CN109060918A - Hydroquinone biosensor and its preparation and application based on the compound horseradish peroxidase of nitrogen-doped graphene - Google Patents
Hydroquinone biosensor and its preparation and application based on the compound horseradish peroxidase of nitrogen-doped graphene Download PDFInfo
- Publication number
- CN109060918A CN109060918A CN201811006266.1A CN201811006266A CN109060918A CN 109060918 A CN109060918 A CN 109060918A CN 201811006266 A CN201811006266 A CN 201811006266A CN 109060918 A CN109060918 A CN 109060918A
- Authority
- CN
- China
- Prior art keywords
- nitrogen
- horseradish peroxidase
- hydroquinone
- doped graphene
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
- G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
Abstract
The invention belongs to electrochemica biological sensor technical field, the hydroquinone biosensor based on the compound horseradish peroxidase of nitrogen-doped graphene and its preparation and application are disclosed.The biosensor is formed by reference electrode, to the working electrode after electrode and modification, working electrode after modification is by basal electrode and is solidificated in the material identification film on basal electrode surface and forms, wherein, the material identification film is mainly mixed by nitrogen-doped graphene dispersion liquid, horseradish peroxidase solution and chitosan solution, and film forming is prepared.The invention also discloses the preparation methods of sensor.Preparation method of the invention is simple and at low cost；The sensor of acquisition has good selectivity, sensitivity and stability, has preferable electrocatalysis characteristic；Hydroquinone is accurately detected, strong antijamming capability；There is wider detection range, lower detection limit simultaneously.
Description
Technical field
The invention belongs to electrochemica biological sensor technical fields, and in particular to one kind is compound peppery based on nitrogen-doped graphene
The biosensor and the preparation method and application thereof of the detection hydroquinone of root peroxidase.
Background technique
Phenolic compound is a kind of widely used industrial chemicals, wherein hydroquinone is as the basic of fine chemicals
Raw material is widely used for cosmetics, dyestuff, pesticide, medicine, leather industry etc..But hydroquinone has high toxicity and difficult drop
Xie Xing, therefore, it is identified as environmental contaminants.Influence of the hydroquinone to human body can not be ignored, the hydroquinone of low concentration
It can lead to the symptoms such as headache and tachycardia.Therefore, the method for developing quick sensitive detection hydroquinone has weight
The meaning wanted.There are many kind methods at present for detecting hydroquinone, such as high performance liquid chromatography, gas chromatography,
PH flow injection analysis, synchronous fluorimetry, spectrophotometry etc..But the above-mentioned method referred to mostly has that consuming time is long,
Expensive equipment, the disadvantages of sensitivity is low, sample pre-treatments are cumbersome, in contrast, electrochemical sensor have and have to object
Higher recognition capability, amount of samples is few, response is fast, at low cost, small in size, and convenient for universal advantage, and electrochemical enzymatic is biological
Sensor is widely used again with the features such as its catalytic capability is high, reaction condition is mild, specificity is good, and therefore, research and development are easy, fast
Speed, accurate, high sensitivity hydroquinone electrochemical enzymatic biosensor have major application prospect.
In the preparation process of electrochemical enzymatic biosensor, an extremely important problem for influencing its performance is exactly enzyme
The redox active centre of catalyst is surrounded by one layer of nonconducting protein coat, this protein coat prevents
Enzyme active center and electrode surface carry out electron transmission, affect the performance of electrode；Influence enzyme biologic sensor performance in addition
One key factor is the stability and life problems of enzyme modified electrode, closely related with the fixed form of enzyme catalyst, is adopted
The hot issue that stable enzyme modified electrode is always biosensor researcher research is prepared with suitable enzyme fixing means.
Summary of the invention
In order to solve the disadvantage that the above prior art and shortcoming, the primary purpose of the present invention is that providing one kind has
The hydroquinone based on the compound horseradish peroxidase of nitrogen-doped graphene of good selectivity, sensitivity and stability is biological
Sensor.
Another object of the present invention is to provide above-mentioned based on the compound horseradish peroxidase of nitrogen-doped graphene to benzene
The preparation method of diphenol biosensor.
A further object of the present invention is to provide above-mentioned based on the compound horseradish peroxidase of nitrogen-doped graphene to benzene
Application of the diphenol biosensor in hydroquinone detection.
The object of the invention is achieved through the following technical solutions:
A kind of hydroquinone biosensor based on the compound horseradish peroxidase of nitrogen-doped graphene, by reference electricity
Pole forms the working electrode after electrode and modification, and the working electrode after the modification is by basal electrode and is solidificated in substrate electricity
The material identification film of pole surface forms, wherein the material identification film is mainly by nitrogen-doped graphene dispersion liquid (N-GN), horseradish
Peroxidase Solution (HRP) and chitosan solution (CS) mixing, film forming are prepared；
The nitrogen-doped graphene dispersion liquid (N-GN) is to be dispersed in water to obtain by nitrogen-doped graphene；Horseradish peroxidating
Object enzyme solutions (HRP) are that horseradish peroxidase is dissolved in PBS solution to obtain；Chitosan solution is by chitosan wiring solution-forming
It obtains.
The nitrogen-doped graphene: horseradish peroxidase: the mass ratio of chitosan is (2~20): (15~35): 10.
The nitrogen-doped graphene material is prepared especially by following methods:
(a) graphene oxide and aniline are uniformly dispersed；Under the action of oxidant and in acid medium, aniline occurs poly-
Reaction is closed, is placed in hydrothermal reaction kettle and carries out hydro-thermal reaction, obtain polyaniline-graphene oxide compound；
(b) polyaniline-graphene oxide compound is calcined, obtains nitrogen-doped graphene composite material.
The mass volume ratio of the graphene oxide and aniline is (50~200) mg:0.5mL；
The oxidant is ammonium persulfate, and the acid medium is hydrochloric acid, the molar ratio of the oxidant and acid medium
For (2~2.5): 1, the volume ratio of aniline and acid medium is 1:(10~20)；Acid medium is added in form of an aqueous solutions, acid
Property medium concentration be 0.5mol/L；The temperature of the polymerization reaction is 4~10 DEG C, and the time of polymerization reaction is 6~12h；Water
The temperature of thermal response is 160~200 DEG C, and the hydro-thermal reaction time is 12~20h.
The atmosphere of calcining described in step (b) is protectiveness atmosphere, and the protectiveness atmosphere is nitrogen or inert atmosphere, institute
It states calcining and refers to that elder generation calcines at 400~500 DEG C, the time of the calcining is 1.5~2.5 hours；Afterwards 700~800 DEG C after
Continuous calcining, the time for continuing calcining are 0.5~1.5 hour.
Step (a) the specific steps are by graphene oxide in water ultrasound remove, be added aniline ultrasonic disperse it is uniform；So
Afterwards under low temperature, the mixture of oxidant and acid medium, polymerization reaction is added, hydro-thermal reaction obtains polyaniline-graphene oxide
Compound.The mass volume ratio of the graphene oxide and water be (50~200) mg:50mL, ultrasound removing time be 1~
4h, the time of reaction are 6~12 hours.
After the reaction was completed, it filters, washs, be dried in vacuo, grinding obtains polyaniline-graphene oxide compound.
The preparation method of the above-mentioned hydroquinone biosensor based on the compound horseradish peroxidase of nitrogen-doped graphene,
The following steps are included:
(1) surface preparation is carried out to basal electrode；
(2) nitrogen-doped graphene composite material is dispersed in water, obtains dispersion liquid；Horseradish peroxidase is dissolved in
In PBS solution, horseradish peroxidase solution is obtained；By chitosan wiring solution-forming, chitosan solution is obtained；By dispersion liquid, peppery
Root Peroxidase Solution is uniformly mixed with chitosan solution, obtains composite solution；
(3) composite solution is added drop-wise to the basal electrode surface through surface preparation, room temperature is dried, and is obtained based on N doping
The enzyme modification working electrode of the compound horseradish peroxidase of graphene；
(4) three-electrode system is formed by the working electrode of enzyme modification and reference electrode and to electrode, obtains detecting to benzene two
The biosensor of phenol.
Surface preparation described in step (1) specifically refers to: by the surface of basal electrode successively use diameter be 0.3 μm and
0.05 μm of Al2O3Powder is polished to mirror surface, then is rinsed with water；Then successively it is cleaned by ultrasonic in dehydrated alcohol and water, takes out and use
Water is cleaned, and is dried.
The concentration of doped graphene material dispersion liquid described in step (2) is 2-20mg/mL.
The concentration of horseradish peroxidase solution described in step (2) is 15~35mg/mL, and enzyme solutions are molten using PBS
(pH 6.5,0.1M) that liquid is prepared.
Chitosan solution described in step (2) is by 0.1-2wt% acetic acid solutions, concentration 10mg/mL.
Doped graphene dispersion liquid described in step (2), horseradish peroxidase solution and chitosan solution volume ratio are
1:1:1。
Composite solution dripping quantity described in step (3) is 3-10 μ L.
The above-mentioned biosensor based on the compound horseradish peroxidase detection hydroquinone enzyme of nitrogen-doped graphene is right
Application in benzenediol quantitative detection.
The principle of the present invention:
The present invention is to prepare doped graphene material first, which overcomes graphene by the synergistic effect of different component
Then the deficiency of bad dispersibility in curling itself, interlayer stacking and solvent using the film forming of chitosan, and utilizes doping stone
The carrier characteristics of black alkene, the strong enzyme catalyst that increases is in the fixed amount and stability of electrode surface, in favor of the catalysis to substrate；
Finally, taking appropriate mixing drop in the working electrode on the working electrode of surface preparation, after being modified；Described in recycling
Modification after working electrode, cooperation reference electrode forms three-electrode system with to electrode, a kind of detection hydroquinone is made
Enzyme biologic sensor.
Nitrogen-doped graphene, horseradish peroxidase are applied to enzyme biologic sensor, the detection being prepared by the present invention
The sensor detection performance of hydroquinone is good, and detection range is 9 × 10-5~5.075 × 10-3Mol/L, linear equation are I (μ
A)=- 11.37-5.89C (mmol/L), related coefficient R2=0.998.Detection is limited to 1 × 10-5Mol/L (S/N=3).
Preparation method of the invention and obtained product have the following advantages that and the utility model has the advantages that
(1) biosensor of detection hydroquinone of the present invention has good electron transmission, can will react
The electronics of generation carries out good transfer, is able to achieve the selective enumeration method of biomolecule, improves the reaction of the biosensor
Speed.
(2) biosensor of detection hydroquinone of the present invention have good selectivity, reproducibility and stabilization
Property, hydroquinone can accurately be detected, strong antijamming capability.
(3) biosensor of detection hydroquinone of the present invention can be used in water in hydroquinone or soil to benzene
The detection of diphenol, preparation is simple, has wider detection range, and lower detection limit is reacted and carried out under property environment at room temperature,
Performance is stablized, and has a good application prospect.
Detailed description of the invention
Fig. 1 is to modify working electrode different molten based on the compound horseradish peroxidase of nitrogen-doped graphene in embodiment 3
Cyclic voltammogram in liquid；Curve a is cyclic voltammetry curve of the enzyme modification working electrode in PBS, and curve b is enzyme modification work
Cyclic voltammetry curve of the electrode in 0.5mM hydroquinone (HQ), curve c are enzyme modification working electrode in 0.5mM hydroquinone
With 0.5mM H2O2Cyclic voltammetry curve in mixed solution；
Fig. 2 is the life that hydroquinone is detected based on the compound horseradish peroxidase of nitrogen-doped graphene prepared by embodiment 3
Cyclic voltammogram of the object sensor in the quinol solution of various concentration is dense for corresponding hydroquinone in box in figure
Degree；
Fig. 3 is the life that hydroquinone is detected based on the compound horseradish peroxidase of nitrogen-doped graphene prepared by embodiment 3
Canonical plotting of the object sensor to the response current of various concentration hydroquinone.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment 1
A kind of electrochemical enzymatic bio-sensing based on the compound horseradish peroxidase detection hydroquinone of nitrogen-doped graphene
The preparation method of device, comprising the following steps:
(1) be successively with diameter by the glass-carbon electrode that diameter is 3mm 0.3 μm and 0.05 μm Al2O3Powder is polished to mirror
Then face is successively cleaned by ultrasonic 1min with distilled water flushing in dehydrated alcohol and distilled water, take out and use distilled water flushing, room
Temperature dries to obtain pretreated glass-carbon electrode；
(2) 100mg graphene oxide is mixed with 50mL deionized water, 0.5mL aniline, stirring is added in ultrasound removing 4h
Continue ultrasound 30min after uniformly, the 0.5M of (10 DEG C or less) additions 5mL ammonium persulfate containing 1.25g (APS) under low temperature stirring
HCl, continue low temperature stir 6 hours after be transferred in reaction kettle, 180 DEG C hydro-thermal 16 hours, the solution obtained after reaction sand core mistake
Filter filtering, and is washed with distilled water for several times, 80 DEG C be dried in vacuum overnight after grind, grind powder is placed in quartz boat, in pipe
In formula furnace nitrogen atmosphere calcine, first calcine 2 hours at 400 DEG C, after be continuously heating to 700 DEG C calcine 1 hour, take out grind is
Obtain nitrogen-doped graphene material；
(3) nitrogen-doped graphene material is separated into the dispersion liquid that concentration is 10mg/mL in water, using PBS solution
(0.1mol/L, pH6.5) compound concentration is the horseradish peroxidase solution of 15mg/mL, using 1wt% acetic acid solutions
Three kinds of solution are mixed to get mixed solution with 1:1:1 volume ratio by 10mg/ml chitosan solution, take 6 μ L mixed solutions drop in step
Suddenly the electrode surface of (1), dries at room temperature, obtains the modification work based on the compound horseradish peroxidase of nitrogen-doped graphene
Electrode；
(4) working electrode and reference electrode will be modified and (platinum electrode is to electrode, saturation to electrode composition three-electrode system
Calomel is reference electrode), obtain the biosensor of detection hydroquinone.
Electrochemical test is carried out at room temperature, in 10mL H containing 0.5mM2O2Phosphate buffer solution (0.1mol/L,
PH6.5 it is carried out in), logical N before test2, cyclic voltammetry is used in test process.Wherein hydroquinone is not added dropwise for blank control
50 μ L quinol solutions are added dropwise after stable testing in solution.
For the present embodiment when hydroquinone concentration is 0.5mmol/L, the reduction peak catalytic current of test is 7.921 μ A.
Embodiment 2
A kind of preparation side of the hydroquinone enzyme biologic sensor based on the compound horseradish peroxidase of nitrogen-doped graphene
Method, comprising the following steps:
(1) be successively with diameter by the glass-carbon electrode that diameter is 3mm 0.3 μm and 0.05 μm Al2O3Powder is polished to mirror
Then face is successively cleaned by ultrasonic 1min with distilled water flushing in dehydrated alcohol and distilled water, take out and use distilled water flushing, room
Temperature dries to obtain pretreated glass-carbon electrode；
(2) 100mg graphene oxide is mixed with 50mL deionized water, 0.5mL aniline, stirring is added in ultrasound removing 4h
Continue ultrasound 30min after uniformly, the 0.5M of (10 DEG C or less) additions 5mL ammonium persulfate containing 1.25g (APS) under low temperature stirring
HCl, continue low temperature stir 6 hours after be transferred in reaction kettle, 180 DEG C hydro-thermal 16 hours, the solution obtained after reaction sand core mistake
Filter filtering, and is washed with distilled water for several times, 80 DEG C be dried in vacuum overnight after grind, grind powder is placed in quartz boat, in pipe
In formula furnace nitrogen atmosphere calcine, first calcine 2 hours at 400 DEG C, after be continuously heating to 700 DEG C calcine 1 hour, take out grind is
Obtain doped graphene material；
(3) doped graphene material is dispersed in water, the dispersion liquid that concentration is 10mg/mL is obtained, using PBS solution
(0.1mol/L, pH 6.5) compound concentration is the horseradish peroxidase solution of 20mg/mL, using 1wt% acetic acid solutions
Three kinds of solution are mixed to get mixed solution with 1:1:1 volume ratio by 10mg/ml chitosan solution, take 6 μ L mixed solutions drop in step
Suddenly the electrode surface of (1), dries at room temperature, obtains the modification work based on the compound horseradish peroxidase of nitrogen-doped graphene
Electrode；
(4) by after modification working electrode and reference electrode and to electrode composition three-electrode system (platinum electrode be to electrode,
Saturation calomel is reference electrode), obtain the biosensor of detection hydroquinone.
Electrochemical test is carried out at room temperature, in 10mL H containing 0.5mM2O2Phosphate buffer solution (0.1mol/L, pH
6.5) it is carried out in, logical N before test2, cyclic voltammetry is used in test process.Wherein it is molten that hydroquinone is not added dropwise for blank control
50 μ L quinol solutions are successively added dropwise after stable testing in liquid.
For the present embodiment when hydroquinone concentration is 0.5mmol/L, the reduction peak catalytic current of test is 9.819 μ A.
Embodiment 3
A kind of preparation side of the hydroquinone enzyme biologic sensor based on the compound horseradish peroxidase of nitrogen-doped graphene
Method, comprising the following steps:
(1) be successively with diameter by the glass-carbon electrode that diameter is 3mm 0.3 μm and 0.05 μm Al2O3Powder is polished to mirror
Then face is successively cleaned by ultrasonic 1min with distilled water flushing in dehydrated alcohol and distilled water, take out and use distilled water flushing, room
Temperature dries to obtain pretreated glass-carbon electrode；
(2) 100mg graphene oxide is mixed with 50mL deionized water, 0.5mL aniline, stirring is added in ultrasound removing 4h
Continue ultrasound 30min after uniformly, the 0.5M of (10 DEG C or less) additions 5mL ammonium persulfate containing 1.25g (APS) under low temperature stirring
HCl, continue low temperature stir 6 hours after be transferred in reaction kettle, 180 DEG C hydro-thermal 16 hours, the solution obtained after reaction sand core mistake
Filter filtering, and is washed with distilled water for several times, 80 DEG C be dried in vacuum overnight after grind, grind powder is placed in quartz boat, in pipe
In formula furnace nitrogen atmosphere calcine, first calcine 2 hours at 400 DEG C, after be continuously heating to 700 DEG C calcine 1 hour, take out grind is
Obtain doped graphene material；
(3) doped graphene material is separated into the dispersion liquid that concentration is 10mg/mL in water, using PBS solution
(0.1mol/L, pH 6.5) compound concentration is the horseradish peroxidase solution of 25mg/mL, using 1% acetic acid solutions
Three kinds of solution are mixed to get mixed solution with 1:1:1 volume ratio by 10mg/ml chitosan solution, take 6 μ L mixed solutions drop in step
Suddenly the electrode surface of (1), dries at room temperature, obtains the modification work based on the compound horseradish peroxidase of nitrogen-doped graphene
Electrode；
(4) by after modification working electrode and reference electrode and to electrode composition three-electrode system (platinum electrode be to electrode,
Saturation calomel is reference electrode), obtain the biosensor of detection hydroquinone.
Electrochemical test is carried out at room temperature, in 10mL H containing 0.5mM2O2Phosphate buffer solution (0.1mol/L, pH
6.5) it is carried out in, logical N before test2, cyclic voltammetry is used in test process.Wherein it is molten that hydroquinone is not added dropwise for blank control
50 μ L quinol solutions are successively added dropwise after stable testing in liquid.
The biosensor of the present embodiment is when hydroquinone concentration is 0.5mmol/L, the reduction peak catalytic current of test
For 10.89 μ A.
Cyclic voltammogram of the enzyme modification working electrode in different solutions is as shown in Figure 1 in the present embodiment.Curve a repairs for enzyme
Cyclic voltammetry curve of the working electrode in PBS is adornd, curve b is circulation of the enzyme modification working electrode in 0.5mM hydroquinone
Volt-ampere curve, curve c is enzyme modification working electrode in 0.5mM hydroquinone and 0.5mM H2O2Cyclic voltammetric in mixed solution
Curve.From fig. 1, it can be seen that there are a pair of apparent redox peaks when being individually added into quinol solution, when hydroquinone with
H2O2It exists simultaneously, obviously increasing occurs in reduction peak, and oxidation peak is substantially reduced, and illustrates that H is added2O2, be conducive to enzyme to benzene two
The catalytic action of phenol.
Biology manufactured in the present embodiment based on the compound horseradish peroxidase detection hydroquinone of nitrogen-doped graphene passes
Cyclic voltammogram of the sensor in various concentration quinol solution is as shown in Figure 2.In 0.1mo1/L, the phosphate that pH is 6.5
In buffer solution, it is continuously added the cyclic voltammetry curve that hydroquinone obtains, wherein oxidation curve right end is successively right from top to bottom
The hydroquinone concentration answered be 0.09mmo1/L, 0.5mmo1/L, 1.5mmo1/L, 2mmo1/L, 3mmo1/L, 4mmo1/L,
5.075mmo1/L.The carrier characteristics of doped graphene are utilized, the strong enzyme catalyst that increases is in the fixed amount of electrode surface and steady
It is qualitative, in favor of the catalysis to substrate.
Biology manufactured in the present embodiment based on the compound horseradish peroxidase detection hydroquinone of nitrogen-doped graphene passes
Sensor is as shown in Figure 3 to the canonical plotting of the response current of various concentration hydroquinone.The modified electrode detects model to substrate
Enclose is 9 × 10-5~5.075 × 10-3Mol/L, linear equation are I (μ A)=- 11.37-5.89C (mmol/L), and related coefficient is
R2=0.998.Detection is limited to 1 × 10-5Mol/L (S/N=3).
Embodiment 4
A kind of preparation side of the hydroquinone enzyme biologic sensor based on the compound horseradish peroxidase of nitrogen-doped graphene
Method, comprising the following steps:
(1) be successively with diameter by the glass-carbon electrode that diameter is 3mm 0.3 μm and 0.05 μm Al2O3Powder is polished to mirror
Then face is successively cleaned by ultrasonic 1min with distilled water flushing in dehydrated alcohol and distilled water, take out and use distilled water flushing, room
Temperature dries to obtain pretreated glass-carbon electrode；
(2) 100mg graphene oxide is mixed with 50mL deionized water, 0.5mL aniline, stirring is added in ultrasound removing 4h
Continue ultrasound 30min after uniformly, the 0.5M of (10 DEG C or less) additions 5mL ammonium persulfate containing 1.25g (APS) under low temperature stirring
HCl, continue low temperature stir 6 hours after be transferred in reaction kettle, 180 DEG C hydro-thermal 16 hours, the solution obtained after reaction sand core mistake
Filter filtering, and is washed with distilled water for several times, 80 DEG C be dried in vacuum overnight after grind, grind powder is placed in quartz boat, in pipe
In formula furnace nitrogen atmosphere calcine, first calcine 2 hours at 400 DEG C, after be continuously heating to 700 DEG C calcine 1 hour, take out grind is
Obtain doped graphene material；
(3) doped graphene material is separated into the dispersion liquid that concentration is 10mg/mL in water, using PBS solution
(0.1mol/L, pH 6.5) compound concentration is the horseradish peroxidase solution of 30mg/mL, using 1% acetic acid solutions
Three kinds of solution are mixed to get mixed solution with 1:1:1 volume ratio by 10mg/ml chitosan solution, take 6 μ L mixtures drop in step
(1) electrode surface, dries at room temperature, obtains the modification work electricity based on the compound horseradish peroxidase of nitrogen-doped graphene
Pole；
(4) by after modification working electrode and reference electrode and to electrode composition three-electrode system (platinum electrode be to electrode,
Saturation calomel is reference electrode), obtain the biosensor of detection hydroquinone.
Electrochemical test is carried out at room temperature, in 10mL H containing 0.5mM2O2Phosphate buffer solution (0.1mol/L, pH
6.5) it is carried out in, logical N before test2, cyclic voltammetry is used in test process.Wherein it is molten that hydroquinone is not added dropwise for blank control
50 μ L quinol solutions are successively added dropwise after stable testing in liquid.
For the present embodiment when hydroquinone concentration is 0.5mmol/L, the reduction peak catalytic current of test is 9.466 μ A.
Embodiment 5
A kind of preparation side of the hydroquinone enzyme biologic sensor based on the compound horseradish peroxidase of nitrogen-doped graphene
Method, comprising the following steps:
(1) be successively with diameter by the glass-carbon electrode that diameter is 3mm 0.3 μm and 0.05 μm Al2O3Powder is polished to mirror
Then face is successively cleaned by ultrasonic 1min with distilled water flushing in dehydrated alcohol and distilled water, then glass-carbon electrode is placed in 10mL
(5mmol/L K in potassium ferricyanide solution3Fe(CN)6+ 0.1mol/L KCl) it is enclosed at 0~0.8V using cyclic voltammetry scanning 6
Electrode activation is carried out, takes out and uses distilled water flushing, room temperature dries to obtain pretreated glass-carbon electrode；
(2) 100mg graphene oxide is mixed with 50mL deionized water, ultrasound 1~4h of removing is added 0.5mL aniline, stirs
Continue ultrasound 30min after mixing uniformly, the 0.5M of (10 DEG C or less) additions 5mL ammonium persulfate containing 1.25g (APS) under low temperature stirring
HCl, continue low temperature stir 6 hours after be transferred in reaction kettle, 180 DEG C hydro-thermal 16 hours, the solution obtained after reaction sand core mistake
Filter filtering, and is washed with distilled water for several times, 80 DEG C be dried in vacuum overnight after grind, grind powder is placed in quartz boat, in pipe
In formula furnace nitrogen atmosphere calcine, first calcine 2 hours at 400 DEG C, after be continuously heating to 700 DEG C calcine 1 hour, take out grind is
Obtain doped graphene material；
(3) doped graphene composite material is separated into the dispersion liquid that concentration is 10mg/mL in water, using PBS solution
(0.1mol/L, pH 6.5) compound concentration is the horseradish peroxidase solution of 35mg/mL, using 1% acetic acid solutions
Three kinds of solution are mixed to get mixed solution with 1:1:1 volume ratio by 10mg/ml chitosan solution, take 6 μ L mixtures drop in step
(1) electrode surface, dries at room temperature, obtains the modification work electricity based on the compound horseradish peroxidase of nitrogen-doped graphene
Pole；
(4) by after the modification working electrode and reference electrode and to electrode composition three-electrode system (platinum electrode be pair
Electrode, saturation calomel are reference electrode), obtain the biosensor of detection hydroquinone.
Electrochemical test is carried out at room temperature, in 10mL H containing 0.5mM2O2Phosphate buffer solution (0.1mol/L, pH
6.5) in carry out, logical N before test2, cyclic voltammetry is used in test process.Wherein it is molten that hydroquinone is not added dropwise for blank control
50 μ L quinol solutions are successively added dropwise after stable testing in liquid.
For the present embodiment when hydroquinone concentration is 0.5mmol/L, the reduction peak catalytic current of test is 7.235 μ A.
The preparation of the graphene oxide:
(a) graphite powder is cleaned；
(b) graphite powder after taking cleaning, prepares graphene oxide using modified Hummers method.
The operation of graphite powder cleaning process described in step (a) is as follows: weighing 2.5~10g graphite powder in large beaker, is added
100mL distilled water, then 100mL concentrated hydrochloric acid is added in ventilating kitchen, 2h is heated and stirred in 60~80 DEG C of water-baths, is filtered by vacuum
Afterwards, it is successively cleaned with distilled water, acetone, ethyl alcohol, after cleaning, 100 DEG C of drying in vacuum oven is put into, later with quartz
Mortar grinder powdering is spare.
The process of graphene oxide preparation manipulation described in step (b) is as follows: (1) low-temp reaction (0~4 DEG C): in ice-water bath
In be put into 1000mL large beaker, add the dense H of 110mL2SO4, while stirring so that its temperature is down to low-temp reaction section, then according to
It is secondary that cleaned 2.5~10g graphite powder, 2.5g NaNO is added3, 15g KMnO4.Rear timing is added, 90min is stirred to react,
Solution is in purple green；(2) medium temperature reaction (30~40 DEG C): temperature being increased, its temperature is made to be maintained at 35 DEG C or so under stiring,
Clock reaction 90min, solution are still in purple green；(3) pyroreaction (70~100 DEG C): after medium temperature reaction, slowly into beaker
220mL deionized water is added, then heats and controls temperature at 85 DEG C, being then slowly added to a certain amount of hydrogen peroxide, (5% about
13mL) golden yellow is gradually become to solution；(4) after completion of the reaction to the preparation, when taking advantage of reaction solution warm, after reaction
Solution be washed with deionized and filter, and be centrifuged repeatedly with the centrifuge that revolving speed is 4000rpm and clean filtering again, until filter
It can't detect SO in liquid4 2-Until (use BaCl2It examines).Obtained black product is cleaned by ultrasonic with deionized water at this time
After 40min, for 24 hours, dark brown graphene oxide is can be obtained in drying at 40 DEG C.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of hydroquinone biosensor based on the compound horseradish peroxidase of nitrogen-doped graphene, it is characterised in that:
It is formed by reference electrode, to the working electrode after electrode and modification, the working electrode after the modification is by basal electrode and solidification
Material identification film on basal electrode surface forms, wherein the material identification film is mainly by nitrogen-doped graphene dispersion liquid, peppery
Root Peroxidase Solution and chitosan solution mixing, film forming are prepared；
The nitrogen-doped graphene dispersion liquid is to be dispersed in water to obtain by nitrogen-doped graphene；Horseradish peroxidase solution is
Horseradish peroxidase is dissolved in PBS solution and is obtained；Chitosan solution is to obtain chitosan wiring solution-forming；The N doping
Graphene: horseradish peroxidase: the mass ratio of chitosan is (2~20): (15~35): 10；
The nitrogen-doped graphene is prepared especially by following methods:
(a) graphene oxide and aniline are uniformly dispersed；Under the action of oxidant and in acid medium, it is anti-that polymerization occurs for aniline
It answers, is placed in hydrothermal reaction kettle and carries out hydro-thermal reaction, obtain polyaniline-graphene oxide compound；
(b) polyaniline-graphene oxide compound is calcined, obtains nitrogen-doped graphene；The calcining refers to first 400
Calcined at~500 DEG C, after continue to calcine at 700~800 DEG C.
2. the hydroquinone bio-sensing according to claim 1 based on the compound horseradish peroxidase of nitrogen-doped graphene
Device, it is characterised in that: the mass volume ratio of graphene oxide described in step (a) and aniline is (50~200) mg:0.5mL；
The molar ratio of the oxidant and acid medium is (2~2.5): 1, the volume ratio of aniline and acid medium be 1:(10~
20)；The temperature of the polymerization reaction is 4~10 DEG C；
The atmosphere of calcining described in step (b) is protectiveness atmosphere, and the time of calcining described in step (b) is 1.5~2.5 small
When；The time for continuing calcining is 0.5~1.5 hour.
3. the hydroquinone bio-sensing according to claim 1 based on the compound horseradish peroxidase of nitrogen-doped graphene
Device, it is characterised in that: step (a) oxidant is ammonium persulfate, and the acid medium is hydrochloric acid, and acid medium is with aqueous solution
Form be added, the concentration of acid medium is 0.5mol/L；The time of polymerization reaction is 6~12h；The temperature of hydro-thermal reaction is
160~200 DEG C, the hydro-thermal reaction time is 12~20h.
4. the hydroquinone bio-sensing according to claim 1 based on the compound horseradish peroxidase of nitrogen-doped graphene
Device, it is characterised in that: step (a) the specific steps are by graphene oxide in water ultrasound remove, be added aniline ultrasonic disperse
Uniformly；Then under low temperature, the mixture of oxidant and acid medium, polymerization reaction is added, hydro-thermal reaction obtains polyaniline-oxygen
Graphite alkene compound.
5. the hydroquinone bio-sensing according to claim 4 based on the compound horseradish peroxidase of nitrogen-doped graphene
Device, it is characterised in that: the mass volume ratio of the graphene oxide and water is (50~200) mg:50mL, the time of ultrasound removing
For 1~4h.
6. the hydroquinone based on the compound horseradish peroxidase of nitrogen-doped graphene described in any one according to claim 1~5
The preparation method of biosensor, it is characterised in that: the following steps are included:
(1) surface preparation is carried out to basal electrode；
(2) nitrogen-doped graphene composite material is dispersed in water, obtains dispersion liquid；It is molten that horseradish peroxidase is dissolved in PBS
In liquid, horseradish peroxidase solution is obtained；By chitosan wiring solution-forming, chitosan solution is obtained；By dispersion liquid, horseradish peroxide
Compound enzyme solutions are uniformly mixed with chitosan solution, obtain composite solution；
(3) composite solution is added drop-wise to the basal electrode surface through surface preparation, room temperature is dried, and is obtained based on N doping graphite
The enzyme modification working electrode of the compound horseradish peroxidase of alkene；
(4) three-electrode system is formed by the working electrode of enzyme modification and reference electrode and to electrode, obtains detection hydroquinone
Biosensor.
7. the hydroquinone biosensor according to claim 6 based on the compound horseradish peroxidase of nitrogen-doped graphene
Preparation method, it is characterised in that: the concentration of doped graphene material dispersion liquid described in step (2) be 2-20mg/mL；
The concentration of horseradish peroxidase solution described in step (2) is 15~35mg/mL, and enzyme solutions are matched using PBS solution
System；
Chitosan solution described in step (2) is by 0.1-2wt% acetic acid solutions, concentration 10mg/mL.
8. the hydroquinone biosensor according to claim 7 based on the compound horseradish peroxidase of nitrogen-doped graphene
Preparation method, it is characterised in that: the PBS solution be pH 6.5, the PBS solution of 0.1M.
9. the hydroquinone biosensor according to claim 6 based on the compound horseradish peroxidase of nitrogen-doped graphene
Preparation method, it is characterised in that: doped graphene dispersion liquid described in step (2), horseradish peroxidase solution and shell are poly-
Sugar juice volume ratio is 1:1:1.
10. the hydroquinone based on the compound horseradish peroxidase of nitrogen-doped graphene described in any one according to claim 1~5
Application of the biosensor in hydroquinone detection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811006266.1A CN109060918B (en) | 2018-08-30 | 2018-08-30 | Hydroquinone biosensor based on nitrogen-doped graphene composite horseradish peroxidase and preparation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811006266.1A CN109060918B (en) | 2018-08-30 | 2018-08-30 | Hydroquinone biosensor based on nitrogen-doped graphene composite horseradish peroxidase and preparation and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109060918A true CN109060918A (en) | 2018-12-21 |
| CN109060918B CN109060918B (en) | 2020-08-18 |
Family
ID=64758105
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811006266.1A Active CN109060918B (en) | 2018-08-30 | 2018-08-30 | Hydroquinone biosensor based on nitrogen-doped graphene composite horseradish peroxidase and preparation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109060918B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115112744A (en) * | 2022-06-15 | 2022-09-27 | 华南理工大学 | Electrochemical sensor and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105645403A (en) * | 2016-03-28 | 2016-06-08 | 南京邮电大学 | Preparation method of high-performance nitrogen-doped three-dimensional graphene |
| CN107132259A (en) * | 2017-04-10 | 2017-09-05 | 华南理工大学 | A kind of cholesterol sensor and preparation and application based on doped graphene |
| CN107290418A (en) * | 2017-05-05 | 2017-10-24 | 华南理工大学 | It is a kind of based on nitrogen-doped graphene without enzyme sensor and its preparation method and application |
| CN108051490A (en) * | 2017-11-08 | 2018-05-18 | 韶关学院 | L-lysine electrochemical sensor based on grapheme material and preparation method thereof |
-
2018
- 2018-08-30 CN CN201811006266.1A patent/CN109060918B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105645403A (en) * | 2016-03-28 | 2016-06-08 | 南京邮电大学 | Preparation method of high-performance nitrogen-doped three-dimensional graphene |
| CN107132259A (en) * | 2017-04-10 | 2017-09-05 | 华南理工大学 | A kind of cholesterol sensor and preparation and application based on doped graphene |
| CN107290418A (en) * | 2017-05-05 | 2017-10-24 | 华南理工大学 | It is a kind of based on nitrogen-doped graphene without enzyme sensor and its preparation method and application |
| CN108051490A (en) * | 2017-11-08 | 2018-05-18 | 韶关学院 | L-lysine electrochemical sensor based on grapheme material and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| JIYANG LIU等: "Functionalization of Monolithic and Porous Three-Dimensional Graphene by One-Step Chitosan Electrodeposition for Enzymatic Biosensor", 《ACS APPL. MATER. INTERFACES》 * |
| KAI WAN等: "pH Effect on Electrochemistry of Nitrogen-Doped Carbon Catalyst for Oxygen Reduction Reaction", 《ACS CATALYSIS》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115112744A (en) * | 2022-06-15 | 2022-09-27 | 华南理工大学 | Electrochemical sensor and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109060918B (en) | 2020-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110455896B (en) | Preparation method of metal organic framework composite ratio electrochemical miR3123 aptamer sensor | |
| Marini et al. | Non‐enzymatic Glucose Sensor Based on Nickel/Carbon Composite | |
| Liu et al. | Using ruthenium polypyridyl functionalized ZnO mesocrystals and gold nanoparticle dotted graphene composite for biological recognition and electrochemiluminescence biosensing | |
| CN108007998B (en) | Nickel oxide non-enzymatic glucose electrochemical sensor | |
| CN106770562B (en) | A kind of CoS2/ nitrogen-doped graphene composite material constructs application of the electrochemical sensor in glucose electrochemical analysis | |
| CN109444230B (en) | Au/CeO2/g-C3N4Composite material, electrochemical sensor, preparation method and application thereof | |
| CN108872343A (en) | A kind of Dopamine Sensor and its preparation and application based on nitrogen-doped graphene | |
| CN108918624A (en) | It is a kind of detect dopamine enzyme biologic sensor and its preparation and application | |
| CN103808779A (en) | Method for detecting p-nitrophenol by using PDDA (Poly Diallyldimethylammonium) functionalized graphene modified glassy carbon electrode | |
| CN110412095B (en) | Nitrogen-doped graphene composite material loaded with flower-ball-shaped copper sulfide-palladium core-shell structure and preparation method and application thereof | |
| Asadpour‐Zeynali et al. | Nanostructured hexacyanoferrate intercalated Ni/Al layered double hydroxide modified electrode as a sensitive electrochemical sensor for paracetamol determination | |
| CN109557138A (en) | A kind of graphene-based gas sensing materials of Metal Palladium load and preparation and application | |
| Sarika et al. | A novel amperometric catechol biosensor based on α-Fe2O3 nanocrystals-modified carbon paste electrode | |
| Li et al. | An electrochemiluminescence aptasensor based on Ru (bpy) 3 2+ encapsulated titanium-MIL-125 metal-organic framework for bisphenol A assay | |
| Li et al. | Facile synthesis of NiO/CuO/reduced graphene oxide nanocomposites for use in enzyme-free glucose sensing | |
| Revathi et al. | MWCNT based non-enzymatic H2O2 sensor: influence of amine functionalization on the electrochemical H2O2 sensing | |
| CN110237865B (en) | Silver phosphate-loaded keratin nanoflower material and preparation and application thereof | |
| Aguila et al. | A biosensor based on Coriolopsis gallica laccase immobilized on nitrogen-doped multiwalled carbon nanotubes and graphene oxide for polyphenol detection | |
| Li et al. | Electrochemical immunosensor for carcinoembryonic antigen detection based on Mo–Mn3O4/MWCNTs/Chits nanocomposite modified ITO electrode | |
| CN109060918A (en) | Hydroquinone biosensor and its preparation and application based on the compound horseradish peroxidase of nitrogen-doped graphene | |
| Cai et al. | Detection of CYFRA 21-1 in human serum by an electrochemical immunosensor based on UiO-66-NH2@ CMWCNTs and CS@ AuNPs | |
| CN105717178A (en) | Preparation method and application of electrochemical diethylstilbestrol sensor based on titanium-dioxide-based two-dimensional composite nanomaterial | |
| Bu et al. | A novel three-dimensional molecularly imprinted polypyrrole electrochemical sensor based on MOF derived porous carbon and nitrogen doped graphene for ultrasensitive determination of dopamine | |
| CN103969307A (en) | Preparation method for titanium dioxide-graphene/screen-printed electrode for detecting parachlorophenol | |
| Bu et al. | A novel molecularly imprinting polypyrrole electrochemiluminescence sensor based on MIL-101-g-C3N4 for supersensitive determination of ciprofloxacin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |