CN102183560B - Methods for preparing and using peroxidase electrode - Google Patents
Methods for preparing and using peroxidase electrode Download PDFInfo
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- CN102183560B CN102183560B CN 201110020051 CN201110020051A CN102183560B CN 102183560 B CN102183560 B CN 102183560B CN 201110020051 CN201110020051 CN 201110020051 CN 201110020051 A CN201110020051 A CN 201110020051A CN 102183560 B CN102183560 B CN 102183560B
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Abstract
The invention discloses methods for preparing and using a peroxidase electrode, belonging to the fields of chemical analysis, environment monitoring and environment protection and particularly relating to methods for preparing and using the peroxidase electrode for determining the content of phenol compounds in water. The method for preparing a horseradish peroxidase immobilization electrode for determining the content of phenol compounds in a water sample is characterized in that dried red chilli is used as a raw material for extraction of horseradish peroxidase, a platinum sheet is used as a carrier, a carbon powder nanotube is used as an electronic carrier, microcrystalline cellulose is used as an immobilized mesh carrier, and D-sorbierite serving as an effector is added. The electrode prepared by the method disclosed by the invention has the advantages of interference resistance, long service life, high activity and strong stability, can be used for measuring cyclic voltammetric curves of the concentrations of different phenol compounds under the same scanning speed to obtain the relation between the magnitude of anodic peak current and the concentration of the phenol compounds, and has the advantages of being capable of directly determining the concentrations of the phenol compounds in a water solution, and the method for using the peroxidase electrode has the advantages of rapidness, accuracy, good repeability and the like.
Description
Technical field
Preparation and the using method thereof of a kind of superoxide enzyme electrode of the present invention; belong to chemical analysis, environmental monitoring and field of environment protection category, specifically relate to a kind of preparation and using method thereof for the superoxide enzyme electrode of measuring the water content of phenolic compounds.
Background technology
Phenolic compound is ubiquity in sanitary sewage, industrial waste water, natural water and potable water, and biosome is had to certain toxicity, is the major pollutants of environment.Both at home and abroad the detection of phenol in water is paid much attention to, strictly controlled the content of the phenolic compound in various water.The standard method that the department of environmental monitoring at present measures volatile phenol in water mainly contains 4-AA spectrophotometric method and extraction spectrophotometric method, the phenolic compound assay method of having reported has multiple, mainly UV-VIS spectrophotometry, atomic absorption method, high performance liquid chromatography, gas phase and liquid phase chromatography etc., these assay methods need to carry out pre-service to water sample, eliminate and disturb, operation is wasted time and energy; In various assay methods, the biology enzyme sensor has high selectivity and high sensitivity, and can on-line analysis.Therefore biology enzyme sensor method fast detecting content of phenolic compounds has a wide range of applications in Industry Control, Study on Environmental Protection.
Peroxidase is a kind of blood red albumen consisted of single peptide chain and porphyrin, can be used as with H
2o
2biocatalyst for the reaction of electron accepter oxidation phenolic compound, there are the characteristics such as high efficiency and selectivity as catalyzer due to enzyme, can prepare a kind of good enzyme electrode, due to enzyme electrode easily be subject to each condition to affect the life-span short, less stable, in order to improve the utilization factor of enzyme, mostly adopt at present the method fixed biologically zymotechnic of electrochemical polymerization conduction high polymer film, that this immobilization technology not only has is reproducible, be convenient to control and the characteristics such as micro-processing, film has good biocompatibility, and peroxidase is fixed on electrode and is applied to measure H
2o
2with the analysis of organic hydroperoxide, also can be used for identifying the materials such as glucose, ethanol, glutamic acid and vitamin; The multiple-wall carbon nanotube that report has to be fixed on glass-carbon electrode is substrate absorption horseradish peroxidase; Also utilize shitosan to fix as carrier, more fixing nano-gold powder, carry out the research of Hydrogen Peroxide Biosensor; In fact also can be for the analysis of phenolic compound, having of report is embedded in horseradish peroxidase in the porous silica material film and builds enzyme sensor at present, but this method need to be combined the mensuration of ultraviolet-visible spectrophotometer method for phenol compound.Although the enzyme electrode of preparation improves the utilization factor of enzyme, need further to improve vigor and the stability of enzyme, can directly utilize the electrode of preparation to be measured phenolic compound.
Summary of the invention
Preparation and the using method thereof of a kind of superoxide enzyme electrode of the present invention, purpose is: in order to overcome above-mentioned problems of the prior art, we study and find to be rich in peroxidase in chilli, cellulose is that the abundantest natural polymer organism is the good carrier of immobilised enzymes, the cyclic voltammetry scanning technique can be observed the variation of electrode in a relatively wide potential range in a short period of time, can judge the stability of electrode by electric current-electromotive force (CV) curve shape, according to directly judgement concentration of substrate variation of peak current size in the CV curve of different concentration of substrate under same sweep velocity, so the present invention is the vigor of raising enzyme and the stability of electrode, provide a kind of and take chilli as raw material extraction capsicum peroxidase, take platinized platinum as matrix, the carbon powder nano pipe is electron carrier, N, dinethylformamide is as spreading agent, microcrystalline cellulose is as the grid carrier of embedding capsicum peroxidase, add the effector molecules D-glucitol, a kind of method for preparing immobilized electrode of the peroxidase of the capsicum for detection of the content of phenolic compounds in water sample.
The preparation method of a kind of superoxide enzyme electrode of the present invention, it is characterized in that a kind ofly take chilli and extracting the capsicum peroxidase as raw material, the carbon powder nano pipe is electron carrier, microcrystalline cellulose is as immobilization grid carrier, add the effector molecules D-glucitol to make a kind of method of capsicum peroxidase immobilization electrode of the content for detection of the phenolic compound in water sample, its concrete preparation method's step is:
The preparation of I, capsicum peroxidase
Take the chilli of market sale as raw material through pulverizing 80～100 orders, take and pulverized 80～100 purpose 20～50g chilli powder, add the distilled water of 200～500mL in 0 ℃ of lower ultrasonic echography 20～40min, suction filtration, obtain crude extract after centrifugal, the ammonium sulfate that is first 0.4 by saturation degree is dialysed to the crude extract extracted, obtain supernatant after centrifugal, by saturation degree, be that 0.8 ammonium sulfate is dialysed to supernatant again, precipitation after centrifugal, adding ℃ acetone classification of 1～2 times of volume-14～-16 to separate the gained precipitation is dialysed, the ultrafiltration membrance filter that soln using molecular cut off after dialysis is 5000～10000 obtains the capsicum peroxidase, 4～6 ℃ of preservations.
The preparation of II, superoxide enzyme electrode
By 0.5～1.5cm
2the size platinized platinum carries out pre-service: soak 0.5～1h in 1moL/LNaOH solution, then take out with cotton balls and dip the distilled water scrub surfaces, at 1moL/L H
2sO
4in solution, in the electrode potential scope of-0.675～1.675V with the sweep velocity of 40～60mV/s, circulate after 40～50 times, take out again water rinse well dry stand-by, get 5～10mL N, dinethylformamide is that solvent adds 20～40mg microcrystalline cellulose and the ultrasonic 10～20min of 20～40mg carbon powder nano pipe to mix, get pretreated platinized platinum, take its diagonal line as separatrix, evenly smear one deck microcrystalline cellulose-carbon powder nano pipe film, natural drying 40～50h is standby, the capsicum peroxidase of 50～150 μ L of above-mentioned preparation and 10～50 μ L D-glucitols are dropped in to dry microcrystalline cellulose-carbon powder nano pipe film surface equably, it is infiltrated lentamente fully, be placed on 20～24h airing in 4～6 ℃ of refrigerators, obtain capsicum superoxide enzyme electrode of the present invention, with distilled water, repeatedly rinse for several times standby.
The using method of above-mentioned a kind of superoxide enzyme electrode, get the prepared capsicum superoxide enzyme electrode of said method while it is characterized in that using as working electrode, saturated calomel electrode is contrast electrode, platinum electrode is that electrode is formed to three-electrode system, the pH value of solution that the Tris-HCl buffer solution of take is regulated phenolic compound is 5～8.0, add 0.5～1% (v/v) hydrogen peroxide, adopt Princeton VMP3 electro-chemical systems to carry out the CV curve determination, sweep velocity is 50～100mV/s, electrode potential is at-0.2～0.6V scope interscan 40～60 circles, the CV curve is along with the increase of scanning times, peak current is almost constant, the stability that shows this electrode is better, this electrode is placed to 5～7d in 4～6 ℃ of refrigerators, the intermittent use between resting period, the variable quantity of its response signal does not surpass 3～5% of initialize signal, illustrate that this capsicum superoxide enzyme electrode has good stability, measure the CV curve of different phenolic compound concentration under same sweep velocity, obtain the relation of peak current size and phenolic compound concentration, for Direct Analysis phenol in aqueous solution compounds to be measured concentration.
Preparation and the using method thereof of a kind of superoxide enzyme electrode of the present invention, advantage is to utilize the chilli that is rich in peroxidase be easy to get to extract peroxidase for raw material, utilization has stronger film forming ability, high osmosis, physical strength is high, avirulent, the microcrystalline cellulose that biocompatibility is large and carbon powder nano pipe are carrier, add the effector molecules D-glucitol vigor of enzyme can be improved to 3～4 times and anodic peak current increase 0.1～0.3mA, due to enzymatic selectivity, the characteristics such as high efficiency and mild condition, so it is anti-interference that electrode prepared by the present invention has, long service life, vigor is high, the advantage that stability is strong, measure the CV curve of different phenolic compound concentration under same sweep velocity, obtain the relation of anodic peak current size and phenol compound concentration, advantage is directly to measure phenol in aqueous solution compounds concentration, this method has fast, accurately, the advantage such as reproducible.
The accompanying drawing explanation
Fig. 1 is capsicum antioxidant enzyme extraction process process flow diagram of the present invention.
Fig. 2 is capsicum superoxide enzyme electrode schematic diagram prepared by the present invention.In figure: c means the smear zone of electrode
Fig. 3 is fixedly capsicum peroxidase schematic diagram of microcrystalline cellulose of the present invention-carbon powder nano pipe film.
In figure: ● mean that carbon powder nano pipe zero means the capsicum peroxidase
Fig. 4 is the electrode for preparing of the present invention (160X) form comparison diagram under the microscope.
In figure:
the carbon powder nano pipe
microcrystalline cellulose
the capsicum peroxidase
Fig. 5 is the electrode for preparing of the present invention and the cyclic voltammogram of bare electrode.
In figure: a: the cyclic voltammetry curve b that is electrode of the present invention: the cyclic voltammetry curve that is bare electrode
Fig. 6 be the electrode for preparing of the present invention with without effector molecules electrode cyclic voltammogram.
In figure: a: be the cyclic voltammetry curve b without the effector molecules electrode: be electrode cyclic voltammetry curve of the present invention
Fig. 7 is the canonical plotting of phenol concentration of the present invention and anodic peak current.
Embodiment
Embodiment 1
Take commercially available chilli as raw material through pulverizing 80 orders, take 20.0g chilli powder, add the water of distillation (0 ℃) ultrasound wave under low temperature of 200mL to process the 20min suction filtration, with the centrifugal 15min of 4000r/min speed, obtain supernatant.It is 0.4 ammonium sulfate (in every 1000mL filtrate, adding 226.0g ammonium sulfate) that the supernatant of said extracted is slowly added to saturation degree under 0 ℃ is constantly stirred, and puts 24h in refrigerator.By the supernatant sucking-off, following dirty solution, with the centrifugal 15min of the centrifugal speed of 3000r/min, is discarded precipitation and merges supernatant.When in supernatant, limit adds 0 ℃, saturation degree is that stir until 24h in refrigerator is put in all dissolvings on 0.8 ammonium sulfate (every 1000mL supernatant adds 258g ammonium sulfate) limit.Draw supernatant, will be deposited under the 13000r/min centrifugal speed after centrifugal 20min abandoning supernatant, collecting precipitation; Then precipitation is suspended in 50mL distilled water, is sub-packed in bag filter, be placed under the tap water that flows and dialysed 24h to remove ammonium sulfate.For avoiding chlorion in water to produce and disturb the peroxidase extracted, need continue dialysis 24h with distilled water, during change 2 distilled water, until check that with the 0.1mol/L liquor argenti nitratis ophthalmicus extracellular fluid dialysis is without chlorion, the merging dislysate.Dislysate after being combined, with the centrifugal 15min of 4000r/min centrifugal speed, discards precipitation, metering supernatant volume.Pour the final supernatant of said extracted into beaker and be placed in the cryosel bath, under constantly stirring, add in advance the acetone of 3 times that is chilled to-14 ℃ of volumes, with the centrifugal 15min of 4000r/min centrifugal speed, discard precipitation; Add-14 ℃ of acetone in supernatant, its amount continues centrifugal collecting precipitation for after 0.8 times of former supernatant volume again; Distilled water dissolves to be loaded in bag filter dialyses to remove acetone, after 5000～10000 ultra filtration membrane ultrafiltration, extract is put to 4 ℃ of preservations of refrigerator, and recording the capsicum peroxidase concn is 8.32g/L, and vigor is 16.77u/g.In order to make extract capsicum peroxidase effectively bring into play vigor, during use, need to add the effector molecules D-glucitol can improve 3 times of enzyme activities.By 0.5cm
2the size platinized platinum soaks 0.5h in 1moL/L NaOH solution, then takes out with cotton balls and dips the distilled water scrub surfaces, at 1moL/L H
2sO
4in solution, in the electrode potential scope of-0.675～1.675V with the sweep velocity of 40mV/s, circulate after 40 times, take out again water rinse well dry stand-by, get 5mLN, dinethylformamide is that solvent adds 20mg microcrystalline cellulose and the ultrasonic 10min of 20mg carbon powder nano pipe to mix, get pretreated platinized platinum, take its diagonal line as separatrix, evenly smear one deck microcrystalline cellulose-carbon powder nano pipe film, natural drying 40h is standby, the capsicum peroxidase of 50 μ L of above-mentioned preparation and 10 μ L D-glucitols are dropped in to dry microcrystalline cellulose-carbon powder nano pipe film surface equably, it is infiltrated lentamente fully, be placed on 20h airing in 4 ℃ of refrigerators, obtain capsicum superoxide enzyme electrode of the present invention, with distilled water, repeatedly rinse for several times standby.Fixedly capsicum peroxidase schematic diagram is as shown in Figure 3 for microcrystalline cellulose-carbon powder nano pipe film.Microscope (160x) is observed electrode shape shown in Fig. 4 (a, b), and figure a is the bare electrode form, figure b is electrode of the present invention, from figure a and figure b contrast, see, the capsicum peroxidase can be immobilized in electrode surface preferably, thereby guarantee the normal use of enzyme electrode.Electrode prepared by the present invention is working electrode, take platinum electrode as to electrode, and mercurous chloride electrode is contrast electrode, adopts the PrincetonVMP3 electro-chemical systems to be measured.Get the solution that contains phenol compound, it is 5.0 that the Tris-HCl buffer solution of take is regulated the pH value, add the hydrogen peroxide that volume ratio is 0.5%, at room temperature can urge lower generation redox reaction at the capsicum peroxidase; get phenol; regulate pH to 7.0 with the Tris-HCl damping fluid, add by volume 0.5%H
2o
2, take sweep velocity as 50mV/s, electrode potential exists--the interscan of 0.2～0.6V scope, with bare electrode, contrasted simultaneously, cyclic voltammogram is as shown in Figure 5.Occurred a pair of redox peak in Fig. 5, negative electrode peak electromotive force and anode peak electromotive force are in-0.025V and 0.2V left and right, and this is the redox character peak that capsicum peroxidase redox center produces.To above-mentioned electrode 40 circles that all circulate, along with the increase of scanning times, peak current is almost constant, shows that the stability of this electrode is better.This electrode is placed to 5d in 4 ℃ of refrigerators, the intermittent use between resting period, the variable quantity of its response signal does not surpass 3% of initialize signal, illustrates that electrode of the present invention has stability preferably.Effect for the electrode that confirms to add the effector molecules D-glucitol, have effector molecules and electrode cyclic voltammetry curve of the present invention to compare, result as shown in Figure 6 wherein a be without the effector molecules electrode, b is electrode of the present invention.As can be seen from Figure 6: add effector molecules D-glucitol rear electrode to go out the peak situation all than not adding the effector molecules fashion, anodic peak current has increased 0.3mA.Adopt electrode system of the present invention, change the concentration of phenol, it is 7.0 that the Tris-HCl damping fluid of take is regulated pH, adds in proportion 0.5% (v/v) H
2o
2, sweep velocity is 50mV/s, and electrode potential is in the interscan of-0.2～0.6V scope, and the anodic peak current in the CV curve and the concentration relationship of phenol are as shown in Figure 7.As can be seen from Figure 7 linear in concentration is 2mmol/L, meet relational expression: I (mA)=0.101369+0.01505C, r by matching anodic peak current value I (mA) and phenol concentration C (mmol/L)
2=0.985 detection is limited to 22mmol/L.Get the sewage 25mL of discharge after processing, the Tris-HCl damping fluid is regulated pH and is preserved 5 days under 5,4 ℃, adds in proportion 0.5% (v/v) hydrogen peroxide, utilizes electrode system of the present invention to be measured.In sweep velocity, be 50mV/s, electrode potential is in-0.2～0.6V scope, carry out three parallel sweeps and measure the CV curve, read the anodic peak current value, calculate the concentration of phenol compound in sewage effluent by the matching relational expression of anodic peak current value and phenol concentration.
Take the chilli of market sale as raw material through pulverizing 85 orders, take and pulverized 85 purpose 25g chilli powder, add the distilled water of 250mL in 0 ℃ of lower ultrasonic echography 30min, suction filtration, obtain crude extract after centrifugal, the ammonium sulfate that is first 0.4 by saturation degree is dialysed to the crude extract extracted, obtain supernatant after centrifugal, by saturation degree, be that 0.8 ammonium sulfate is dialysed to supernatant again, precipitation after centrifugal, adding-15 ℃ of acetone classifications of 1 times of volume to separate the gained precipitation is dialysed, the ultrafiltration membrance filter that soln using molecular cut off after dialysis is 5000～10000, the capsicum peroxidase obtained under 4 ℃, recording the capsicum peroxidase concn is 8.56g/L, and vigor is 15.53u/g.By 1.0cm
2the size platinized platinum soaks 0.5h in 1moL/LNaOH solution, then takes out with cotton balls and dips the distilled water scrub surfaces, at 1moL/L H
2sO
4in solution, in the electrode potential scope of-0.675～1.675V with the sweep velocity of 40mv/s, circulate after 40 times, take out again water rinse well dry stand-by, get 5mLN, dinethylformamide is that solvent adds 20mg microcrystalline cellulose and the ultrasonic 10min of 20mg carbon powder nano pipe to mix, get pretreated platinized platinum, take its diagonal line as separatrix, evenly smear one deck microcrystalline cellulose-carbon powder nano pipe film, natural drying 40h is standby, the capsicum peroxidase of 50 μ L of above-mentioned preparation and 10 μ L D-glucitols are dropped in to dry microcrystalline cellulose-carbon powder nano pipe film surface equably, it is infiltrated lentamente fully, be placed on 20h airing in 4 ℃ of refrigerators, obtain capsicum superoxide enzyme electrode of the present invention, with distilled water, repeatedly rinse for several times standby.Gather after Sewage Plant 1 is processed the sewage 25mL discharged, the Tris-HCl damping fluid is regulated pH and is preserved 5 days under 5.0 in 4 ℃, adds the hydrogen peroxide that volume ratio is 0.5% during mensuration, utilizes electrode system of the present invention to be measured.In sweep velocity, be 50mV/s, electrode potential is in-0.2～0.6V scope, carry out three parallel sweeps and measure the CV curve, read the anodic peak current value, it is 15.4 ± 0.23mmol/L that the matching relational expression by anodic peak current value and phenol concentration calculates content of phenolic compounds in the sewage of Sewage Plant 1 discharge.Other is with embodiment 1.
Embodiment 3.
Take the chilli of market sale as raw material through pulverizing 100 orders, take and pulverized 100 purpose 50g chilli powder, add the distilled water of 500mL in 0 ℃ of lower ultrasonic echography 40min, suction filtration, obtain crude extract after centrifugal, the ammonium sulfate that is first 0.4 by saturation degree is dialysed to the crude extract extracted, obtain supernatant after centrifugal, by saturation degree, be that 0.8 ammonium sulfate is dialysed to supernatant again, precipitation after centrifugal, adding ℃ acetone classification of 2 times of volume-16 to separate the gained precipitation is dialysed, the ultrafiltration membrance filter that soln using molecular cut off after dialysis is 10000, the capsicum peroxidase obtained under 6 ℃, recording concentration is 10.25g/L, vigor is 23.3u/g.By 1.5cm
2the size platinized platinum soaks 1h in 1moL/LNaOH solution, then takes out with cotton balls and dips the distilled water scrub surfaces, at 1moL/L H
2sO
4in solution, in the electrode potential scope of-0.675～1.675V with the sweep velocity of 60mV/s, circulate after 50 times, take out again water rinse well dry stand-by, get 10mLN, dinethylformamide is that solvent adds 40mg microcrystalline cellulose and the ultrasonic 20min of 40mg carbon powder nano pipe to mix, get pretreated platinized platinum, take its diagonal line as separatrix, evenly smear one deck microcrystalline cellulose-carbon powder nano pipe film, natural drying 50h is standby, the capsicum peroxidase of 150 μ L of above-mentioned preparation and 50 μ L D-glucitols are dropped in to dry microcrystalline cellulose-carbon powder nano pipe film surface equably, it is infiltrated lentamente fully, be placed on 24h airing in 6 ℃ of refrigerators, obtain capsicum superoxide enzyme electrode of the present invention, with distilled water, repeatedly rinse for several times standby.Gather the sewage 25mL of Sewage Plant 2 discharges, the Tris-HCl damping fluid is regulated pH and is preserved 5 days under 8.0,6 ℃, and adding volume ratio is 1.0% hydrogen peroxide, utilizes electrode system of the present invention to be measured.In sweep velocity, be 100mV/s, electrode potential is in-0.2～0.6V scope, carry out three parallel sweeps and measure the CV curve, read the anodic peak current value, it is 14.5 ± 0.12mmol/L that the matching relational expression by anodic peak current value and phenol concentration calculates content of phenolic compounds in the sewage of Sewage Plant 2 discharges.Other is with embodiment 1.
Take the chilli of market sale as raw material through pulverizing 90 orders, take and pulverized 90 purpose 35g chilli powder, add the distilled water of 350mL in 0 ℃ of lower ultrasonic echography 30min, suction filtration, obtain crude extract after centrifugal, the ammonium sulfate that is first 0.4 by saturation degree is dialysed to the crude extract extracted, obtain supernatant after centrifugal, by saturation degree, be that 0.8 ammonium sulfate is dialysed to supernatant again, precipitation after centrifugal, adding ℃ acetone classification of 1.5 times of volume-15 to separate the gained precipitation is dialysed, the ultrafiltration membrance filter that soln using molecular cut off after dialysis is 5000, the capsicum peroxidase obtained under 5 ℃, recording its concentration is 21.5g/L, vigor is 20.53u/g.By 0.5cm
2the size platinized platinum soaks 0.8h in 1moL/LNaOH solution, then takes out with cotton balls and dips the distilled water scrub surfaces, at 1moL/L H
2sO
4in solution, in the electrode potential scope of-0.675～1.675V with the sweep velocity of 50mV/s, circulate after 50 times, take out again water rinse well dry stand-by, get 8mLN, dinethylformamide is that solvent adds 32mg microcrystalline cellulose and the ultrasonic 15min of 32mg carbon powder nano pipe to mix, get pretreated platinized platinum, take its diagonal line as separatrix, evenly smear one deck microcrystalline cellulose-carbon powder nano pipe film, natural drying 48h is standby, the capsicum peroxidase of 100 μ L of above-mentioned preparation and 40 μ LD-sorbierites are dropped in to dry microcrystalline cellulose-carbon powder nano pipe film surface equably, it is infiltrated lentamente fully, be placed on 24h airing in 5 ℃ of refrigerators, obtain capsicum superoxide enzyme electrode of the present invention, with distilled water, repeatedly rinse for several times standby.Gather the sewage 25mL of Sewage Plant 3 discharges, the Tris-HCl damping fluid is regulated pH and is preserved 5 days under 7.0,5 ℃, adds the hydrogen peroxide that volume ratio is 0.8%, utilizes electrode system of the present invention to be measured.In sweep velocity, be 60mV/s, electrode potential is in-0.2～0.6V scope, carry out three parallel sweeps and measure the CV curve, read the anodic peak current value, it is 16.8 ± 0.31mmol/L that the matching relational expression by anodic peak current value and phenol concentration calculates content of phenolic compounds in the sewage of Sewage Plant 3 discharges.Other is with embodiment 1.
Embodiment 5.
Take the chilli of market sale as raw material through pulverizing 95 orders, take and pulverized 95 purpose 40g chilli powder, add the distilled water of 400mL in 0 ℃ of lower ultrasonic echography 35min, suction filtration, obtain crude extract after centrifugal, the ammonium sulfate that is first 0.4 by saturation degree is dialysed to the crude extract extracted, obtain supernatant after centrifugal, by saturation degree, be that 0.8 ammonium sulfate is dialysed to supernatant again, precipitation after centrifugal, adding ℃ acetone classification of 1 times of volume-16 to separate the gained precipitation is dialysed, the ultrafiltration membrance filter that soln using molecular cut off after dialysis is 5000～10000, the capsicum peroxidase obtained under 4 ℃, recording its concentration is 30.23g/L, vigor is 25.53u/g.By 1cm
2the size platinized platinum soaks 1h in 1moL/L NaOH solution, then takes out with cotton balls and dips the distilled water scrub surfaces, at 1moL/LH
2sO
4in solution, in the voltage range of-0.675～1.675V with the sweep velocity of 50mv/s, circulate after 48 times, take out again water rinse well dry stand-by, get 6mLN, dinethylformamide is that solvent adds 24mg microcrystalline cellulose and the ultrasonic 15min of 24mg carbon powder nano pipe to mix, get pretreated platinized platinum, take its diagonal line as separatrix, evenly smear one deck microcrystalline cellulose-carbon powder nano pipe film, natural drying 40h is standby, the capsicum peroxidase of 100 μ L of above-mentioned preparation and 30 μ L D-glucitols are dropped in to dry microcrystalline cellulose-carbon powder nano pipe film surface equably, it is infiltrated lentamente fully, be placed on 20h airing in 4 ℃ of refrigerators, obtain capsicum superoxide enzyme electrode of the present invention, with distilled water, repeatedly rinse for several times standby.Gather the sewage 25mL of Sewage Plant 4, the Tris-HCl damping fluid is regulated pH and is preserved 5 days under 5.0,4 ℃, and adding volume ratio is 0.9% hydrogen peroxide, utilizes electrode system of the present invention to be measured.In sweep velocity, be 70mV/s, electrode potential is in-0.2～0.6V scope, carry out three parallel sweeps and measure the CV curve, read the anodic peak current value, it is 14.8 ± 0.14mmol/L that the matching relational expression by anodic peak current value and phenol concentration calculates content of phenolic compounds in the sewage of Sewage Plant 4 discharges.Other is with embodiment 1.
Claims (1)
1. the preparation method of a superoxide enzyme electrode, it is characterized in that a kind ofly take chilli and extracting the capsicum peroxidase as raw material, platinized platinum is matrix, the carbon powder nano pipe is electron carrier, microcrystalline cellulose is as immobilization grid carrier, add the effector molecules D-glucitol to make a kind of method of capsicum peroxidase immobilization electrode of the content for detection of the phenolic compound in water sample, its concrete preparation method's step is:
Take the chilli of market sale as raw material through pulverizing 80 ~ 100 orders, take and pulverized 80 ~ 100 purpose 20 ~ 50g chilli powder, add the distilled water of 200 ~ 500mL in 0 ℃ of lower ultrasonic echography 20 ~ 40min, suction filtration, obtain crude extract after centrifugal, the ammonium sulfate that is first 0. 4 by saturation degree is dialysed to the crude extract extracted, obtain supernatant after centrifugal, by saturation degree, be that 0.8 ammonium sulfate is dialysed to supernatant again, precipitation after centrifugal, adding ℃ acetone classification of 1 ~ 2 times of volume-14 ~-16 to separate the gained precipitation is dialysed, the ultrafiltration membrance filter that soln using molecular cut off after dialysis is 5000 ~ 10000 obtains the capsicum peroxidase, 4 ~ 6 ℃ of preservations,
By 0.5 ~ 1.5cm
2the size platinized platinum carries out pre-service: soak 0.5 ~ 1h in 1moL/L NaOH solution, then take out with cotton balls and dip the distilled water scrub surfaces, at 1moL/L H
2sO
4in solution, in the electrode potential scope of-0.675 ~ 1.675V, with the sweep velocity of 40 ~ 60mV/s, circulate after 40 ~ 50 times, take out again water rinse well dry stand-by
,get 5 ~ 10mL N, dinethylformamide is that solvent adds 20 ~ 40mg microcrystalline cellulose and the ultrasonic 10 ~ 20min of 20 ~ 40mg carbon powder nano pipe to mix, get pretreated platinized platinum, take its diagonal line as separatrix, evenly smear one deck microcrystalline cellulose-carbon powder nano pipe film, natural drying 40 ~ 50h is standby, by 50 ~ 150 of above-mentioned preparation
the capsicum peroxidase and 10 ~ 50 of L
the L D-glucitol drops in dry microcrystalline cellulose-carbon powder nano pipe film surface equably, and it is infiltrated lentamente fully, is placed on 20 ~ 24h airing in 4 ~ 6 ℃ of refrigerators, obtains capsicum superoxide enzyme electrode, with distilled water, repeatedly rinses for several times standby.
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| JP4462395B2 (en) * | 2000-04-24 | 2010-05-12 | 東洋紡績株式会社 | Polyphenol sensor |
| CN101625334B (en) * | 2009-08-05 | 2012-09-05 | 西北师范大学 | Enzyme modified electrode based on sol gel layer and preparation method thereof |
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2011
- 2011-01-14 CN CN 201110020051 patent/CN102183560B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| 于洋.基于有机-无机杂化材料的硝酸还原酶和辣根过氧化物酶电极的研究.《中国优秀硕士学位论文全文数据库》.2009,(第4期),第55、77页. |
| 基于有机-无机杂化材料的硝酸还原酶和辣根过氧化物酶电极的研究;于洋;《中国优秀硕士学位论文全文数据库》;20090415(第4期);第55、77页 * |
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