CN102183560A - Methods for preparing and using peroxidase electrode - Google Patents
Methods for preparing and using peroxidase electrode Download PDFInfo
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- CN102183560A CN102183560A CN2011100200517A CN201110020051A CN102183560A CN 102183560 A CN102183560 A CN 102183560A CN 2011100200517 A CN2011100200517 A CN 2011100200517A CN 201110020051 A CN201110020051 A CN 201110020051A CN 102183560 A CN102183560 A CN 102183560A
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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
The 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 that is used for measuring the superoxide enzyme electrode of 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 certain toxicity, is the major pollutants of environment.The detection of phenol in the water is paid much attention to the content of the phenolic compound in the various water of strict control both at home and abroad.The standard method of volatile phenol mainly contains 4-amino-antipyrine spectrophotometric method and extraction spectrophotometric method in the environmental monitoring department mensuration water at present, the phenolic compound assay method of having reported has multiple, mainly be UV-VIS spectrophotometry, atomic absorption method, high performance liquid chromatography, gas phase and liquid phase chromatography etc., these assay methods need 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 that is made of single peptide chain and porphyrin, can be used as with H
2O
2Biocatalyst for the reaction of electron accepter oxidation phenolic compound, because enzyme has characteristics such as high efficiency and selectivity as catalyzer, can prepare a kind of good enzyme electrode, because enzyme electrode is subjected to each condition effect life-span short easily, less stable, in order to improve the utilization factor of enzyme, adopt the method fixed biologically zymotechnic of electrochemical polymerization conduction high polymer film at present mostly, this immobilization technology not only has good reproducibility, is convenient to characteristics such as control and little processing, film has good biocompatibility, and peroxidase is fixed on and is applied to measure H on the electrode
2O
2With the analysis of organic hydroperoxide, also can be used for identifying materials such as glucose, ethanol, glutamic acid and vitamin; It is substrate absorption horseradish peroxidase that report has the multiple-wall carbon nanotube to be fixed on the glass-carbon electrode; Also utilize shitosan to fix as carrier, fixing nanometer bronze carries out the hydrogen peroxide Study on Biosensor again; Also can be used for the analysis of phenolic compound in fact, at present report has horseradish peroxidase is embedded in the porous silica material film and makes up enzyme sensor, but this method need be united the mensuration that the ultraviolet-visible spectrophotometer method is used for phenol compound.Although the enzyme electrode of preparation improves the utilization factor of enzyme, need further to improve the vigor and the stability of enzyme, can directly utilize the electrode of preparation to measure phenolic compound.
Summary of the invention
The 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 discover is rich in peroxidase in the 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 the interior electrode of potential range of a relative broad in a short period of time, can judge the stability of electrode by electric current-electromotive force (CV) curve shape, judge directly that according to peak current size in the CV curve of different concentration of substrate under the same sweep velocity concentration of substrate changes, so the present invention is the vigor of raising enzyme and the stability of electrode, providing a kind of is that raw material extracts the capsicum peroxidase with the chilli, with the platinized platinum is matrix, the carbon powder nano pipe is an electron carrier, N, dinethylformamide is as spreading agent, microcrystalline cellulose is as the grid carrier of embedding capsicum peroxidase, add effector molecules D-sorbierite, prepare a kind of method of immobilized electrode of capsicum peroxidase of the content of phenolic compounds that is used for detecting water sample.
The preparation method of a kind of superoxide enzyme electrode of the present invention, it is characterized in that be a kind of be that raw material extracts the capsicum peroxidase with the chilli, the carbon powder nano pipe is an electron carrier, microcrystalline cellulose is as immobilization grid carrier, add effector molecules D-sorbierite and make a kind of method of capsicum peroxidase immobilization electrode of content of the phenolic compound that is used for detecting water sample, its concrete preparation method's step is:
The preparation of I, capsicum peroxidase
Chilli with market sale is that raw material was through pulverizing 80～100 orders, take by weighing and pulverized 80～100 purposes, 20～50g chilli powder, the distilled water that adds 200～500mL is in 0 ℃ of following ultrasonic echography 20～40min, suction filtration, get crude extract after centrifugal, be that 0.4 ammonium sulfate is dialysed to the crude extract of extraction earlier with saturation degree, get supernatant after centrifugal, be that 0.8 ammonium sulfate is dialysed to supernatant with saturation degree again, precipitation after centrifugal, the gained precipitation is added 1～2 times of volume-14～-16 ℃ acetone fractionated dialyses, soln using molecular cut off after the dialysis is that 5000～10000 ultrafiltration membrance filter obtains the capsicum peroxidase, 4～6 ℃ of preservations.
The preparation of II, superoxide enzyme electrode
With 0.5～1.5cm
2The size platinized platinum carries out pre-service: soak 0.5～1h in 1moL/LNaOH solution, take out then to dip in cotton balls and get the distilled water scrub surfaces, at 1moL/L H
2SO
4In the 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 mixes, get pretreated platinized platinum, with its diagonal line is the separatrix, evenly smear one deck microcrystalline cellulose-carbon powder nano pipe film, air dry 40～50h is standby, capsicum peroxidase and 10～50 μ L D-sorbierites of 50～150 μ L of above-mentioned preparation are dropped in 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, wash for several times standby with distilled water repeatedly.
The using method of above-mentioned a kind of superoxide enzyme electrode, get the prepared capsicum superoxide enzyme electrode of said method when it is characterized in that using as working electrode, saturated calomel electrode is a contrast electrode, platinum electrode is that electrode is constituted three-electrode system, the pH value of solution of regulating phenolic compound with Tris-HCl buffer solution 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 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 stable, measure the CV curve of different phenolic compound concentration under the same sweep velocity, obtain the relation of peak current size and phenolic compound concentration, be used for directly analyzing aqueous solution phenolic compound concentration to be measured.
The 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 that is easy to get to extract peroxidase for raw material, utilization has stronger film forming ability, high osmosis, the physical strength height, avirulence, microcrystalline cellulose that biocompatibility is big and carbon powder nano pipe are carrier, add effector molecules D-sorbierite and the vigor of enzyme can be improved 3～4 times and anodic peak current increase 0.1～0.3mA, because enzymatic selectivity, characteristics such as high efficiency and mild condition, so the electrode of the present invention's preparation has anti-interference, long service life, the vigor height, the advantage that stability is strong, measure the CV curve of different phenolic compound concentration under the same sweep velocity, obtain the relation of anodic peak current size and phenol compound concentration, advantage is directly to measure phenolic compound concentration in the aqueous solution, and this method has fast, accurately, advantages such as good reproducibility.
Description of drawings
Fig. 1 is a capsicum antioxidant enzyme extraction process process flow diagram of the present invention.
Fig. 2 is the capsicum superoxide enzyme electrode synoptic diagram that the present invention prepares.Among the figure: c represents the smear zone of electrode
Fig. 3 is a fixedly capsicum peroxidase synoptic diagram of microcrystalline cellulose of the present invention-carbon powder nano pipe film.
Among the figure: ● expression carbon powder nano pipe zero expression capsicum peroxidase
Fig. 4 is that the electrode for preparing of the present invention is at microscopically (160X) form comparison diagram.
Among the 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.
Among the figure: a: the cyclic voltammetry curve b that is electrode of the present invention: the cyclic voltammetry curve that is bare electrode
Fig. 6 is the electrode and no effector molecules electrode cyclic voltammogram that the present invention prepares.
Among the figure: a: the cyclic voltammetry curve b that is no 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
With commercially available chilli be raw material through pulverizing 80 orders, take by weighing 20.0g chilli powder, add water (0 ℃) ultrasonic Treatment 20min suction filtration under low temperature of the distillation of 200mL, with the centrifugal 15min of 4000r/min speed, supernatant.The supernatant of said extracted is slowly added saturation degree under 0 ℃ is constantly stirred be 0.4 ammonium sulfate (adding 226.0g ammonium sulfate in every 1000mL filtrate), puts 24h in the refrigerator.With the supernatant sucking-off, following muddy liquid with the centrifugal 15min of the centrifugal speed of 3000r/min, is discarded precipitation and merges supernatant.Saturation degree was that stir until whole dissolvings on 0.8 ammonium sulfate (every 1000mL supernatant adds 258g ammonium sulfate) limit when the limit added 0 ℃ in supernatant, put 24h in the refrigerator.Draw supernatant, will be deposited under the 13000r/min centrifugal speed behind the centrifugal 20min abandoning supernatant, collecting precipitation; Then precipitation is suspended in the 50mL distilled water, is sub-packed in bag filter, place under the tap water that flows and dialyse 24h to remove ammonium sulfate.Disturb for avoiding chlorion in the water that the peroxidase that extracts is produced, need to continue dialysis 24h with distilled water, during change 2 distilled water, till checking that with the 0.1mol/L liquor argenti nitratis ophthalmicus extracellular fluid dialysis does not have chlorion, the merging dislysate.Dislysate after being combined discards precipitation with the centrifugal 15min of 4000r/min centrifugal speed, metering supernatant volume.Pour the final supernatant of said extracted into beaker and place cryosel to bathe, under constantly stirring, add 3 times the acetone that is chilled to-14 ℃ of volumes in advance,, discard precipitation with the centrifugal 15min of 4000r/min centrifugal speed; Add-14 ℃ of acetone again in supernatant, its amount continues centrifugal collecting precipitation for behind 0.8 times of former supernatant volume; Dissolved in distilled water is loaded in the bag filter and dialyses to remove acetone, after 5000～10000 the ultra filtration membrane ultrafiltration extract is put 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, need add effector molecules D-sorbierite during use and can improve 3 times of enzyme activities.With 0.5cm
2The size platinized platinum soaks 0.5h in 1moL/L NaOH solution, take out then to dip in cotton balls and get the distilled water scrub surfaces, at 1moL/L H
2SO
4In the 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 the 20mg microcrystalline cellulose and the ultrasonic 10min of 20mg carbon powder nano pipe mixes, get pretreated platinized platinum, with its diagonal line is the separatrix, evenly smear one deck microcrystalline cellulose-carbon powder nano pipe film, air dry 40h is standby, capsicum peroxidase and the 10 μ L D-sorbierites of 50 μ L of above-mentioned preparation are dropped in 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, wash for several times standby with distilled water repeatedly.Fixedly capsicum peroxidase synoptic diagram is as shown in Figure 3 for microcrystalline cellulose-carbon powder nano pipe film.Microscope (160x) observe electrode shape such as Fig. 4 (a, b) shown in, figure a is the bare electrode form, figure b is an electrode of the present invention, sees that the capsicum peroxidase can be immobilized preferably in electrode surface from figure a and figure b contrast, thereby guarantees the normal use of enzyme electrode.The electrode of the present invention's preparation is a working electrode, is to electrode with platinum electrode, and mercurous chloride electrode is a contrast electrode, adopts the PrincetonVMP3 electro-chemical systems to measure.Get the solution that contains phenol compound, regulating the pH value with Tris-HCl buffer solution is 5.0, the adding volume ratio is 0.5% hydrogen peroxide, at room temperature can urge redox reaction takes place down; get phenol; regulate pH to 7.0 with the Tris-HCl damping fluid, add 0.5%H by volume at the capsicum peroxidase
2O
2, be 50mV/s with the sweep velocity, electrode potential exists--the interscan of 0.2～0.6V scope, contrast with bare electrode simultaneously, cyclic voltammogram is as shown in Figure 5.Occurred a pair of redox peak among Fig. 5, negative electrode peak electromotive force and anode peak electromotive force are about-0.025V and 0.2V, and this is the redox characteristic 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, and the stability that shows this electrode better.This electrode is placed 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 effector molecules D-sorbierite has effector molecules and electrode cyclic voltammetry curve of the present invention to compare, the result as shown in Figure 6 wherein a be no effector molecules electrode, b is an electrode of the present invention.As can be seen from Figure 6: add effector molecules D-sorbierite rear electrode and 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, regulating pH with the Tris-HCl damping fluid is 7.0, adds 0.5% (v/v) H in proportion
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 match 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 that handles the back discharging, the Tris-HCl damping fluid is regulated pH and was preserved 5 days under 5,4 ℃, adds 0.5% (v/v) hydrogen peroxide in proportion, utilizes electrode system of the present invention to measure.In sweep velocity is 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 the sewage effluent by the match relational expression of anodic peak current value and phenol concentration.
Chilli with market sale is that raw material was through pulverizing 85 orders, take by weighing and pulverized 85 purpose 25g chilli powder, the distilled water that adds 250mL is in 0 ℃ of following ultrasonic echography 30min, suction filtration, get crude extract after centrifugal, be that 0.4 ammonium sulfate is dialysed to the crude extract of extraction earlier with saturation degree, get supernatant after centrifugal, be that 0.8 ammonium sulfate is dialysed to supernatant with saturation degree again, precipitation after centrifugal,-15 ℃ of acetone fractionated that gained precipitation added 1 times of volume are dialysed, and the soln using molecular cut off after the dialysis is 5000～10000 ultrafiltration membrance filter, the capsicum peroxidase that obtains under 4 ℃; Recording the capsicum peroxidase concn is 8.56g/L, and vigor is 15.53u/g.With 1.0cm
2The size platinized platinum soaks 0.5h in 1moL/LNaOH solution, take out then to dip in cotton balls and get the distilled water scrub surfaces, at 1moL/L H
2SO
4In the 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 the 20mg microcrystalline cellulose and the ultrasonic 10min of 20mg carbon powder nano pipe mixes, get pretreated platinized platinum, with its diagonal line is the separatrix, evenly smear one deck microcrystalline cellulose-carbon powder nano pipe film, air dry 40h is standby, capsicum peroxidase and the 10 μ L D-sorbierites of 50 μ L of above-mentioned preparation are dropped in 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, wash for several times standby with distilled water repeatedly.The sewage 25mL of discharging after collection Sewage Plant 1 is handled, the Tris-HCl damping fluid is regulated pH and was preserved 5 days under 5.0 in 4 ℃, and the adding volume ratio is 0.5% hydrogen peroxide during mensuration, utilizes electrode system of the present invention to measure.In sweep velocity is 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, the match relational expression by anodic peak current value and phenol concentration calculates that content of phenolic compounds is 15.4 ± 0.23mmol/L in the sewage of Sewage Plant 1 discharging.Other is with embodiment 1.
Embodiment 3.
Chilli with market sale is that raw material was through pulverizing 100 orders, take by weighing and pulverized 100 purpose 50g chilli powder, the distilled water that adds 500mL is in 0 ℃ of following ultrasonic echography 40min, suction filtration, get crude extract after centrifugal, be that 0.4 ammonium sulfate is dialysed to the crude extract of extraction earlier with saturation degree, get supernatant after centrifugal, be that 0.8 ammonium sulfate is dialysed to supernatant with saturation degree again, precipitation after centrifugal, the gained precipitation is added 2 times of volume-16 ℃ acetone fractionated dialyses, soln using molecular cut off after the dialysis is 10000 ultrafiltration membrance filter, the capsicum peroxidase that obtains under 6 ℃, recording concentration is 10.25g/L, vigor is 23.3u/g.With 1.5cm
2The size platinized platinum soaks 1h in 1moL/LNaOH solution, take out then to dip in cotton balls and get the distilled water scrub surfaces, at 1moL/L H
2SO
4In the 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 the 40mg microcrystalline cellulose and the ultrasonic 20min of 40mg carbon powder nano pipe mixes, get pretreated platinized platinum, with its diagonal line is the separatrix, evenly smear one deck microcrystalline cellulose-carbon powder nano pipe film, air dry 50h is standby, capsicum peroxidase and the 50 μ L D-sorbierites of 150 μ L of above-mentioned preparation are dropped in 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, wash for several times standby with distilled water repeatedly.Gather the sewage 25mL of Sewage Plant 2 dischargings, the Tris-HCl damping fluid is regulated pH and was preserved 5 days under 8.0,6 ℃, and adding volume ratio is 1.0% hydrogen peroxide, utilizes electrode system of the present invention to measure.In sweep velocity is 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, the match relational expression by anodic peak current value and phenol concentration calculates that content of phenolic compounds is 14.5 ± 0.12mmol/L in the sewage of Sewage Plant 2 dischargings.Other is with embodiment 1.
Chilli with market sale is that raw material was through pulverizing 90 orders, take by weighing and pulverized 90 purpose 35g chilli powder, the distilled water that adds 350mL is in 0 ℃ of following ultrasonic echography 30min, suction filtration, get crude extract after centrifugal, be that 0.4 ammonium sulfate is dialysed to the crude extract of extraction earlier with saturation degree, get supernatant after centrifugal, be that 0.8 ammonium sulfate is dialysed to supernatant with saturation degree again, precipitation after centrifugal, the gained precipitation is added 1.5 times of volume-15 ℃ acetone fractionated dialyses, soln using molecular cut off after the dialysis is 5000 ultrafiltration membrance filter, the capsicum peroxidase that obtains under 5 ℃, recording its concentration is 21.5g/L, vigor is 20.53u/g.With 0.5cm
2The size platinized platinum soaks 0.8h in 1moL/LNaOH solution, take out then to dip in cotton balls and get the distilled water scrub surfaces, at 1moL/L H
2SO
4In the 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 the 32mg microcrystalline cellulose and the ultrasonic 15min of 32mg carbon powder nano pipe mixes, get pretreated platinized platinum, with its diagonal line is the separatrix, evenly smear one deck microcrystalline cellulose-carbon powder nano pipe film, air dry 48h is standby, capsicum peroxidase and the 40 μ LD-sorbierites of 100 μ L of above-mentioned preparation are dropped in 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, wash for several times standby with distilled water repeatedly.The sewage 25mL of collection Sewage Plant 3 dischargings, the Tris-HCl damping fluid is regulated pH and was preserved 5 days under 7.0,5 ℃, and the adding volume ratio is 0.8% hydrogen peroxide, utilizes electrode system of the present invention to measure.In sweep velocity is 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, the match relational expression by anodic peak current value and phenol concentration calculates that content of phenolic compounds is 16.8 ± 0.31mmol/L in the sewage of Sewage Plant 3 dischargings.Other is with embodiment 1.
Embodiment 5.
Chilli with market sale is that raw material was through pulverizing 95 orders, take by weighing and pulverized 95 purpose 40g chilli powder, the distilled water that adds 400mL is in 0 ℃ of following ultrasonic echography 35min, suction filtration, get crude extract after centrifugal, be that 0.4 ammonium sulfate is dialysed to the crude extract of extraction earlier with saturation degree, get supernatant after centrifugal, be that 0.8 ammonium sulfate is dialysed to supernatant with saturation degree again, precipitation after centrifugal, the gained precipitation is added 1 times of volume-16 ℃ acetone fractionated dialyses, soln using molecular cut off after the dialysis is 5000～10000 ultrafiltration membrance filter, the capsicum peroxidase that obtains under 4 ℃, recording its concentration is 30.23g/L, vigor is 25.53u/g.With 1cm
2The size platinized platinum soaks 1h in 1moL/L NaOH solution, take out then to dip in cotton balls and get the distilled water scrub surfaces, at 1moL/LH
2SO
4In the 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 the 24mg microcrystalline cellulose and the ultrasonic 15min of 24mg carbon powder nano pipe mixes, get pretreated platinized platinum, with its diagonal line is the separatrix, evenly smear one deck microcrystalline cellulose-carbon powder nano pipe film, air dry 40h is standby, capsicum peroxidase and the 30 μ L D-sorbierites of 100 μ L of above-mentioned preparation are dropped in 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, wash for several times standby with distilled water repeatedly.Gather the sewage 25mL of Sewage Plant 4, the Tris-HCl damping fluid is regulated pH and was preserved 5 days under 5.0,4 ℃, and adding volume ratio is 0.9% hydrogen peroxide, utilizes electrode system of the present invention to measure.In sweep velocity is 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, the match relational expression by anodic peak current value and phenol concentration calculates that content of phenolic compounds is 14.8 ± 0.14mmol/L in the sewage of Sewage Plant 4 dischargings.Other is with embodiment 1.
Claims (2)
1. the preparation method of a superoxide enzyme electrode, it is characterized in that be a kind of be that raw material extracts the capsicum peroxidase with the chilli, platinized platinum is a matrix, the carbon powder nano pipe is an electron carrier, microcrystalline cellulose is as immobilization grid carrier, add effector molecules D-sorbierite and make a kind of method of capsicum peroxidase immobilization electrode of content of the phenolic compound that is used for detecting water sample, its concrete preparation method's step is:
The preparation of I, capsicum peroxidase
Chilli with market sale is that raw material was through pulverizing 80～100 orders, take by weighing and pulverized 80～100 purposes, 20～50g chilli powder, the distilled water that adds 200～500mL is in 0 ℃ of following ultrasonic echography 20～40min, suction filtration, get crude extract after centrifugal, be that 0.4 ammonium sulfate is dialysed to the crude extract of extraction earlier with saturation degree, get supernatant after centrifugal, be that 0.8 ammonium sulfate is dialysed to supernatant with saturation degree again, precipitation after centrifugal, the gained precipitation is added 1～2 times of volume-14～-16 ℃ acetone fractionated dialyses, soln using molecular cut off after the dialysis is that 5000～10000 ultrafiltration membrance filter obtains the capsicum peroxidase, 4～6 ℃ of preservations.
The preparation of II, superoxide enzyme electrode
With 0.5～1.5cm
2The size platinized platinum carries out pre-service: soak 0.5～1h in 1moL/L NaOH solution, take out then to dip in cotton balls and get the distilled water scrub surfaces, at 1moL/L H
2SO
4In the 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 mixes, get pretreated platinized platinum, with its diagonal line is the separatrix, evenly smear one deck microcrystalline cellulose-carbon powder nano pipe film, air dry 40～50h is standby, capsicum peroxidase and 10～50 μ L D-sorbierites of 50～150 μ L of above-mentioned preparation are dropped in 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, wash for several times standby with distilled water repeatedly.
2. with the using method of a kind of superoxide enzyme electrode of the method for claim 1 preparation, get the prepared capsicum superoxide enzyme electrode of said method when it is characterized in that using as working electrode, saturated calomel electrode is a contrast electrode, platinum electrode is that electrode is constituted three-electrode system, the pH value of solution of regulating phenolic compound with Tris-HCl buffer solution is 5.0～8.0, 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 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 stable, measure the CV curve of different phenolic compound concentration under the same sweep velocity, obtain the relation of peak current size and phenolic compound concentration, be used for directly analyzing aqueous solution phenolic compound concentration to be measured.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104973661A (en) * | 2014-04-10 | 2015-10-14 | 中国石油化工股份有限公司 | Composite cathode electrode, preparation method and application thereof |
| CN107290409A (en) * | 2016-03-30 | 2017-10-24 | 上海大学 | Cell release concentration of hydrogen peroxide detecting electrode, detection method and preparation method |
| CN110592171A (en) * | 2019-08-23 | 2019-12-20 | 江苏大学 | Novel immobilized oxidoreductase electrode and preparation method thereof |
| CN113884619A (en) * | 2021-09-30 | 2022-01-04 | 眉山博雅新材料股份有限公司 | Titration method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001305103A (en) * | 2000-04-24 | 2001-10-31 | Toyobo Co Ltd | Polyphenol sensor |
| CN101625334A (en) * | 2009-08-05 | 2010-01-13 | 西北师范大学 | Enzyme modified electrode based on sol gel layer and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001305103A (en) * | 2000-04-24 | 2001-10-31 | Toyobo Co Ltd | Polyphenol sensor |
| CN101625334A (en) * | 2009-08-05 | 2010-01-13 | 西北师范大学 | Enzyme modified electrode based on sol gel layer and preparation method thereof |
Non-Patent Citations (3)
| 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》 * |
| 李燕妮等: "硫酸铵-丙酮协同沉淀法纯化南极假丝酵母产脂肪酶", 《化学与生物工程》 * |
| 田国忠等: "植物过氧化物酶研究进展", 《武汉植物学研究》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104973661A (en) * | 2014-04-10 | 2015-10-14 | 中国石油化工股份有限公司 | Composite cathode electrode, preparation method and application thereof |
| CN104973661B (en) * | 2014-04-10 | 2017-09-29 | 中国石油化工股份有限公司 | A kind of composite cathode electrode and its preparation method and application |
| CN107290409A (en) * | 2016-03-30 | 2017-10-24 | 上海大学 | Cell release concentration of hydrogen peroxide detecting electrode, detection method and preparation method |
| CN110592171A (en) * | 2019-08-23 | 2019-12-20 | 江苏大学 | Novel immobilized oxidoreductase electrode and preparation method thereof |
| CN113884619A (en) * | 2021-09-30 | 2022-01-04 | 眉山博雅新材料股份有限公司 | Titration method |
| CN113884619B (en) * | 2021-09-30 | 2024-02-02 | 眉山博雅新材料股份有限公司 | Titration method |
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| CN102183560B (en) | 2013-11-06 |
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