CN102495115A - Electrochemical method for detecting malic acid in root exudates by utilizing biological enzyme electrode method - Google Patents
Electrochemical method for detecting malic acid in root exudates by utilizing biological enzyme electrode method Download PDFInfo
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- CN102495115A CN102495115A CN2011104391119A CN201110439111A CN102495115A CN 102495115 A CN102495115 A CN 102495115A CN 2011104391119 A CN2011104391119 A CN 2011104391119A CN 201110439111 A CN201110439111 A CN 201110439111A CN 102495115 A CN102495115 A CN 102495115A
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Abstract
The invention discloses an electrochemical method for detecting malic acid in root exudates by utilizing a biological enzyme electrode method. The method comprises the following steps of: firstly, producing a malic dehydrogenase electrode; secondly, connecting the malic dehydrogenase electrode, a saturated calomel electrode and a platinum wire electrode with an electrochemical work station, and researching optimum conditions of the electrode reaction under a sodium pyrophosphate butter system; thirdly, setting an operating voltage under constant-temperature electromagnetic stirring, continuously adding a malic acid solution with a certain concentration into an electrolytic cell after a background current is stabilized, and measuring current change values corresponding to different concentrations of the malic acid so as to obtain a linear range of a catalytic current and the concentrations of the malic acid; and fourthly, adding a root exudates sample into the electrolytic cell, drawing a standard curve with an increased value of the current as a y axis and an addition of the standard solution of the pure malic acid as an x axis under constant-temperature electromagnetic stirring, and calculating an x value when y is equal to 0 according to the standard curve, wherein an absolute value of x is concentration of the malic acid in the diluted sample.
Description
Technical field
The present invention relates to a kind of method of utilizing enzyme electrode to detect malic acid in the root exudates, belong to biochemical analysis field.
Background technology
Root exudates is the one type of material that discharges to rhizosphere in the growing process.The root exudates of broad sense is the general name that root system of plant is discharged into various materials in the rhizospheric environment, and the root exudates of narrow sense is meant that root system of plant is discharged into the general name of various dissolved organic matters in the rhizospheric environment through the exudation effect.Root exudates of a great variety; The organic compound of at present having identified out has kind more than 200; Mainly include machine acid, carbohydrate, amino acid and phenolic acid low-molecular-weight organism such as (allelochemicals), also comprise the high-molecular-weight organic material matter that is similar to viscose and enzyme and so on.In numerous materials of root exudates, low molecular weight organic acid is that to study the most also be of paramount importance one type of material.Low molecular weight organic acid mainly comprises such as formic acid in the root exudates, acetate, oxalic acid, malic acid, succinic acid, maleic acid, citric acid, fumaric acid or the like.Wherein malic acid is the important organic acid in the root exudates; In specific plant; The malic acid amount of root system secretion not only has significant positive correlation with the organic acid total amount that root system is secreted; But also present significant positive correlation with oxalic acid, citric acid etc., therefore, measure the malic acid amount of root system secretion and can estimate a certain species organic acid secretion situation under given conditions.
The complicated component of root exudates; At present; The vapor-phase chromatography (GC) of the malic acid in the common mensuration root exudates, the chromatography of ions (IC) and high performance liquid chromatography methods such as (HPLC); All need numerous and diverse pre-treatment process, and usually occur that sensitivity is low, the unfavorable situation of effect.These methods also exist single sample long analysis time simultaneously, and chromatographic column is prone to pollute, and analyzes expensive defective.
Biology sensor is very an active research and a field of engineering technology, and it is in the intersection region of life science and information science with subjects such as bioinformatics, biochip, biocybernetics, bionics, biocomputers.Biology sensor research originates from the sixties in 20th century, and Updike in 1967 and Hicks fit together glucose oxidase (GOD) fixed film and oxygen electrode, have at first processed first kind of biology sensor, i.e. glucose enzyme electrode.To the eighties biology sensor research field basically form.Its significant incident is: " biology sensor " international publication was started publication in Britain in 1985; Publishing house publishes biology sensor classical works in 1987 in the Oxford; The academic conference of nineteen ninety first world biology sensor is held in Singapore, and whenever holds once at a distance from 2 years after confirming.After this biology sensor that comprises enzyme sensor is studied prosperity gradually, from the sensor of one or more enzymes as molecular recognition elements, develops gradually and designs the sensor of making recognition component with other biomolecule.The advantage of enzyme sensor has good stability, and analysis precision is high, analysis cost is minimum, wide in variety, and applied range, analysis speed are fast.To in the root exudates of numerous composition coexistences, measure malic acid, utilize the selectivity of malic acid and malic dehydrogenase thereof, make biology sensor and detect,, can reach easy, quick, sensitive effect simultaneously improving the specificity that detects.
Graphene (Graphene), it is that Coase Dalmatia Nuo Woxiefu and An Deliegaimu group by University of Manchester in 2004 at first find.Since finding, Graphene has received whole world scientist's extensive concern.Pure Graphene is a kind of crystalline solid that has only an atom thick, has the characteristics such as electricity, calorifics and mechanical property of ultra firm, superpower electric conductivity, excellence.Graphene nanometer composite is as a kind of novel high performance material; The a series of premium properties that not only kept Graphene; Also have other premium properties such as catalytic activity and easily disperse etc. that pure grapheme material does not have, thereby precondition is provided for the widespread use of Graphene.Because the graphene nanometer composite good electrical conductivity, be applied to a series of performances such as sensitivity, response speed and antijamming capability that bio-sensing can improve device, this is that the biology sensor of present many material is incomparable.Using mineral carbon alkene nano-complex is made material malate dehydrogenase enzyme electrode, sets up the malic acid in the enzyme electrode sensor root exudates, can reach highly sensitive, detectability is low, specificity good, instrument simple cheap, effect fast easy and simple to handle.
Summary of the invention
The technical matters that the present invention will solve is, a kind of electrochemical method that utilizes the biologic enzyme electrode method to detect malic acid in the root exudates is provided, this method with graphene nanometer composite as a kind of novel high performance material; Modified electrode prepares the malate dehydrogenase enzyme electrode; Directly measure the malic acid in the root exudates, comparatively complicated with the processing that overcomes sample in other method, single sample is long analysis time; Chromatographic column is prone to pollute, the shortcoming that disturbing factor is many.
The present invention takes following technical scheme, and it comprises following process: the first, make malic dehydrogenase (MDH) electrode; The second, malate dehydrogenase enzyme electrode, saturated calomel electrode and platinum electrode are connected with electrochemical workstation, the optimal conditions of this electrode reaction of research under the sodium pyrophosphate buffer system; The 3rd, under the constant temperature electromagnetic agitation, setting operation voltage; After treating that background current is stable; Add certain density malic acid solution continuously in electrolytic cell, utilize the constant potential chronoamperometry, measure corresponding electric current changing value under the different malic acid concentrations; Obtain the range of linearity of a catalytic current and malic acid concentration, the result must be in this range of linearity in the actual detected process in requirement; The 4th, add the root exudates sample in electrolytic cell, utilize the constant potential chronoamperometry; Under the constant temperature electromagnetic agitation; As the y axle, pure malic acid standard solution addition is as the x axle with the added value of electric current, the drawing standard curve; X value when the establishing criteria curve is asked y=0, this moment, the absolute value of x was exactly the concentration of the malic acid in the sample after the dilution certain multiple.
In first process, include following steps: (1) makes cadmium sulfide-graphene composite material; (2) prepare modified electrode with cadmium sulfide-graphene composite material; (3) make the malate dehydrogenase enzyme electrode.
In the step (1) of described first process, the at first synthetic oxidized graphite flake of functional groups such as carboxyl, hydroxyl that is rich in.Be the further synthetic CdS/G nano-complex of original material then with the oxidized graphite flake; Its preparation method is following; Take by weighing that 17 mg oxidized graphite flakes (GO) are ultrasonic to be scattered in the 10 mL redistilled waters; Be mono-layer graphite oxide alkene through ultrasonic oxidized graphite flake is peeled off, obtain the homogeneous phase brown solution after 30 minutes, i.e. the WS of graphene oxide.Under stirring state, dropwise add 50 mL, 0.035 mol/LCd (NO to this solution
3)
2Solution, and stir the Cd that made in the mixed liquor in 3 hours at normal temperatures
2+With graphite oxide-COOH ,-ion-exchange fully takes place in functional groups such as OH.In mixed liquor, feed H then
2S gas obtained green precipitate after one hour, and the solid of gained is used redistilled water respectively through centrifuging, and acetone respectively washs 3 times, and at last promptly obtaining the Graphene amount of mixing in 24 hours 45 ℃ of following vacuum drying is 4.6% CdS/G nano-complex.
In the step (2) of described first process, glass-carbon electrode (GCE, Φ=3.0 mm) is at first polished on abrasive paper for metallograph before using, and uses 1.0 m then successively, 0.3 m Al
2O
3Burnishing powder is polished to minute surface on polishing cloth, ultrasonic cleaning one minute in 0.1 mol/L HCl, 1.0 mol/L NaOH and absolute ethyl alcohol was successively used the redistilled water ultrasonic cleaning two minutes again after rinsing well with redistilled water, dried subsequent use.
Take by weighing 1.0mg CdS/G nano-complex and be dispersed in 1.0mL 0.5% chitosan solution, obtain the unit for uniform suspension of CdS-G nano-complex after the ultrasonic dispersion.Get this hanging drop of 6 L and be coated in the glass-carbon electrode surface of anticipating, dry under the room temperature, make CdS-G nano-complex modified electrode (being designated as CdS/G/CHIT/GCE).
In the step (3) of described first process, malic dehydrogenase (MDH) drips of solution of getting 5 L 10mg/mL is on CdS-G nano-complex modified electrode surface, and room temperature is dried, and treats that electrode surface puts into 4 ℃ of refrigerators after drying and preserve for use.Obtained the malate dehydrogenase enzyme electrode like this.
In described second process, the reaction conditions in the electrolytic cell is: the pH value 9.0 of sodium pyrophosphate buffer solution, oxidizing potential are 0.7 V, the oxidation state coenzyme NAD
+Concentration is 5 * 10
-3MoL L
-1, select 30 ℃ as temperature of reaction.
In described the 3rd process, under 30 ℃ of electromagnetic agitation of constant temperature, the pH value 9.0 of sodium pyrophosphate buffer solution, oxidizing potential is 0.7 V, the oxidation state coenzyme NAD
+Concentration is 5 * 10
-3MoL L
-1, treat that background current is stable after, add certain density malic acid solution continuously in electrolytic cell, utilize the constant potential chronoamperometry, measure corresponding electric current changing value under the different malic acid concentrations, obtain the range of linearity figure of a catalytic current and malic acid concentration.
In described the 4th process, temperature is controlled to be 30 ℃, and operating voltage is controlled to be 0.7V, gets pH=9.0, coenzyme NAD that 1 mL testing sample joins 4 mL
+Concentration is 5 * 10
-3MoL L
-1Sodium pyrophosphate buffer solution in, electromagnetic agitation is following, treat that background current is stable after, per 50 s sample introductions once, add 5 L concentration is 0.01 moL L at every turn
-1The standard malic acid solution totally 4 times, is measured corresponding electric current changing value in electrolytic cell; As the y axle, pure malic acid standard solution addition is as the x axle with the added value of electric current, the drawing standard curve; Make y=0, obtain the value of x, the absolute value of x is exactly the concentration of the malic acid of sample after having diluted 5 times.
Advantage of the present invention is following:
(1) the malic dehydrogenase electrode stability is good, and analysis precision is high.
(2) the present invention can directly measure malic acid in the complicated root exudates of constituent, and need not numerous and diverse pre-treatment process.
(3) the present invention is easy and simple to handle fast, and detectability is low, selectivity is strong, highly sensitive, instrument simple cheap.
Description of drawings
Fig. 1 a is a malic acid constant potential timing current curve diagram in the rape root secretion handled of full phosphorus;
Fig. 1 b is malic acid-electric current linear relationship chart in the rape root secretion handled of full phosphorus;
Accompanying drawing 2a is a malic acid constant potential timing current curve diagram in the rape root secretion of without phosphorus processing;
Accompanying drawing 2b is malic acid-electric current linear relationship chart in the rape root secretion of without phosphorus processing;
Accompanying drawing 3a is a malic acid constant potential timing current curve diagram in the rape root secretion handled of polyglycol;
Accompanying drawing 3b is malic acid-electric current linear relationship chart in the rape root secretion handled of polyglycol.
Embodiment:
Embodiments of the invention: it comprises following process: the first, make malic dehydrogenase (MDH) electrode; The second, malate dehydrogenase enzyme electrode, saturated calomel electrode and platinum electrode are connected with electrochemical workstation, the optimal conditions of this electrode reaction of research under the sodium pyrophosphate buffer system; The 3rd, under the constant temperature electromagnetic agitation, setting operation voltage; After treating that background current is stable; Add certain density malic acid solution continuously in electrolytic cell, utilize the constant potential chronoamperometry, measure corresponding electric current changing value under the different malic acid concentrations; Obtain the range of linearity of a catalytic current and malic acid concentration, the result must be in this range of linearity in the actual detected process in requirement; The 4th, add the root exudates sample in electrolytic cell, utilize the constant potential chronoamperometry; Under the constant temperature electromagnetic agitation; As the y axle, pure malic acid standard solution addition is as the x axle with the added value of electric current, the drawing standard curve; X value when the establishing criteria curve is asked y=0, this moment, the absolute value of x was exactly the concentration of the malic acid in the sample after the dilution certain multiple.
In first process, include following steps: (1) makes cadmium sulfide-graphene composite material; (2) prepare modified electrode with cadmium sulfide-graphene composite material; (3) make the malate dehydrogenase enzyme electrode.
In the step (1) of described first process, the at first synthetic oxidized graphite flake of functional groups such as carboxyl, hydroxyl that is rich in.Be the further synthetic CdS/G nano-complex of original material then with the oxidized graphite flake; Its preparation method is following; Take by weighing that 17 mg oxidized graphite flakes (GO) are ultrasonic to be scattered in the 10 mL redistilled waters; Be mono-layer graphite oxide alkene through ultrasonic oxidized graphite flake is peeled off, obtain the homogeneous phase brown solution after 30 minutes, i.e. the WS of graphene oxide.Under stirring state, dropwise add 50 mL, 0.035 mol/LCd (NO to this solution
3)
2Solution, and stir the Cd that made in the mixed liquor in 3 hours at normal temperatures
2+With graphite oxide-COOH ,-ion-exchange fully takes place in functional groups such as OH.In mixed liquor, feed H then
2S gas obtained green precipitate after one hour, and the solid of gained is used redistilled water respectively through centrifuging, and acetone respectively washs 3 times, and at last promptly obtaining the Graphene amount of mixing in 24 hours 45 ℃ of following vacuum drying is 4.6% CdS/G nano-complex.
In the step (2) of described first process, glass-carbon electrode (GCE, Φ=3.0 mm) is at first polished on abrasive paper for metallograph before using, and uses 1.0 m then successively, 0.3 m Al
2O
3Burnishing powder is polished to minute surface on polishing cloth, ultrasonic cleaning one minute in 0.1 mol/L HCl, 1.0 mol/L NaOH and absolute ethyl alcohol was successively used the redistilled water ultrasonic cleaning two minutes again after rinsing well with redistilled water, dried subsequent use.
Take by weighing 1.0mg CdS/G nano-complex and be dispersed in 1.0mL 0.5% chitosan solution, obtain the unit for uniform suspension of CdS-G nano-complex after the ultrasonic dispersion.Get this hanging drop of 6 L and be coated in the glass-carbon electrode surface of anticipating, dry under the room temperature, make CdS-G nano-complex modified electrode (being designated as CdS/G/CHIT/GCE).
In the step (3) of described first process, malic dehydrogenase (MDH) drips of solution of getting 5 L 10mg/mL is on CdS-G nano-complex modified electrode surface, and room temperature is dried, and treats that electrode surface puts into 4 ℃ of refrigerators after drying and preserve for use.Obtained the malate dehydrogenase enzyme electrode like this.
In described second process, the reaction conditions in the electrolytic cell is: the pH value 9.0 of sodium pyrophosphate buffer solution, oxidizing potential are 0.7 V, the oxidation state coenzyme NAD
+Concentration is 5 * 10
-3MoL L
-1, select 30 ℃ as temperature of reaction.
In described the 3rd process, under 30 ℃ of electromagnetic agitation of constant temperature, the pH value 9.0 of sodium pyrophosphate buffer solution, oxidizing potential is 0.7 V, the oxidation state coenzyme NAD
+Concentration is 5 * 10
-3MoL L
-1, treat that background current is stable after, add certain density malic acid solution continuously in electrolytic cell, utilize the constant potential chronoamperometry, measure corresponding electric current changing value under the different malic acid concentrations, obtain the range of linearity figure of a catalytic current and malic acid concentration.
In described the 4th process, temperature is controlled to be 30 ℃, and operating voltage is controlled to be 0.7V, gets pH=9.0, coenzyme NAD that 1 mL testing sample joins 4 mL
+Concentration is 5 * 10
-3MoL L
-1Sodium pyrophosphate buffer solution in, electromagnetic agitation is following, treat that background current is stable after, per 50 s sample introductions once, add 5 L concentration is 0.01 moL L at every turn
-1The standard malic acid solution totally 4 times, is measured corresponding electric current changing value in electrolytic cell; As the y axle, pure malic acid standard solution addition is as the x axle with the added value of electric current, the drawing standard curve; Make y=0, obtain the value of x, the absolute value of x is exactly the concentration of the malic acid of sample after having diluted 5 times.
Application example 1: in order to prove implementation result of the present invention; With the rape of growth after 30 days; Handle with full phosphorus (Hoagland nutrient solution), after 15 days rape root secretion is collected and constant volume, get pH=9.0, coenzyme NAD that this root exudates of 1mL joins 4 mL
+Concentration is 5 * 10
-3MoL L
-1Sodium pyrophosphate buffer solution, temperature is controlled at 30 ℃, electromagnetic agitation and operating voltage are set to 0.7 V, after background current reached stationary value, add 5 L concentration was 0.01 moL L at every turn
-1The standard malic acid solution in electrolytic cell, per 50 s sample introductions once, totally 4 times; Measure corresponding electric current changing value; As the y axle, pure malic acid standard solution addition is as the x axle with the added value of electric current, the drawing standard curve; X value when the establishing criteria curve is asked y=0; This moment, the absolute value of x was exactly the malic acid concentration that has diluted in 5 times the full phosphorus rape root secretion, again with the absolute value of X multiply by 5 promptly be collect and constant volume after malic acid concentration in the full phosphorus rape root secretion, like accompanying drawing 1a, accompanying drawing 1b.
Application example 2: in order to prove implementation result of the present invention,, handle, after 15 days rape root secretion is collected and constant volume, get pH=9.0, coenzyme NAD that this root exudates of 1mL joins 4 mL with without phosphorus nutrient solution with the rape of growth after 30 days
+Concentration is 5 * 10
-3MoL L
-1Sodium pyrophosphate buffer solution, temperature is controlled at 30 ℃, electromagnetic agitation and operating voltage are set to 0.7 V, after background current reached stationary value, add 5 L concentration was 0.01 moL L at every turn
-1The standard malic acid solution in electrolytic cell, per 50 s sample introductions once, totally 4 times; Measure corresponding electric current changing value; As the y axle, pure malic acid standard solution addition is as the x axle with the added value of electric current, the drawing standard curve; X value when the establishing criteria curve is asked y=0; This moment, the absolute value of x was exactly the malic acid concentration that has diluted in 5 times the without phosphorus rape root secretion, again with the absolute value of x multiply by 5 promptly be collect and constant volume after malic acid concentration in the without phosphorus rape root secretion, like accompanying drawing 2 a, accompanying drawing 2b.
Application example 3:
In order to prove implementation result of the present invention, with the rape of growth after 30 days, handle with the Polyethylene Glycol Simulated arid, after 15 days rape root secretion is collected and constant volume, get pH=9.0, coenzyme NAD that this root exudates of 1mL joins 4 mL
+Concentration is 5 * 10
-3MoL L
-1Sodium pyrophosphate buffer solution, temperature is controlled at 30 ℃, electromagnetic agitation and operating voltage are set to 0.7 V, after background current reached stationary value, add 5 L concentration was 0.01 moL L at every turn
-1The standard malic acid solution in electrolytic cell, per 50 s sample introductions once, totally 4 times; Measure corresponding electric current changing value; As the y axle, pure malic acid standard solution addition is as the x axle with the added value of electric current, the drawing standard curve; X value when the establishing criteria curve is asked y=0; This moment, the absolute value of x was exactly the malic acid concentration that has diluted in the rape root secretion that 5 times Polyethylene Glycol Simulated arid handles, again with the absolute value of x multiply by 5 promptly be collect and constant volume after malic acid concentration in the rape root secretion handled of Polyethylene Glycol Simulated arid, like accompanying drawing 3 a, accompanying drawing 3b.
Through above application example, implementation result such as the table 1 of three embodiment.Can find out that from table 1 rape does not receive environment or nutrition when coercing, malic acid content is minimum, and the shortage of phosphorus has promoted rape secretion malic acid, causes the malic acid of without phosphorus processing root system secretion maximum.Arid also promotes root system secretion organic acid, has wherein also promoted the secretion of malic acid.This is consistent with root system secretion organic acid rule.
Claims (7)
1. electrochemical method that utilizes the biologic enzyme electrode method to detect malic acid in the root exudates, it is characterized in that: it comprises following process: the first, make the malate dehydrogenase enzyme electrode; The second, malate dehydrogenase enzyme electrode, saturated calomel electrode and platinum electrode are connected with electrochemical workstation, the optimal conditions of this electrode reaction of research under the sodium pyrophosphate buffer system; The 3rd, under the constant temperature electromagnetic agitation, setting operation voltage; After treating that background current is stable; Add certain density malic acid solution continuously in electrolytic cell, utilize the constant potential chronoamperometry, measure corresponding electric current changing value under the different malic acid concentrations; Obtain the range of linearity of catalytic current and malic acid concentration, require result in the actual detected process in this range of linearity; The 4th, add the root exudates sample in electrolytic cell, utilize the constant potential chronoamperometry; Under the constant temperature electromagnetic agitation; As the y axle, pure malic acid standard solution addition is as the x axle with the added value of electric current, the drawing standard curve; X value when the establishing criteria curve is asked y=0, this moment, the absolute value of x was exactly the concentration of the malic acid in the sample after the dilution certain multiple.
2. the electrochemical method that utilizes the biologic enzyme electrode method to detect malic acid in the root exudates according to claim 1 is characterized in that: in described first process, include following steps: (1) makes cadmium sulfide-graphene composite material; (2) prepare modified electrode with cadmium sulfide-graphene composite material; (3) make the malate dehydrogenase enzyme electrode.
3. the electrochemical method that utilizes the biologic enzyme electrode method to detect malic acid in the root exudates according to claim 2; It is characterized in that: in the step (1) of described first process; The at first synthetic oxidized graphite flake of functional groups such as carboxyl, hydroxyl that is rich in; Be the further synthetic CdS/G nano-complex of original material then with the oxidized graphite flake, its preparation method is following: take by weighing that 17 mg oxidized graphite flakes are ultrasonic to be scattered in the 10 mL redistilled waters, be mono-layer graphite oxide alkene through ultrasonic oxidized graphite flake is peeled off; Obtain the homogeneous phase brown solution after 30 minutes, i.e. the WS of graphene oxide; Under stirring state, dropwise add 50 mL, 0.035 mol/LCd (NO to this solution
3)
2Solution, and stir the Cd that made in the mixed liquor in 3 hours at normal temperatures
2+With graphite oxide-COOH ,-ion-exchange fully takes place in functional groups such as OH; In mixed liquor, feed H then
2S gas obtained green precipitate after one hour, and the solid of gained is used redistilled water respectively through centrifuging, and acetone respectively washs 3 times, and at last promptly obtaining the Graphene amount of mixing in 24 hours 45 ℃ of following vacuum drying is 4.6% CdS/G nano-complex.
4. the electrochemical method that utilizes the biologic enzyme electrode method to detect malic acid in the root exudates according to claim 2; It is characterized in that: in the step (2) of described first process, glass-carbon electrode is at first polished on abrasive paper for metallograph before using; Use 1.0 m then successively, 0.3 m Al
2O
3Burnishing powder is polished to minute surface on polishing cloth, ultrasonic cleaning one minute in 0.1 mol/L HCl, 1.0 mol/L NaOH and absolute ethyl alcohol was successively used the redistilled water ultrasonic cleaning two minutes again after rinsing well with redistilled water, dried subsequent use; Take by weighing 1.0mg CdS/G nano-complex and be dispersed in 1.0mL 0.5% chitosan solution, obtain the unit for uniform suspension of CdS-G nano-complex after the ultrasonic dispersion; Get this hanging drop of 6 L again and be coated in the glass-carbon electrode surface of anticipating, dry under the room temperature, make CdS-G nano-complex modified electrode.
5. the electrochemical method that utilizes the biologic enzyme electrode method to detect malic acid in the root exudates according to claim 2; It is characterized in that: in the step (3) of described first process; The malic dehydrogenase drips of solution of getting 5 L 10mg/mL is on CdS-G nano-complex modified electrode surface; Room temperature is dried, and treats that electrode surface puts into 4 ℃ of refrigerators after drying and preserve for usely, has obtained the malate dehydrogenase enzyme electrode like this.
6. the electrochemical method that utilizes the biologic enzyme electrode method to detect malic acid in the root exudates according to claim 1; It is characterized in that: in described second process; Reaction conditions in the electrolytic cell is: the pH value 9.0 of sodium pyrophosphate buffer solution, oxidizing potential are 0.7 V, the oxidation state coenzyme NAD
+Concentration is 5 * 10
-3MoL L
-1, select 30 ℃ as temperature of reaction.
7. the electrochemical method that utilizes the biologic enzyme electrode method to detect malic acid in the root exudates according to claim 1; It is characterized in that: in described the 3rd process, under 30 ℃ of electromagnetic agitation of constant temperature, the pH value 9.0 of sodium pyrophosphate buffer solution; Oxidizing potential is 0.7 V, the oxidation state coenzyme NAD
+Concentration is 5 * 10
-3MoL L
-1, treat that background current is stable after, add certain density malic acid solution continuously in electrolytic cell, utilize the constant potential chronoamperometry, measure corresponding electric current changing value under the different malic acid concentrations, obtain the range of linearity figure of catalytic current and malic acid concentration.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103163238A (en) * | 2013-02-22 | 2013-06-19 | 江苏大学 | Method of using organic acid secretion characteristics to detect phosphorus deficiency stress resistance ability of plants |
| CN104353469A (en) * | 2014-10-28 | 2015-02-18 | 江苏大学 | Method for preparing nanocomposite photocatalyst and application of nanocomposite photocatalyst |
| CN111307900A (en) * | 2020-02-07 | 2020-06-19 | 山东省科学院生物研究所 | Coenzyme factor compound, enzyme electrode, enzyme sensor, preparation method and application thereof |
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|---|---|---|---|---|
| JPS5513860A (en) * | 1978-07-14 | 1980-01-31 | Matsushita Electric Ind Co Ltd | Enzyme electrode |
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| CN111896599B (en) * | 2020-02-07 | 2022-12-30 | 山东省科学院生物研究所 | Malic dehydrogenase electrode and preparation method and application thereof |
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