CN103207224A - Electrochemical biosensor electrode for detection of hydrogen peroxide and preparation method thereof - Google Patents
Electrochemical biosensor electrode for detection of hydrogen peroxide and preparation method thereof Download PDFInfo
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Abstract
The invention provides an enzyme biosensor for detection of hydrogen peroxide and a preparation method thereof. The biosensor employs a classic three-electrode system, wherein a reference electrode is a saturated calomel electrode, a counter electrode is a platinum electrode and a working electrode is a glassy carbon electrode. The biosensor provided by the invention promotes electron transfer between hemoglobin and the electrodes mainly by using a composite material of a helical carbon nanofiber and a colloidal gold nanoparticle as a carrier of hemoglobin, so sensitivity of detection of hydrogen peroxide is effectively improved and a satisfactory result is achieved.
Description
Technical field
The invention belongs to the hydrogen peroxide detection range, relate in particular to a kind of sensor that detects hydrogen peroxide and preparation method thereof.
Technical background
Along with the continuous popularization of hydrogen peroxide in the application of electronics, light industry, food, environmental protection, medicine and other fields, for example be applied to the cleaning of bacterium virus killing, textile bleaching, Industrial Wastewater Treatment, cosmetics, food and dairy produce production equipment and production flow line etc. that disappears, but meanwhile, hydrogen peroxide also may cause various diseases, bring hidden danger for people's life and health, therefore, the fast detecting for hydrogen peroxide seems particularly important.The detection technique of relevant hydrogen peroxide has a lot, and wherein, the advantage that detects hydrogen peroxide with electrochemical method is simple, quick, highly sensitive.
In recent years, enzyme biologic sensor caused many researchists' attention.Because the catalytic active center of most of enzymes is buried in zymoprotein inside, and can not obtains the electrode surface that the reasons such as absorption sex change of suitable orientation or protein itself make the big molecule of zymoprotein directly modify at electrode surface and be difficult to the realization direct electron transfer.Therefore, the center of gravity of development is to seek the fixing means of suitable carriers and enzyme at present, improves the detection performance of enzyme biologic sensor afterwards.The researchist has attempted the carrier of all kinds of modified electrodes and the fixing means of enzyme.Such as using surfactant, carbon black, polyeletrolyte, biological high polymer, quantum dot as carrier.[reference: (a) Dong, S.J.; Che, G.L.; Xie, Y.W.Chemical modified electrodes.Beijing:Science Press, 2003,456-570. (b) X.Y.He, L.Zhu, Electrochem.Commun.2006,8,615-620. (c) G.X.Ma, T.H.Lu, Y.Y.Xia, bioelectrochemistry.2007,71,80. (d) H.Sun, N.F.Hu, J.Electroanal.Chem.2006,588,207-217. (e) Q.Lu, T.Zhou, S.S.Hu, Biosens.Bioelectron.2007,22,899-904. (f) D.Shan, G.Cheng, D.Zhu, H.Xue, S.Cosnier, S.Ding, Sensors Actuators B:Chem.2009,137,259-265. (g) Q.Lu, S.S.Hu, D.W.Pang, Z.K.He, Chem.Commun.2005,20,2584-2585.].Golden nanometer particle is widely used in fixedly matrix of protein because having a kind of good mediation ability, to improve protein or enzyme and interelectrode electronics transfer rate.It not only can provide bigger specific surface area, but also can keep excellent biological compatibility with protein or enzyme.[referring to: (h) C.X.Lei, S.Q.Hu, G.L.Shen, R.Q.Yu, Talanta, 2003,59,981. (i) Dequarie, M.; Degrand, C.; Limoges, B.Anal.Chem.2000,72,5521-5528. (j) Shi Wentao, Di Jing, Ma Zhanfang, [J]. chemical progress, 2012,24,569. (k) Yu, A.; Liang, Z.; Cho, J.; Caruso, F.Nano Lett, 2003,3,1203, (l) Zhang, S.-X.; Wang, N.; Niu, Y.-M.Sens.Actuators B, 2005,109,367].In addition, the compound substance with novel nano-materials such as physics, carbon nanomaterial Graphene that chemical property is good, carbon nano-tube and gold nano-material is applied in the enzyme biologic sensor, and current-responsive sensitivity is improved a lot.[reference: (m) C.Y.Li, D.S, D.B.Tian, J.R Zhang, J.J.Zhu.J.Mater, Chem, 2011,21,7604. (n) Y.C.Gao, K.Xi, W.N.Wang, X.D.Jia, J.J.Zhu, Anal.Methods, 2011,3,2387.].The spiral carbon nano-fiber is as a kind of novel nano-material, because special helical structure, makes it have bigger specific surface area, and water-soluble better, excellent conductivity.But the application of spiral carbon nano-fiber aspect electrochemica biological sensor also do not reported as yet.The present invention is applied to enzyme biologic sensor with spiral carbon nano-fiber and gold size compound substance, this compound substance has had the biocompatibility that spiral carbon tube good electric conductivity has had gold size again on the one hand, make the sensor detection performance of preparation that large increase arranged, it is 1.00 μ M～3157 μ M that hydrogen peroxide is detected the range of linearity, detects to be limited to 0.46 μ M.
Summary of the invention
The purpose of this invention is to provide a kind of bioelectrochemical sensor that detects hydrogen peroxide and preparation method thereof.This sensor is that spiral carbon nano-fiber and gold size nano composition are made carrier, greatly promotes enzyme and interelectrode electronics transmission, thereby reaches the purpose that improves transducer sensitivity.
Technical scheme of the present invention is as follows:
A kind of electrochemica biological sensor that detects hydrogen peroxide, comprise working electrode, platinum electrode, saturated calomel electrode, it is characterized in that the spiral carbon nano-fiber of self assembly and compound substance and haemoglobin or myoglobins or the horseradish peroxidase of gold size nano particle, and make the work electrode with the fixing modified glassy carbon electrode of nafion.
Mass ratio between the spiral carbon nano-fiber of described self assembly and compound substance, haemoglobin or the myoglobins of gold size nano particle or peroxidase nafion is 1.5: 2.25: 0.7;
The mass ratio of the spiral carbon nano-fiber of self assembly and gold size nano particle is 50: 7 in the spiral carbon nano-fiber of described self assembly and the compound substance of gold size nano particle.
The technical scheme that the preparation method of described electrochemica biological sensor adopts is as follows:
It is made up of the following step:
The preparation of step 1 spiral carbon nano-fiber: after the NiO powder that takes by weighing 51mg is put into the porcelain handleless cup, insert in the quartz glass tube, then under the environment of 375 ℃ of logical hydrogen, reacted 1 hour, in 415 ℃ of acetylene atmosphere, reacted 0.5 hour at last, can obtain 0.364g spiral carbon nano-fiber, the product cool to room temperature can use.
The preparation of step 2 gold/spiral carbon nano-fiber composite material: at first with spiral carbon nano-fiber polydiene propyl-dimethyl amine hydrochlorate (PDDA) functionalization that obtains, specific practice be with 20mg spiral carbon nano-fiber be distributed to the 10mL mass percent concentration be 0.25% PDDA (NaCl that contains 0.5M) aqueous solution in, obtain uniform black suspension behind the ultrasonic 10min, through centrifuging, it is neutral cleaning to pH with distilled water.Then, get 1mL by the above spiral carbon nano-fiber solution that obtains, the gold size of the about 10～30nm of particle diameter of 10mL is scattered in wherein, ultrasonic 30min, afterwards, the gold size that adds 1mL successively, whenever add the ultrasonic 10min of gold size one time, extremely the gold size that adds is total to till the 15mL, and is last, remove the upper strata stillness of night, obtain mass percent concentration and be the solution of gold size/spiral carbon nano-fiber composite material of 0.5%.
Step 4 glass-carbon electrode (diameter the is 3mm) alumina powder with 1.0,0.3,0.05 μ m at first respectively polishes smooth at chamois leather, wash with redistilled water then, ultrasonic cleaning, place 1mM potassium ferricyanide solution (KCl that contains 0.1M) to do cyclic voltammetry scan, potential range is-0.2～0.6V, sweeps speed and is made as 100mV/s, obtain the difference of oxidation-reduction potential in 60～80mV, illustrate that electrode surface is treated clean, ultrasonic cleaning, room temperature is dried.
Step 5 is got solution that 3 μ L obtain by step 2 and is dripped on the glass-carbon electrode that step 4 obtains, after at room temperature treating solvent evaporates, drip last 4.5 μ L mass percent concentrations and be 0.5% haemoglobin aqueous solution, after at room temperature treating solvent evaporates then, drip that to be coated with 2 μ L mass percent concentrations be that 0.5% nafion solution is fixed, room temperature is dried, the modified electrode that obtains, be placed in 4 ℃ of refrigerators preserve standby.
The modified electrode that step 6 step 5 obtains and platinum electrode and saturated calomel electrode are formed the bioelectrochemical sensor of three-electrode system.
Compound substance to spiral carbon nano-fiber and gold size nano particle carries out scanning electron microscope (SEM) sign.
Adopt cyclic voltammetry and two kinds of electrochemical methods of time current method that the enzyme biologic sensor of above preparation is tested.Concrete steps are: under the room temperature, in electrolyte solution, feed after nitrogen 15min removes oxygen in the solution, the top keeps feeding nitrogen, it is in 7.0 the phosphate buffered solution that three electrodes are immersed 100mM, pH, adopt cyclic voltammetry or time current method to carry out electro-chemical test, the speed of sweeping when adopting cyclic voltammetry is 100mV/s, scan potential range to be-0.8V～-0.2V; The operating potential of choosing when adopting the time current method is-0.5V.
SEM characterizes: spiral carbon nano-fiber and gold size nano composition are carried out the SEM sign.The result shows that golden nanometer particle is adsorbed on (see figure 1) on the spiral carbon nano-fiber uniformly.
The cyclic voltammetry result: the reduction peak of the compound substance of spiral carbon nano-fiber and gold size nano particle and the cyclic voltammogram (see figure 2) of the glass-carbon electrode of modified hemoglobin in the phosphate buffered solution that contains 200 μ M hydrogen peroxide is obviously than not containing the big of superoxol, oxidation peak reduces, show the catalytic activity owing to haemoglobin, body series can produce sensitive current-responsive to the hydrogen peroxide in the solution.
Time current method measurement result: Fig. 3 is under the operating potential of-0.5V, is in 7 phosphate buffered solution to 100mM pH, adds hydrogen peroxide (concentration is 1.00 μ M～3157 μ M) continuously, obtain the time current curve.As seen from the figure, with the increase of concentration of hydrogen peroxide, current-responsive is also in continuous increase, and the response time in 10 seconds, show at electrode surface and realized that electronics shifts fast.Shown in Figure 4 is that this sensor is to the typical curve of the response current of variable concentrations hydrogen peroxide.In 1.00 μ M～3157 μ M, response current and concentration of hydrogen peroxide are linear at concentration of hydrogen peroxide.The equation of linear regression formula is: y=0.4718+0.0193x, R
2=0.9987, detect and be limited to 0.46 μ M (signal to noise ratio (S/N ratio) is 3).Therefore, the present invention can be used for the quantitative detection of hydrogen peroxide.
The present invention selects for use spiral carbon nano-fiber and gold size nano composition as the carrier of haemoglobin, make haemoglobin obtain suitable orientation, accelerate enzyme and interelectrode electronics transmission, greatly improved the sensitivity that detects hydrogen peroxide, widened sensing range, can be used for the quantitative detection of hydrogen peroxide, method is simple, detects rapidly.
Description of drawings
Fig. 1 is the shape appearance figure of the scanning electron microscope of spiral carbon nano-fiber and gold size nano composition.
Fig. 2 is that gold/spiral carbon nano-fiber composite material and modified hemoglobin electrode are not containing and containing 200 μ M H
2O
2The phosphate buffered solution of 100mM pH=7 in cyclic voltammogram.
Fig. 3 is gold/spiral carbon nano-fiber composite material enzyme biologic sensor at current potential during for-0.5V, to H
2O
2Timing ampere response diagram.
Fig. 4 is H in the phosphate buffered solution of 100mM pH=7
2O
2Concentration and the linear relationship of response current.
Embodiment
Embodiment one: the preparation of enzyme biologic sensor
After the NiO powder that takes by weighing 51mg is put into the porcelain handleless cup, insert in the quartz glass tube, then under the environment of 375 ℃ of logical hydrogen, reacted 1 hour, reacted 0.5 hour in 415 ℃ of acetylene atmosphere at last, can obtain 0.364g spiral carbon nano-fiber, the product cool to room temperature can use.
Taking by weighing 20mg spiral carbon nano-fiber, to be distributed to the 10mL mass percent concentration be in 0.25% PDDA (NaCl that the contains 0.5M) aqueous solution, to obtain uniform black suspension behind the ultrasonic 10min, and through centrifuging, it is neutral cleaning to pH with distilled water.Then, get 1mL by the above spiral carbon nano-fiber solution that obtains, the gold size of the about 10～30nm of particle diameter of 10mL is scattered in wherein, ultrasonic, afterwards, the gold size that adds 1mL successively, whenever add the ultrasonic 10min of gold size one time, extremely the gold size that adds is total to till the 15mL, and is last, remove the upper strata stillness of night, obtain mass percent concentration and be the solution of gold/spiral carbon nano-fiber composite material of 0.5%.
The mass percent concentration of getting 20 μ L is that 5% Nafion solution adds 180 μ L absolute ethyl alcohols, and it is 0.5% Nafion solution that ultrasonic mixing can obtain mass percent concentration.
Glass-carbon electrode polishes smooth at chamois leather with the alumina powder of 1.0,0.3,0.05 μ m at first respectively, wash with redistilled water then, ultrasonic cleaning, place 1mM potassium ferricyanide solution (KCl that contains 0.1M) to do cyclic voltammetry scan, potential range is-0.2～0.6V, sweeps speed and is made as 100mV/s, obtains the difference of oxidation-reduction potential in 60～80mV, ultrasonic cleaning, room temperature is dried.Getting 3 μ L gold/spiral carbon nano-fiber composite material solution drips on the glass-carbon electrode, after at room temperature treating solvent evaporates, drip last 4.5 μ L mass percent concentrations and be 0.5% haemoglobin aqueous solution, after at room temperature treating solvent evaporates then, drip that to be coated with 2 μ L mass percent concentrations be that 0.5% nafion solution is fixed, after room temperature is dried, can obtain enzyme biologic sensor, be placed in 4 ℃ of refrigerators preserve standby.
Embodiment two: the preparation of enzyme biologic sensor
" it is 0.25% PDDA that 20mg spiral carbon nano-fiber is distributed to the 10mL mass percent concentration " among the embodiment one changed into " it is 0.25% PDDA that 20～30mg spiral carbon nano-fiber is distributed to 5～30mL mass percent concentration ", other conditions of preparation are with embodiment one, the performance classes of the sensor that obtains is similar to embodiment one, can be used for the quantitative detection of hydrogen peroxide.
Embodiment three: the preparation of enzyme biologic sensor
With among the embodiment one " mass percent concentration be 0.25% PDDA " change into mass percent concentration be 0.05～0.5% PDDA "; other conditions of preparation are with embodiment one; performance classes of the sensor that obtains is similar to embodiment one, can be used for the quantitative detection of hydrogen peroxide.
Embodiment four: the preparation of enzyme biologic sensor
" mass percent concentration is the solution of gold/spiral carbon nano-fiber composite material of 0.5% " among the embodiment one changed into " solution that mass percent concentration is gold/spiral carbon nano-fiber composite material of 0.5%～2.0% ", other conditions of preparation are with embodiment one, the performance classes of the sensor that obtains is similar to embodiment one, can be used for the quantitative detection of hydrogen peroxide.
Embodiment five: the preparation of enzyme biologic sensor
" ultrasonic 10min " among the embodiment one changed into " ultrasonic 10～30min ", and other conditions of preparation are with embodiment one, and the performance classes of the sensor that obtains is similar to embodiment one, can be used for the quantitative detection of hydrogen peroxide.
Embodiment six: the preparation of enzyme biologic sensor
" gold size of the about 20nm of particle diameter of 10mL " among the embodiment one changed into " gold size of the about 10～30nm of particle diameter of 10mL ", and other conditions of preparation are with embodiment one, and the performance classes of the sensor that obtains is similar to embodiment one, can be used for the quantitative detection of hydrogen peroxide.
Embodiment seven: the preparation of enzyme biologic sensor
" to the gold size that adds altogether till the 15mL " among the embodiment one changed into " to the gold size that adds till totally 15～20mL ", other conditions of preparation are with embodiment one, and the performance classes of the sensor that obtains is similar to embodiment one, can be used for the quantitative detection of hydrogen peroxide.
Embodiment eight: the preparation of enzyme biologic sensor
" 4.5 μ L mass percent concentrations are 0.5% haemoglobin aqueous solution " among the embodiment one changed into " mass percent concentration of 3～8 μ L is 0.5%～1.0% haemoglobin aqueous solution ", other conditions of preparation are with embodiment one, the performance classes of the sensor that obtains is similar to embodiment one, can be used for the quantitative detection of hydrogen peroxide.
Embodiment nine: the preparation of enzyme biologic sensor
" haemoglobin " among the embodiment one changed into " myoglobins " or " horseradish peroxidase ", and other conditions of preparation are with embodiment one, and the performance classes of the sensor that obtains is similar to embodiment one, can be used for the quantitative detection of hydrogen peroxide.
Embodiment ten: the preparation of enzyme biologic sensor
" 2.0 μ L mass percent concentrations are that 0.5% nafion solution is fixed " among the embodiment one changed into " mass percent concentration of 1.0～3.0 μ L is that 0.1～0.5% nafion solution is fixed ", other conditions of preparation are with embodiment one, the performance classes of the sensor that obtains is similar to embodiment one, can be used for the quantitative detection of hydrogen peroxide.
Enzyme biologic sensor with above preparation adopts cyclic voltammetry and two kinds of electrochemical methods of time current method to test.Concrete steps are: under the room temperature, after removing the oxygen in the solution, above electrolytic solution, keep atmosphere of inert gases, it is in 7.0 the phosphate buffered solution that three electrodes are immersed 100mM, pH, adopt cyclic voltammetry or time current method to carry out electro-chemical test, the speed of sweeping when adopting cyclic voltammetry is 100mV/s, scan potential range to be-0.8V～-0.2V; The operating potential of choosing when adopting the time current method is-0.5V.
Claims (4)
1. electrochemica biological sensor that detects hydrogen peroxide, comprise working electrode, platinum electrode, saturated calomel electrode, it is characterized in that the spiral carbon nano-fiber of self assembly and compound substance and haemoglobin or myoglobins or the horseradish peroxidase of gold size nano particle, and make the work electrode with the fixing modified glassy carbon electrode of nafion.
2. the electrochemica biological sensor of detection hydrogen peroxide according to claim 1 is characterized in that the spiral carbon nano-fiber of self assembly and compound substance, haemoglobin or myoglobins or the mass ratio between peroxidase nafion of gold size nano particle are 1.5: 2.25: 0.7.
3. the electrochemica biological sensor of detection hydrogen peroxide according to claim 1 and 2 is characterized in that the spiral carbon nano-fiber of self assembly in the compound substance of the spiral carbon nano-fiber of self assembly and gold size nano particle and the mass ratio of gold size nano particle are 50: 7.
4. method for preparing as the electrochemica biological sensor of the described detection hydrogen peroxide of each claim among the claim 1-3 comprises:
The preparation of step 1 spiral carbon nano-fiber: after the NiO powder that takes by weighing 51mg is put into the porcelain handleless cup, insert in the quartz glass tube, under the environment of 375 ℃ of logical hydrogen, reacted 1 hour then, reacted 0.5 hour in 415 ℃ of acetylene atmosphere at last, the product cool to room temperature can use;
The preparation of step 2 gold/spiral carbon nano-fiber composite material: at first with spiral carbon nano-fiber polydiene propyl-dimethyl amine hydrochlorate (PDDA) functionalization that obtains, specific practice be the spiral carbon nano-fiber that takes by weighing 20～30mg be distributed to 5～30mL mass percent concentration be 0.05%～0.5% PDDA (NaCl that contains 0.5M) aqueous solution in, obtain uniform black suspension behind ultrasonic 10～30min, through centrifuging, it is neutral cleaning to pH with distilled water: then, get 1mL by the above spiral carbon nano-fiber solution that obtains, the gold size of the about 10～30nm of particle diameter of 10mL is scattered in wherein, ultrasonic, afterwards, the gold size that adds 1mL successively, whenever add the ultrasonic 10～30min of gold size one time, be 15～20mL to adding the gold size cumulative volume, at last, remove the upper strata stillness of night, obtain mass percent concentration and be the solution of gold/spiral carbon nano-fiber composite material of 0.5%～2.0%;
Step 3 configuration quality percent concentration is 0.1%～0.5% Nafion solution;
Step 4 glass-carbon electrode cleans;
Step 5 is got solution that 2～5 μ L obtain by step 2 and is dripped on the glass-carbon electrode that step 4 obtains, after at room temperature treating solvent evaporates, drip last 3～8 μ L mass percent concentrations and be 0.5%～1.0% hemoglobin solutions, after at room temperature treating solvent evaporates then, drip that to be coated with 1～3 μ L mass percent concentration be that 0.1%～0.5% nafion solution is fixed, after room temperature is dried, the modified electrode that obtains, be placed in 4 ℃ of refrigerators preserve standby;
The modified electrode that step 6 step 5 obtains and platinum electrode and saturated calomel electrode are formed the bioelectrochemical sensor of three-electrode system.
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Application publication date: 20130717 |