Embodiment 4
Electro-chemical test.
The enzyme modified electrode that embodiment 3 is obtained carries out electro-chemical test:
(1) cyclic voltammetry of enzyme modified electrode.
As working electrode, reference electrode is ag/agcl electrode to enzyme modified electrode, and auxiliary electrode is platinum electrode；Bottom liquid is
0.2 mol/l pbs (ph 7.0) and the hydrogen peroxide having added 125 μm of ol/l；Scanning speed is 0.1 v/s.Cyclic voltammogram
See Fig. 1, it can be seen from figure 1 that after adding hydrogen peroxide, the peak current of reduction peak increases, the peak current of oxidation peak reduces, and illustrates that enzyme is repaiied
Decorations electrode pair hydrogen peroxide creates catalysis reduction, and catalytic current is to be reduced by the horseradish peroxidase enzyme catalytic in enzyme electrode
Hydrogen oxide produces.
(2) cyclic voltammetric of different modifying electrode characterizes.
Respectively by brucite modified electrode, brucite/carbon nano tube modified electrode, horseradish peroxidase/CNT
Modified electrode and brucite-horseradish peroxidase/carbon nano tube modified electrode are working electrode, and reference electrode is ag/agcl
Electrode, auxiliary electrode is platinum electrode；Bottom liquid is 0.2 mol/l pbs (ph 7.0), and potential range is 0.4 ~ -0.8 v, sweeps
Speed is 100m v/s；Cyclic voltammetry result is shown in Fig. 2.It can be seen that the direct electro-deposition modification electricity of brucite
(curve a) is not have obvious redox peaks to occur in this potential region for pole；And brucite/carbon nano tube modified electrode is (bent
Line b) occurs in that a pair less redox peaks, and this is one layer of CNT and the peak that occurs in electrode face finish, this
When because the effect of CNT is so as to charging current becomes big；Horseradish peroxidase/carbon nano tube modified electrode (curve c)
After being one layer of horseradish peroxidase in carbon nano tube surface Deca, -0.097v and -0.293v occur a pair obvious
Reversible redox peak, this is produced by fe () in pbs solution for the horseradish peroxidase/fe () oxidoreduction
Reversible peak, but make horseradish peroxidase be fixed on the method for carbon nano tube surface when carrying out Electrochemical Detection by drop-coating
Easily come off, the stability tested can be led to poor；(curve d) leads to brucite-horseradish peroxidase/carbon nano tube modified electrode
Cross the fixing horseradish peroxidase of electro-deposition brucite method in carbon nano tube modified electrode surface, make the oxidoreduction peak current of enzyme
Become much larger, and also ensure that the stability of experiment.
(3) the chrono-amperometric test of enzyme modified electrode
Brucite-horseradish peroxidase/carbon nano tube modified electrode is working electrode, reference electrode is ag/agcl
Electrode, auxiliary electrode is platinum electrode；Bottom liquid is 0.2 mol/l pbs (ph 7.0)；Mensure current potential is -0.3 v, stirs in magnetic force
Mix down, the certain density hydrogen peroxide of continuous Deca, measurement result is shown in Fig. 3, Fig. 4.The range of linearity measuring hydrogen peroxide is 2.5
×10-6～7.85 × 10-4Mol/l, detection is limited to 1.6 × 10-6Mol/l, sensitivity is 0.00881 a l/mol.Parallel
The relative standard deviation measuring 5 times is 2.1%, and the response current measuring hydrogen peroxide after surrounding is maintained at more than 83%.
Claims (4)
1. a kind of preparation method of enzyme sensor is it is characterised in that the concretely comprising the following steps of methods described:
(1) take the scattered uniform Deca of 0.3～2.0 mg/ml carbon nano tube suspension of 2～15 μ l with microsyringe
In clean glassy carbon electrode surface, naturally dry at room temperature, deionized water is rinsed well, obtain carbon nano tube modified electricity
Pole；
(2) first prepare containing cobalt nitrate 15～25 mmol/l, aluminum nitrate 5～10 mmol/l, potassium nitrate 0.1～0.5 mol/l and
The electrodeposit liquid of horseradish peroxidase 0.3～1.5 mg/ml, then carbon nano tube modified electrode is placed in electrodeposit liquid
Carry out potentiostatic electrodeposition under -0.6～-1.2 v, the time is 10～80s, and after deposition terminates, deionized water is rinsed well,
Obtain brucite-horseradish peroxidase/carbon nano tube modified electrode, this horseradish peroxidase modified electrode as measured
The enzyme sensor of hydrogen oxide.
2. a kind of preparation method of enzyme sensor according to claim 1 is it is characterised in that described electrodeposit liquid is containing nitre
Sour cobalt 15～25 mmol/l, aluminum nitrate 5～10 mmol/l, potassium nitrate 0.1～0.5 mol/l and horseradish peroxidase 0.3
～1.5 mg/ml.
3. a kind of enzyme sensor according to claim 1 preparation method it is characterised in that described electrodeposition condition be-
Carry out potentiostatic electrodeposition, the time is 10～80s under 0.6～-1.2 v.
4. a kind of enzyme sensor with brucite as carrier detects the method for hydrogen peroxide it is characterised in that methods described utilizes
The catalytic performance to hydrogen peroxide for the horseradish peroxidase, CNT signal enhancing effect, horseradish peroxidase straight
Connect electron transfer characteristic, using horseradish peroxidase modified electrode as working electrode, reference electrode is ag/agcl electrode, auxiliary
Help electrode to be platinum electrode, form three-electrode system, realize the Sensitive Detection to hydrogen peroxide.
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| Title |
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| Ni(OH)2 versus Ni/Al layered double hydroxides as matrices to immobilize glucose oxidase;Lorella Guadagnini,Adriana Mignani,Erika Scavetta, et al;《Electrochimica Acta》;20091105;第55卷(第3期);第1218页左栏第5-8段，即第2.3节 * |
| 碳纳米管生物界面的构筑及其电化学性质的研究;郑卫;《中国优秀博硕士学位论文全文数据库 (博士) 工程科技Ⅰ辑》;20061215;第28页第2段，第29页第1段 * |
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