CN102353713B - Detection methods for activity and substrate concentration of oxidase - Google Patents
Detection methods for activity and substrate concentration of oxidase Download PDFInfo
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- CN102353713B CN102353713B CN201110188451.9A CN201110188451A CN102353713B CN 102353713 B CN102353713 B CN 102353713B CN 201110188451 A CN201110188451 A CN 201110188451A CN 102353713 B CN102353713 B CN 102353713B
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Abstract
The invention provides a detection method for activity of oxidase. The method comprises the following steps: mixing a solution to be detected with an oxidase substrate and arylborane compounds so as to obtain an intermediate solution to be detected containing phenolic compounds; detecting the concentration of the phenolic compounds in the intermediate solution to be detected by using an electrochemical detection method and calculating the activity of oxidase in the solution to be detected according to the concentration of the phenolic compounds. According to the invention, the activity of oxidase is indirectly detected based on electrochemical detection of the concentration of the phenolic compounds; the phenolic compounds have the characteristic of low potential, so it is easy to carry out electrochemical detection; since the electrochemical detection method has the characteristics of simple operation and high sensitivity, the detection method provided in the invention has the characteristics of simple operation and high sensitivity and detection accuracy. The invention also provides three detection methods for substrate concentration of oxidase. The detection approach of the three detection methods is identical with that of the above-mentioned detection method, and the detection methods have the characteristics of simple operation and high sensitivity and detection accuracy.
Description
Technical field
The present invention relates to biological technical field, particularly the detection method of oxidase active and oxidase concentration of substrate.
Background technology
Oxidase is directly to generate water using molecular oxygen as electron accepter, the enzyme of catalytic substrate oxidation.Common oxidase has glucose oxidase, urate oxidase, Lactate Oxidase and monoamine oxidase etc.Oxidase is indispensable material in most of biosome, determine the elimination that results from of oxidative metabolism process in biosome and secondary metabolite, to ensure that biosome has different adaptive states under different growing environments, its activity has vital role for maintaining biosome eubolism.Therefore, the detection of oxidase active and oxidase substrate content is all significant in bioanalysis and clinical assays.
Enzymatic activity is its sign to zymolyte catalytic reaction ability, and unit of enzyme activity is IU, is abbreviated as U, and it is defined as: 1min can transform the enzyme amount of 1 μ mol substrate, i.e. 1IU=1 μ mol/min.Application number is the fluorescence detection method that 200710067620.7 Chinese patent discloses a kind of activity of monoamine oxidase, and the method is to form fluorescent material with monoamine oxidase hydrolysis probes, measures fluorescence intensity, obtains activity of monoamine oxidase data.But adopt this kind of method to measure and need to prepare different types of fluorescence probe the oxidase of other kinds, versatility is lower.
Although various oxidase act on different substrates, its public characteristic is: when oxydasis substrate, hydrogen reduction is become to hydrogen peroxide (H
2o
2).According to this feature, the detection of existing oxidase active and oxidase substrate has the spectrophotometric method of employing to record, and is H as the application number Chinese patent that is 200810107036.4 discloses a kind of product
2o
2oxidase active detection method, it is that oxidase extract and nitrite ion are mixed in buffer solution, so in mixed solution, add oxidase substrate, after a period of time, adopt spectrophotometry wavelength 550nm place optical density value, record thus oxidasic concentration.Though the versatility of this kind of method increases, but spectrophotometry complex operation and sensitivity are not high, have affected thus the precision of test result yet.
Summary of the invention
The technical matters that the present invention solves is to provide a kind of simple to operate, oxidase active detection method that detection sensitivity is high and the detection method of oxidase concentration of substrate.
In view of this, the invention provides a kind of detection method of oxidase active, comprising:
A1), solution to be measured is mixed with oxidase substrate and aryl boron compound, obtain the centre solution to be measured that contains phenolic compound;
A2), adopt electrochemical assay to detect the concentration of phenolic compound in the solution to be measured of described centre, calculate oxidasic activity in solution to be measured according to the densimeter of phenolic compound.
Preferably, described aryl boron compound is the compound of formula (I) structure, R in formula (I)
1for amino or hydroxyl, R
2for boronate or boric acid pinacol ester group;
Preferably, described electrochemical assay is volt-ampere analysis.
Preferably, described volt-ampere analysis is cyclic voltammetry, and voltage scan range is 0～0.4V, and sweep velocity is 0.03V/s～0.1V/s.
The present invention also provides a kind of detection method of oxidase concentration of substrate, comprising:
B1), solution to be measured is mixed with oxidase and aryl boron compound, obtain the centre solution to be measured that contains phenolic compound;
B2), adopt electrochemical assay to detect the concentration of phenolic compound in the solution to be measured of described centre, calculate the concentration of oxidase substrate in described solution to be measured according to the densimeter of phenolic compound.
Preferably, described aryl boron compound is the compound of formula (I) structure, R in formula (I)
1for amino or hydroxyl, R
2for boronate or boric acid pinacol ester group;
The present invention also provides a kind of detection method of oxidase concentration of substrate, comprising:
The carbon paste electrode of modifying using aryl boron compound and oxidase is as working electrode, adopts electrochemical assay to detect the concentration of phenolic compound in solution to be measured, calculates the concentration of oxidase substrate in solution to be measured according to the densimeter of phenolic compound.
Preferably, described aryl boron compound is the compound of formula (I) structure, R in formula (I)
1for amino or hydroxyl, R
2for boronate or boric acid pinacol ester group;
Preferably, described electrochemical assay is potentiostatic scanning method, and scanning current potential is 0.08V～0.15V, and be 10s～18s sweep time.
The present invention also provides a kind of detection method of oxidase concentration of substrate, comprising:
C1), solution to be measured is mixed to solution to be measured in the middle of obtaining with aryl boron compound;
C2), the carbon paste electrolysis of modifying using oxidase is as working electrode, adopts electrochemical assay to detect the concentration of phenolic compound in the solution to be measured of described centre, calculates the concentration of oxidase substrate in solution to be measured according to the densimeter of phenolic compound.
The invention provides a kind of detection method of enzyme, it is that oxidase is mixed with oxidase substrate and hydrogen peroxide, mix rear section oxidase substrate and be oxidized Hydrogen Peroxide under oxidasic catalytic action, the hydrogen peroxide generating generates and is converted into phenolic compound with aryl boron compound again, solution to be measured in the middle of obtaining thus.Because the oxidizing potential of phenolic compound is lower, be easy to adopt electrochemical assay to detect its concentration, therefore adopt in the middle of detecting that electrochemical assay can be simple, sensitive the concentration of phenolic compound in solution to be measured, extrapolated the concentration of hydrogen peroxide by the concentration of phenolic compound, extrapolated the concentration of the oxidase substrate having reacted by the concentration of hydrogen peroxide, calculate oxidasic active concentration according to the oxidase densimeter having reacted, finally extrapolate oxidasic activity according to oxidase activity concentration again.Be that the present invention carrys out indirect detection based on Electrochemical Detection phenolic compound concentration to go out oxidasic activity, because phenolic compound has the advantages that oxidizing potential is low, therefore be easy to into Electrochemical Detection, simultaneously because electrochemical assay has simple to operate, highly sensitive feature, make thus method provided by the invention just can realize the detection to oxidase active at lower current potential, easy operating, sensitivity and accuracy in detection are higher.
The present invention also provides a kind of detection method of oxidase concentration of substrate, it is that first oxidase substrate is converted to hydrogen peroxide, again hydrogen peroxide is converted into the phenolic compound that oxidizing potential is lower, therefore be easy to into Electrochemical Detection, simultaneously because electrochemical assay has simple to operate, highly sensitive feature, make thus method provided by the invention just can realize the detection to oxidase concentration of substrate at lower current potential, easy operating, sensitivity and accuracy in detection are higher.
Brief description of the drawings
Fig. 1 is glucose oxidase activity concentration and strength of current relation curve;
Fig. 2 is concentration of glucose and strength of current relation curve.
Embodiment
In order further to understand the present invention, below in conjunction with embodiment, the preferred embodiment of the invention is described, but should be appreciated that these are described is for further illustrating the features and advantages of the present invention, instead of limiting to the claimed invention.
The detection method that the invention provides a kind of oxidase active, comprises the steps:
A1), solution to be measured is mixed with oxidase substrate and aryl boron compound, obtain the centre solution to be measured that contains phenolic compound;
A2), adopt electrochemical assay to detect the concentration of phenolic compound in the solution to be measured of described centre, calculate oxidasic activity in solution to be measured according to the densimeter of phenolic compound.
From such scheme, the present invention adopts electrochemical assay to detect oxidase active, selects the reason of Electrochemical Detection to be that it has feature highly sensitive, simple to operate.But, be applied to have certain difficulty in content of hydrogen peroxide detection, this is because the oxidizing potential of glucose is higher, the oxidizing potential of the hydrogen oxide self forming after its oxidation is also higher, in view of the oxidizing potential of hydrogen peroxide self is compared with operation easier high and that bring to electrochemical assay, inventor considers hydrogen peroxide to change into the intermediate compound that is easy to carry out Electrochemical Detection, calculates the concentration of hydrogen peroxide by detecting the concentration of intermediate compound.Because phenolic compound has lower oxidizing potential, therefore choice for use aryl boron compound of the present invention reacts with hydrogen peroxide, generate the phenol compound that has certain proportion relation with content of hydrogen peroxide, then adopt electrochemical assay to detect the concentration of phenol compound, and then indirectly draw the concentration of hydrogen peroxide, the concentration of oxidized oxidase substrate, concentration by oxidized oxidase substrate can be extrapolated oxidase concentration of enzymatic activity, and concentration of enzymatic activity is exactly the activity of the enzyme of unit volume; Finally extrapolate oxidase concentration according to concentration of enzymatic activity again.
In step a1, oxidase substrate partial oxidation Hydrogen Peroxide under oxidasic effect in solution to be measured, the hydrogen peroxide of generation reacts with aryl boron compound, generates phenolic compound, has obtained thus the centre solution to be measured that contains phenols chemical combination.
The substrate of oxidase described in the present invention includes but not limited to glucose, uric acid, lactic acid and monoamine etc., and corresponding, oxidase includes but not limited to glucose oxidase, urate oxidase, Lactate Oxidase and monoamine oxidase etc.The oxidation reaction of oxidase substrate is as follows:
Aryl boron compound of the present invention refers to that the boron of boron-containing group is directly connected in the compound of aromatic rings.Aryl boron compound preferably mixes with liquid to be measured with the form of solution.Aryl boron compound is preferably the compound of formula (I) structure.
R in formula (I)
1for amino or hydroxyl, R
2for boronate or boric acid pinacol ester group.It occurs to react as follows with hydrogen peroxide, and the phenolic compound of generation has good chemical property:
The aryl boron compound of formula (I) structure is preferably aminobenzene boric acid, more preferably p-aminophenyl boric acid, and p-aminophenyl boric acid can occur to react as follows with hydrogen peroxide:
The para-aminophenol generating has lower oxidizing potential, and reversible electrochemical behavior is almost pollution-free to electrode surface, and the accuracy that is easy to carry out Electrochemical Detection and detection is higher.The mol ratio of reagent hydrogen peroxide and product para-aminophenol is 1: 1, therefore in the middle of, the concentration of para-aminophenol is the concentration of hydrogen peroxide under equal volume in liquid to be measured, and the concentration of hydrogen peroxide is the concentration of oxidase substrate oxidized in tracer liquid system, oxidasic active concentration can be extrapolated thus, more oxidasic activity can be calculated according to oxidasic volume.
It will be appreciated by those skilled in the art that, in order to ensure that the oxidase in solution to be measured fully participates in catalytic reaction, the oxidase substrate adding in step a1 and aryl boron compound are all excessive, and those skilled in the art can judge according to the source of testing sample the addition of oxidase substrate and aryl boron compound.
Step a2 is the operation that detects phenolic compound concentration, because phenolic compound has lower oxidizing potential, is therefore easy to carry out Electrochemical Detection.Electrochemical assay preferably adopts volt-ampere analysis, and volt-ampere analysis is an alanysis method general name of analyzing composition and the content of electrolytic solution by current-voltage curve in electrolytic solution under mensuration certain condition.More preferably adopt cyclic voltammetry, potentiostatic scanning method, linear sweep voltammetry or square wave voltammetry, adopt above-mentioned volt-ampere analysis to detect operation comparatively easy.The present invention most preferably adopts cyclic voltammetry, and the accuracy of detection of this kind of method is higher.Cyclic voltammetry is that control electrode electromotive force carries out one or many with triangular waveform in time with different speed and repeatedly scans, make, on electrode, different reduction and oxidation reaction alternately to occur, and record current-potential curve, obtain the corresponding relation of testing concentration in electric current and electrolytic solution according to this electric current-potential curve.
Adopt cyclic voltammetry to detect in the process of phenolic compound concentration in middle liquid to be measured, preferred settings voltage scan range is 0～0.4V, and sweep velocity is preferably made as 0.03V/s～0.1V/s.In the three-electrode system adopting: working electrode is preferably glass-carbon electrode, gold electrode, platinum electrode, graphite electrode or pyrolytic graphite electrode; Electrode is preferably to platinum electrode; Contrast electrode is preferably silver/silver chloride electrode.Buffer solution is trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl) buffer solution or phosphate buffered solution.
From such scheme, the present invention is in order to detect oxidasic activity, oxidase is mixed with oxidase substrate and hydrogen peroxide, mix rear section oxidase substrate and be oxidized Hydrogen Peroxide under oxidasic catalytic action, the hydrogen peroxide generating generates and is converted into phenolic compound with aryl boron compound again, solution to be measured in the middle of obtaining thus.Because the oxidizing potential of phenolic compound is lower, be easy to adopt electrochemical assay to detect its concentration, therefore adopt in the middle of detecting that electrochemical assay can be simple, sensitive the concentration of phenolic compound in solution to be measured, extrapolated the concentration of hydrogen peroxide by the concentration of phenolic compound, extrapolated the concentration of the oxidase substrate having reacted by the concentration of hydrogen peroxide, calculate oxidasic active concentration according to the oxidase densimeter having reacted, finally extrapolate oxidasic activity according to oxidase activity concentration again.Be that the present invention carrys out indirect detection based on Electrochemical Detection phenolic compound concentration to go out oxidasic activity, because phenolic compound has the advantages that oxidizing potential is low, therefore be easy to into Electrochemical Detection, simultaneously because electrochemical assay has simple to operate, highly sensitive feature, make thus method provided by the invention just can realize the detection to oxidase active at lower current potential, easy operating, sensitivity and accuracy in detection are higher.
The present invention also provides a kind of detection method of oxidase concentration of substrate, comprises the steps:
B1), solution to be measured is mixed with oxidase and aryl boron compound, obtain the centre solution to be measured that contains phenolic compound;
B2), adopt electrochemical assay to detect the concentration of phenolic compound in the solution to be measured of described centre, calculate the concentration of oxidase substrate in described solution to be measured according to the densimeter of phenolic compound.
This detection method is similar to the thinking that above-mentioned oxidase active detects, it is that the oxidase substrate in solution to be measured is oxidized to hydrogen peroxide, again hydrogen peroxide is converted into phenolic compound, finally detect the concentration of phenolic compound by electrochemical assay, extrapolate the concentration of oxidase substrate in solution to be measured according to the concentration of phenolic compound.
Step b1 is the operation that the glucose in solution to be measured is all converted into phenolic compound.Oxidase substrate in solution to be measured reacts Hydrogen Peroxide under oxidasic effect, and the hydrogen peroxide of generation reacts with aryl boron compound and generates phenolic compound, obtains thus the centre solution to be measured that contains phenolic compound.
The substrate of oxidase described in the present invention includes but not limited to glucose, uric acid, lactic acid and monoamine etc., and corresponding, oxidase includes but not limited to glucose oxidase, urate oxidase, Lactate Oxidase and monoamine oxidase etc.
Aryl boron compound of the present invention refers to that the boron of boron-containing group is directly connected in the compound of aromatic rings.Aryl boron compound preferably mixes with liquid to be measured with the form of solution.Aryl boron compound is preferably the compound of formula (I) structure.
R in formula (I)
1for amino or hydroxyl, R
2for boronate or boric acid pinacol ester group.It occurs to react as follows with hydrogen peroxide, and the phenolic compound of generation has good chemical property:
The aryl boron compound of formula (I) structure is preferably aminobenzene boric acid, more preferably p-aminophenyl boric acid, and p-aminophenyl boric acid can occur to react as follows with hydrogen peroxide:
The para-aminophenol generating has lower oxidizing potential, and reversible electrochemical behavior is almost pollution-free to electrode surface, and the accuracy that is easy to carry out Electrochemical Detection and detection is higher.The mol ratio of reagent hydrogen peroxide and product para-aminophenol is 1: 1, therefore in the middle of, the concentration of para-aminophenol is the concentration of hydrogen peroxide under equal volume in liquid to be measured, and the concentration of hydrogen peroxide is the concentration of oxidase substrate in tracer liquid system, can extrapolate thus the concentration of oxidase substrate in former liquid to be measured.
It will be appreciated by those skilled in the art that, in order to ensure that in solution to be measured, oxidase substrate reactions is complete, the oxidase adding in step b1 of the present invention and aryl boron compound are all excessive, and those skilled in the art can judge according to the source of solution to be measured the addition of oxidase and aryl boron compound.
Step b2 is the operation that detects phenolic compound concentration, because phenolic compound has lower oxidizing potential, is therefore easy to carry out Electrochemical Detection.Electrochemical assay preferably adopts volt-ampere analysis, concrete as cyclic voltammetry, potentiostatic scanning, linear sweep voltammetry or square wave voltammetry, most preferably adopts potentiostatic scanning method.
In the middle liquid to be measured of employing potentiostatic scanning method detection, in phenolic compound concentration process, scanning current potential is 0.08V～0.15V, and sweep velocity is preferably set to 10s～18s.In the three-electrode system adopting: working electrode is preferably glass-carbon electrode, gold electrode, platinum electrode, graphite electrode or pyrolytic graphite electrode; Electrode is preferably to platinum electrode; Contrast electrode is preferably silver/silver chloride electrode.Buffer solution is trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl) buffer solution or phosphate buffered solution.
From such scheme, the present invention is in order to detect the concentration of oxidase substrate, first oxidase substrate is converted to hydrogen peroxide, again hydrogen peroxide is converted into the phenolic compound that oxidizing potential is lower, therefore be easy to into Electrochemical Detection, the while is simple to operate, highly sensitive because electrochemical assay has, and makes thus method provided by the invention just can realize the detection to oxidase concentration of substrate at lower current potential, easy operating, sensitivity and accuracy in detection are higher.
The present invention also provides the detection method of another kind of oxidase concentration of substrate, comprises the steps:
The carbon paste electrode of modifying using aryl boron compound and oxidase is as working electrode, adopts electrochemical assay to detect the concentration of phenolic compound in solution to be measured, calculates the concentration of oxidase substrate in solution to be measured according to the densimeter of phenolic compound.
The detection method that this method provides is identical with a upper Method And Principle, and it is to be also hydrogen peroxide by oxidase substrate conversion, then hydrogen peroxide is converted into phenolic compound, and the concentration that goes out phenolic compound by electrical detection is extrapolated the concentration of oxidase substrate.Difference is: it is that aryl boron compound and oxidase are modified at and on working electrode, make carbon paste electrode, in the time that needs detect, solution to be measured is directly detected, saved the operation that solution to be measured is mixed with aryl boron compound and oxidase before detecting.For the serum sample that needs clinically to detect oxidase concentration of substrate, the error that should detect as early as possible to avoid cell tissue to destroy and bring to detection.Therefore, detection method provided by the invention has not only been simplified operation, can also improve the precision of detection.
The substrate of oxidase described in the present invention includes but not limited to glucose, uric acid, lactic acid and monoamine etc., and corresponding, oxidase includes but not limited to glucose oxidase, urate oxidase, Lactate Oxidase and monoamine oxidase etc.
Aryl boron compound of the present invention refers to that the boron of boron-containing group is directly connected in the compound of aromatic rings.Aryl boron compound preferably mixes with liquid to be measured with the form of solution.Aryl boron compound is preferably the compound of formula (I) structure.
R in formula (I)
1for amino or hydroxyl, R
2for boronate or boric acid pinacol ester group, more preferably aminobenzene boric acid, most preferably is p-aminophenyl boric acid.Select the reason of above-mentioned aryl boron compound identical with said method.
Equally, it will be appreciated by those skilled in the art that, in order to ensure that in solution to be measured, oxidase substrate reactions is complete, the oxidase adding in carbon paste electrode and aryl boron compound are all excessive, and those skilled in the art can judge according to the source of solution to be measured the addition of oxidase and aryl boron compound.The carbon paste electrode that aryl boron compound and oxidase are modified can be prepared as follows:
Aryl boron compound, oxidase, cementing agent and dag grinding are mixed and obtain pastel, described pastel is coated in to electrode bar surface, or is packed in electrode tube, obtain carbon paste electrode.
Chemical measure preferably adopts volt-ampere analysis, concrete as cyclic voltammetry, potentiostatic scanning, linear sweep voltammetry or square wave voltammetry, most preferably adopts potentiostatic scanning method.
In the middle liquid to be measured of employing potentiostatic scanning method detection, in phenolic compound concentration process, scanning current potential is 0.08V～0.15V, and sweep velocity is preferably set to 10s～18s.In the three-electrode system adopting: electrode is preferably to platinum electrode; Contrast electrode is preferably silver/silver chloride electrode.Buffer solution is trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl) buffer solution or phosphate buffered solution.
From such scheme, the present invention, in order to detect the concentration of oxidase substrate, modifies oxidase and aryl boron compound in working electrode, then carries out Electrochemical Detection taking the carbon paste electrode after this oxidase and the modification of aryl boron compound as working electrode.Oxidase substrate in solution to be measured is at oxidasic catalytic action Hydrogen Peroxide, the hydrogen peroxide generating reacts with aryl boron compound again and generates phenolic compound, calculates the concentration of oxidase substrate in solution to be measured by detecting the densimeter of phenolic compound.In the time that needs detect solution to be measured, solution to be measured is directly detected, easy and simple to handle, sensitivity and accuracy in detection are higher.
The present invention also provides a kind of detection method of oxidase concentration of substrate, comprises the steps:
C1), solution to be measured is mixed to solution to be measured in the middle of obtaining with aryl boron compound;
C2), the carbon paste electrode modified using oxidase is as working electrode, adopts electrochemical assay to detect the concentration of phenolic compound in the solution to be measured of described centre, calculates the concentration of oxidase substrate in solution to be measured according to the densimeter of phenolic compound.
The detection method that this method provides is identical with a upper Method And Principle, and it is to be also hydrogen peroxide by oxidase substrate conversion, then hydrogen peroxide is converted into phenolic compound, and the concentration that goes out phenolic compound by electrical detection is extrapolated the concentration of oxidase substrate.Difference is: it is just oxidase to be modified on carbon paste electrode, because oxidase itself does not react, before and after reaction, chemical property is also for changing, during it also can be applicable to detect next time, therefore this carbon paste electrode can be recycled, and has saved testing cost.
Similarly, aryl boron compound preferably adopts the compound of above-mentioned formula (I) structure, R
1for amino or hydroxyl, R
2for boronate or boric acid pinacol ester group, more preferably aminobenzene boric acid, most preferably is p-aminophenyl boric acid.Chemical measure preferably adopts volt-ampere analysis, concrete as cyclic voltammetry, potentiostatic scanning, linear sweep voltammetry or square wave voltammetry, most preferably adopt potentiostatic scanning method, scanning current potential is preferably 0.08V～0.15V, and sweep velocity is preferably set to 10s～18s.In the three-electrode system adopting: electrode is preferably to platinum electrode; Contrast electrode is preferably silver/silver chloride electrode.Buffer solution is trishydroxymethylaminomethane-hydrochloric acid (Tris-HCl) buffer solution or phosphate buffered solution.
In order further to understand the present invention, oxidase active provided by the invention and oxidase concentration of substrate detection method are described as an example of oxidase example below in conjunction with the embodiments, protection scope of the present invention is not limited by the following examples.
Embodiment 1
Detection solution described in the present embodiment comprises the Tris-HCl buffer solution of 2mM p-aminophenyl boric acid and 200mM glucose and surplus, and pH value is 7.4.
1, getting aqueous sample that 100 μ L contain 0.1U/mL glucose oxidase detects solution with 900 μ L and mixes, leave standstill after 2 minutes, adopting pyrolytic graphite electrode is working electrode, adopting platinum electrode is to electrode, silver/silver chloride electrode is contrast electrode, within the scope of 0～0.2 volt, carry out cyclic voltammetry scan, sweep fast 0.05 volt/second, measured oxidation peak current is 0.76 μ A.
2, getting aqueous sample that 100 μ L contain 0.5U/mL glucose oxidase detects solution with 900 μ L and mixes, leave standstill after 2 minutes, adopting pyrolytic graphite electrode is working electrode, adopting platinum electrode is to electrode, silver/silver chloride electrode is contrast electrode, within the scope of 0～0.2 volt, carry out cyclic voltammetry scan, sweep fast 0.05 volt/second, measured oxidation peak current is 1.01 μ A.
3, getting aqueous sample that 100 μ L contain 1U/mL glucose oxidase detects solution with 900 μ L and mixes, leave standstill after 2 minutes, employing gold electrode is working electrode, adopting platinum electrode is to electrode, silver/silver chloride electrode is contrast electrode, within the scope of 0～0.2 volt, carry out cyclic voltammetry scan, sweep fast 0.05 volt/second, measured oxidation peak current is 1.33 μ A.
4, getting aqueous sample that 100 μ L contain 3U/mL glucose oxidase detects solution with 900 μ L and mixes, leave standstill after 2 minutes, adopting pyrolytic graphite electrode is working electrode, adopting platinum electrode is to electrode, silver/silver chloride electrode is contrast electrode, within the scope of 0～0.2 volt, carry out cyclic voltammetry scan, sweep fast 0.05 volt/second, measured oxidation peak current is 2.67 μ A.
5, getting aqueous sample that 100 μ L contain 5U/mL glucose oxidase detects solution with 900 μ L and mixes, leave standstill after 2 minutes, adopting pyrolytic graphite electrode is that working electrode employing platinum electrode is to electrode, silver/silver chloride electrode is contrast electrode, within the scope of 0～0.2 volt, carry out cyclic voltammetry scan, sweep fast 0.05 volt/second, measured oxidation peak current is 3.87 μ A.
6, getting aqueous sample that 100 μ L contain 8U/mL glucose oxidase detects solution with 900 μ L and mixes, leave standstill after 2 minutes, adopting pyrolytic graphite electrode is working electrode, adopting platinum electrode is to electrode, silver/silver chloride electrode is contrast electrode, within the scope of 0～0.2 volt, carry out cyclic voltammetry scan, sweep fast 0.05 volt/second, measured oxidation peak current is 5.22 μ A.
The oxidation current peak value corresponding with it according to the glucose oxidase activity concentration of the variable concentrations obtaining of step 1～6, obtain glucose oxidase activity concentration and strength of current relation curve as shown in Figure 1, in figure, horizontal ordinate is the active concentration of glucose oxidase sample solution, unit is U/mL, ordinate is measured strength of current, and unit is μ A.
When glucose in solutions oxidase concentration to be measured is detected, can be first to the buffer solution that adds p-aminophenyl boric acid and enough glucose oxidases in solution to be measured, then adopt volt-ampere round-robin method to obtain the i-v curve of mixed solution, can in glucose oxidase enzyme concentration and strength of current relation curve working curve, obtain the active concentration of glucose oxidase according to its peak current, then calculate the activity of enzyme according to the volumescope of solution.
Embodiment 2
The preparation of working electrode: the p-aminophenyl pinacol borate of 2mg, the methyl-silicone oil of 40mg and the dag of 48mg are mixed and ground evenly in mortar the inside, then add the glucose oxidase of 10mg, potpourri is ground evenly.This potpourri is loaded in electrode shell inside, be made into carbon paste electrode; Be platinum electrode to electrode, contrast electrode is silver/silver chloride electrode.Buffer solution is Tris-HCl buffer solution.
1, prepared carbon paste working electrode is placed in to the buffer solution that contains 2mM glucose.Under the current potential of 0.12V, carry out potentiostatic scanning 15 seconds, the oxidation current maximal value obtaining is 0.034 μ A.
2, prepared carbon paste working electrode is placed in to the buffer solution that contains 6mM glucose, carries out potentiostatic scanning 15 seconds under the current potential of 0.12V, the oxidation current maximal value obtaining is 0.055 μ A.
3, prepared carbon paste working electrode is placed in to the buffer solution that contains 10mM glucose, carries out potentiostatic scanning 15 seconds under the current potential of 0.12V, the oxidation current maximal value obtaining is 0.074 μ A.
4, prepared carbon paste working electrode is placed in to the buffer solution that contains 14mM glucose, carries out potentiostatic scanning 15 seconds under the current potential of 0.12V, the oxidation current maximal value obtaining is 0.094 μ A.
5, prepared carbon paste working electrode is placed in to the buffer solution that contains 18mM glucose, carries out potentiostatic scanning 15 seconds under the current potential of 0.12V, the oxidation current maximal value obtaining is 0.108 μ A.
The oxidation current peak value that the concentration of glucose of the variable concentrations obtaining according to step 1～5 is corresponding with it, obtain concentration of glucose and strength of current relation curve as shown in Figure 2, in figure, horizontal ordinate is the concentration of glucose sample solution, unit is mM, ordinate is measured strength of current, and unit is μ A.
When glucose in solutions concentration to be measured is detected, the carbon paste electrode that can first use according to the method described above preparation to contain aminobenzene pinacol borate and glucose oxidase, then adopt the i-v curve of the detection solution to be measured that potentiostatic scanning obtains, can in glucose oxidase enzyme concentration and strength of current relation curve working curve, obtain the concentration of glucose oxidase according to its peak current.
The explanation of above embodiment is just for helping to understand method of the present invention and core concept thereof.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of the claims in the present invention.
To the above-mentioned explanation of the disclosed embodiments, make professional and technical personnel in the field can realize or use the present invention.To be apparent for those skilled in the art to the multiple amendment of these embodiment, General Principle as defined herein can, in the situation that not departing from the spirit or scope of the present invention, realize in other embodiments.Therefore, the present invention will can not be restricted to these embodiment shown in this article, but will meet the widest scope consistent with principle disclosed herein and features of novelty.
Claims (7)
1. a detection method for oxidase active, comprising:
A1), solution to be measured is mixed with oxidase substrate and aryl boron compound, obtain the centre solution to be measured that contains phenolic compound; Described aryl boron compound is the compound of formula I structure, R in formula I
1for amino or hydroxyl, R
2for boronate or boric acid pinacol ester group;
A2), adopt electrochemical assay to detect the concentration of phenolic compound in the solution to be measured of described centre, calculate oxidasic activity in solution to be measured according to the densimeter of phenolic compound.
2. detection method according to claim 1, is characterized in that, described electrochemical assay is volt-ampere analysis.
3. detection method according to claim 1, is characterized in that, described volt-ampere analysis is cyclic voltammetry, and voltage scan range is 0～0.4V, and sweep velocity is 0.03V/s～0.1V/s.
4. a detection method for oxidase concentration of substrate, comprising:
B1), solution to be measured is mixed with oxidase and aryl boron compound, obtain the centre solution to be measured that contains phenolic compound; Described aryl boron compound is the compound of formula I structure, R in formula I
1for amino or hydroxyl, R
2for boronate or boric acid pinacol ester group;
B2), adopt electrochemical assay to detect the concentration of phenolic compound in the solution to be measured of described centre, calculate the concentration of oxidase substrate in described solution to be measured according to the densimeter of phenolic compound.
5. a detection method for oxidase concentration of substrate, comprising:
The carbon paste electrode of modifying using aryl boron compound and oxidase is as working electrode, adopts electrochemical assay to detect the concentration of phenolic compound in solution to be measured, calculates the concentration of oxidase substrate in solution to be measured according to the densimeter of phenolic compound;
Described aryl boron compound is the compound of formula I structure, R in formula I
1for amino or hydroxyl, R
2for boronate or boric acid pinacol ester group;
6. detection method according to claim 5, is characterized in that, described electrochemical assay is potentiostatic scanning method, and scanning current potential is 0.08V～0.15V, and be 10s～18s sweep time.
7. a detection method for oxidase concentration of substrate, comprising:
C1), solution to be measured is mixed to solution to be measured in the middle of obtaining with aryl boron compound; Described aryl boron compound is the compound of formula I structure, R in formula I
1for amino or hydroxyl, R
2for boronate or boric acid pinacol ester group;
C2), the carbon paste electrode modified using oxidase is as working electrode, adopts electrochemical assay to detect the concentration of phenolic compound in the solution to be measured of described centre, calculates the concentration of oxidase substrate in solution to be measured according to the densimeter of phenolic compound.
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| SU1027208A1 (en) * | 1981-05-21 | 1983-07-07 | Научно-Исследовательский Институт Биологии При Иркутском Государственном Университете Им.А.А.Жданова | Method for determining activity of phenoloxidases |
| CN87200230U (en) * | 1987-01-19 | 1988-01-20 | 上海电视十一厂 | Enzyme activity measuring instrument |
| CN1847210A (en) * | 2005-04-11 | 2006-10-18 | 临海市永太化工有限公司 | Process of producing pentafluorophenol |
| CN101294191A (en) * | 2007-04-27 | 2008-10-29 | 中国科学院沈阳应用生态研究所 | Analysis method for testing ammonium oxidizing enzyme activity in soil |
| CN101659986A (en) * | 2008-08-27 | 2010-03-03 | 周静 | Method for testing activity of H2O2-producing oxidase |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| SU1027208A1 (en) * | 1981-05-21 | 1983-07-07 | Научно-Исследовательский Институт Биологии При Иркутском Государственном Университете Им.А.А.Жданова | Method for determining activity of phenoloxidases |
| CN87200230U (en) * | 1987-01-19 | 1988-01-20 | 上海电视十一厂 | Enzyme activity measuring instrument |
| CN1847210A (en) * | 2005-04-11 | 2006-10-18 | 临海市永太化工有限公司 | Process of producing pentafluorophenol |
| CN101294191A (en) * | 2007-04-27 | 2008-10-29 | 中国科学院沈阳应用生态研究所 | Analysis method for testing ammonium oxidizing enzyme activity in soil |
| CN101659986A (en) * | 2008-08-27 | 2010-03-03 | 周静 | Method for testing activity of H2O2-producing oxidase |
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