CN111307890A - Manganese oxide/graphene modified electrode and method for detecting salicylic acid in plant tissue based on manganese oxide/graphene modified electrode - Google Patents
Manganese oxide/graphene modified electrode and method for detecting salicylic acid in plant tissue based on manganese oxide/graphene modified electrode Download PDFInfo
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Abstract
The invention discloses a manganese oxide/graphene modified electrode and a method for detecting salicylic acid in plant tissues based on the manganese oxide/graphene modified electrode, which comprises the following steps: and taking the pH value corresponding to the maximum intensity current in the voltage-current curves of the N first salicylic acid mixed solutions as X, configuring M second salicylic acid mixed solutions with the pH value of X, obtaining the salicylic acid response characteristic peak corresponding to each salicylic acid concentration through the current-voltage curves of the M second salicylic acid mixed solutions, drawing a curve, obtaining a linear regression equation, and correcting. Inserting the working electrode, the reference electrode and the counter electrode into the detected plant tissue to obtain a DPV curve of the detected plant tissue, wherein the current value corresponding to the peak of the DPV curve is IDetection ofIs shown byDetection ofSubstitution to obtain CiTo measure the salicylic acid concentration of plant tissues. The invention can directly detect the concentration of the salicylic acid in the plant tissue, the lower limit of the detection can be as low as 2 mu mol/L, and the test sensitivity and the accuracy are high; low cost and is beneficial to civilization.
Description
Technical Field
The invention belongs to the technical field of electrochemical plant sensors, and particularly relates to a manganese oxide/graphene modified electrode and a method for detecting salicylic acid in plant tissues based on the manganese oxide/graphene modified electrode.
Background
Salicylic Acid (SA) is a natural substance widely found in the plant kingdom. It not only has important pharmacological effect, but also has wide regulation and control effect on the growth and development process of plants and the disease resistance of plants. By measuring the change of the endogenous SA level of the tissues in the fruit ripening and senescence process, the ripening and senescence mechanism of the fruits can be better understood, and the accumulation basis of SA and derivatives thereof for regulating the ripening and senescence process of the fruits is provided. However, the very low endogenous SA level in plant tissues and a more sensitive detection method are important contents for researching the regulation of SA on the fruit ripening and senescence.
At present, the detection of salicylic acid in plant tissues needs to extract plant endogenous SA firstly, but the level of the endogenous SA in the plant tissues is very low, the extraction method is complex, multiple steps of extraction, liquid-liquid distribution, evaporation to dryness and the like are required, the recovery rate is low and generally ranges from 34.0% to 80.7%, and the detection is carried out by using High Performance Liquid Chromatography (HPLC) or fluorescence spectroscopy after the extraction. The method is single.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a manganese oxide/graphene modified electrode.
The invention also aims to provide a preparation method of the manganese oxide/graphene modified electrode, which adopts high electrocatalytic activity graphene and manganese oxide as active materials, develops a sensing electrode by optimizing the growth condition of the graphene, can realize direct oxidation of plant tissue salicylic acid, and obtains a response signal.
Another objective of the present invention is to provide a method for detecting salicylic acid in plant tissues, which can perform qualitative and quantitative detection in response to an electric signal generated by oxidation or reduction of an analyte, and has the advantages of fast response, high sensitivity and low cost.
The purpose of the invention is realized by the following technical scheme.
A method of detecting salicylic acid in plant tissue comprising the steps of:
1) preparing N first salicylic acid mixed solutions with different pH values, connecting a working electrode, a reference electrode and a counter electrode with an electrochemical workstation, respectively inserting the working electrode, the reference electrode and the counter electrode into each first salicylic acid mixed solution, testing by adopting a differential pulse voltammetry to obtain voltage-current curves of the N first salicylic acid mixed solutions, and taking a pH value corresponding to the maximum intensity current in the N voltage-current curves as X, wherein N is 6-10, and the preparation method of each first salicylic acid mixed solution comprises the following steps: mixing a phosphoric acid buffer solution with salicylic acid;
in the step 1), the pH values of the N first salicylic acid mixed solutions comprise neutrality, acidity and alkalinity.
In the step 1), N is 7, and the pH of the N first salicylic acid mixed solutions is 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0.
In the step 1), the concentration of salicylic acid in the first salicylic acid mixed solution is 0.1-1 mmol/l.
2) Preparing M second salicylic acid mixed solutions with the pH value of X, wherein M is 10-30, and the concentration of salicylic acid in each second salicylic acid mixed solution is Ci，i＝1……M，CmaxIs the maximum value of the concentration of salicylic acid in M second salicylic acid mixed solutions, CminIs the minimum value of the concentration of the salicylic acid in the M second salicylic acid mixed solutions,
connecting a working electrode, a reference electrode and a counter electrode with an electrochemical workstation and respectively inserting the working electrode, the reference electrode and the counter electrode into each second salicylic acid mixed solution, testing the M second salicylic acid mixed solutions by adopting a differential pulse voltammetry method to obtain current-voltage curves of the M second salicylic acid mixed solutions, and obtaining the concentration C of each salicylic acid through the current-voltage curves of the M second salicylic acid mixed solutionsiCorresponding salicylic acid response characteristic peak, and the current I corresponding to the salicylic acid response characteristic peakiWith salicylic acid concentration CiDrawing curves for horizontal and vertical coordinates, fitting to obtain a working curve and a linear regression equation I of the working curvei＝a+bCiWherein a and b are constants, and the preparation method of the second salicylic acid mixed solution comprises the following steps: mixing a phosphoric acid buffer solution with salicylic acid;
in said step 2), Cmin≤CTo be treated≤CmaxWherein, CTo be treatedTo determine the concentration of salicylic acid in plant tissues.
In the step 2), the M second salicylic acid mixed solutions are tested by adopting a differential pulse voltammetry method according to the sequence of salicylic acid concentration from low to high.
In the step 2), the number of the linear regression equations is at least 1.
3) Obtaining a coefficient K, and linearly regressing equation I with the coefficient K through formula (1)i＝a+bCiAnd (3) correcting:
Ii'＝K(a+bCi) Formula (1) wherein Ii' for the corrected current, the coefficient K is obtained by: preparing a substance of the same kind as the detected plant tissue as a correction plant tissue, and obtaining 2 resistances of the correction substance by using the squeezed liquid of the correction plant tissue and the correction plant tissue as the correction substance respectively, wherein the resistance of each correction substance is obtained by the following method: connecting the working electrode, the reference electrode and the counter electrode with an electrochemical workstation, respectively inserting the working electrode, the reference electrode and the counter electrode into the calibration material, and obtaining an open circuit of the calibration material by adopting a method of an open circuit potential-time curveVoltage, based on the open circuit voltage, measuring by adopting alternating current impedance to obtain the resistor; dividing the resistance of the liquid obtained after the correction of the plant tissue is juiced by the resistance of the correction of the plant tissue to obtain the coefficient K;
4) connecting the working electrode, the reference electrode and the counter electrode with an electrochemical workstation, and inserting the working electrode, the reference electrode and the counter electrode into the detected plant tissue to obtain a DPV curve of the detected plant tissue, wherein the current value corresponding to the peak of the DPV curve is IDetection ofIs shown byDetection ofSubstituting into I in formula (1)i', obtaining CiTo measure the salicylic acid concentration of plant tissues.
In the technical scheme, the potential range of the differential pulse voltammetry is set to be 0-1.4V; the potential increment is set to be 0.001-0.05V, the amplitude is set to be 0.01-0.05V, the pulse width is set to be 0.005-0.25 s, and the pulse period is set to be 0.01-0.5 s.
In the technical scheme, the working electrode is a glassy carbon electrode, a gold electrode or conductive glass, the reference electrode is a saturated calomel electrode, an Ag/AgCl electrode or a mercury/mercurous sulfate electrode, and the counter electrode is a platinum sheet electrode.
In the above technical scheme, the working electrode is a manganese oxide/graphene modified electrode, and the preparation method of the manganese oxide/graphene modified electrode comprises the following steps:
① cleaning the surface of the solid electrode;
in the step ①, the cleaning method comprises polishing the solid electrode with alumina powder to form a mirror surface, and sequentially performing ultrasonic treatment for 5-15 min with 30-80 wt% nitric acid aqueous solution, ultrapure water, absolute ethyl alcohol and ultrapure water for removing organic and inorganic dirt.
In step ①, the solid electrode is a glassy carbon electrode.
② potassium permanganate, potassium chloride, graphite and ultrapure water are uniformly mixed to obtain a mixed solution, a solid electrode, a reference electrode and a counter electrode are inserted into the mixed solution, electrodeposition treatment is carried out by adopting a cyclic voltammetry, the potential range of the cyclic voltammetry is set to-1.5V, the voltage scanning speed is 0.05-0.2V/s, continuous scanning is carried out for 30-100 circles, the solid electrode is taken out and washed by the ultrapure water and dried to obtain the manganese oxide/graphene modified electrode, wherein the concentration of the potassium permanganate in the mixed solution is 0.1-0.5 mol/L, the concentration of the potassium chloride in the mixed solution is 0.1-0.5 mol/L, and the concentration of the graphite in the mixed solution is 5-10 mg/mL.
In the step ②, the drying is blow drying with nitrogen.
In the step ②, the graphite is graphite powder, graphene or carbon nanotubes.
In the step ②, preparing graphite by an electrochemical stripping method, namely connecting a graphite rod to each of the positive electrode and the negative electrode of a voltage-stabilized direct-current power supply, immersing 2 graphite rods into dilute sulfuric acid, setting the voltage to be 5-15V and the current to be 100-400 mA, keeping for 5-20 minutes, carrying out vacuum filtration, and drying to obtain the graphite.
In the technical scheme, the concentration of the dilute sulfuric acid is 3-5 mol/L.
In the technical scheme, the diameter of the graphite rod is 0.5-3 mm.
The preparation method of the manganese oxide/graphene modified electrode is convenient to operate; the electrode can be modified by an electrochemical method, and salicylic acid can be directly oxidized to obtain a response signal; the concentration of salicylic acid in plant tissues can be directly detected; the sensor has good reproducibility, repeatability and stability, two working electrodes prepared by the same method under the same condition are used for detecting the same plant, the difference between the salicylic acid content in the obtained two plant tissues is not more than 0.1 mu mol/L, the concentration of potassium permanganate during electrode preparation is changed, and the sensor can still be used for detecting the salicylic acid content in the plant tissues; the detection limit can be as low as 2 mu mol/L, and the test sensitivity and accuracy are high; low cost and is beneficial to civilization.
Drawings
FIG. 1 is a scanning electron microscope photograph of the manganese oxide/graphene modified electrode obtained in example 1;
FIG. 2 is a graph showing the operating curve and M current-voltage curves in step 2) of example 1, wherein 2a is the operating curve and 2b is the current-voltage curve;
FIG. 3 is a scanning electron microscope photograph of the manganese oxide/graphene modified electrode obtained in example 2;
FIG. 4 is a graph showing the operating curve and M current-voltage curves in step 2) of example 2, in which 4a is the operating curve and 4b is the current-voltage curve;
FIG. 5 is a scanning electron micrograph of the manganese oxide/graphene modified electrode obtained in example 3;
FIG. 6 shows the operating curve and M current-voltage curves in step 2) of example 3, where 6a is the operating curve and 6b is the current-voltage curve.
Detailed Description
The technical scheme of the invention is further explained by combining specific examples.
The method for obtaining the liquid after the correction of the plant tissue is to squeeze the juice from the correction plant tissue, and the adopted instruments are as follows: the portable rechargeable juice extractor that rubs flies, the operating procedure is: cutting the corrected plant tissue (fruit) into small pieces, putting the small pieces into a juicer, pressing a start key, and obtaining the liquid obtained after the corrected plant tissue is juiced after five minutes.
The following examples relate to apparatus having an electronic balance, a pH meter, an ultrasonic cleaner, a regulated DC power supply, an electrochemical workstation (Shanghai Chen Hua CHI660E)
The following examples relate to the purity of the drug and the manufacturer's anhydrous disodium hydrogen phosphate (analytical purity of Guangxi Fine chemical research institute, Tianjin), anhydrous sodium dihydrogen phosphate (analytical purity of Guangxi Fine chemical research institute, Tianjin), salicylic acid (analytical purity of Jiangtian unified science and technology Co., Ltd., Tianjin), potassium permanganate (analytical purity of the second division of Tianjin chemical reagent supply and distribution Co., Ltd.), potassium chloride (superior pure chemical reagent factory, Tianjin), nitric acid (analytical purity of Tianjin Fengshu chemical reagent science and technology Co., Ltd., Tianjin Hangzhou chemical technology Co., Ltd.), and anhydrous ethanol (analytical purity of Tianjin Hui Hangzhou chemical technology Co., Ltd., Tianjin)
Preparing a phosphoric acid buffer solution, firstly preparing 0.1mol/L sodium dihydrogen phosphate solution as a solution A, and 0.1mol/L disodium hydrogen phosphate solution as a solution B, and mixing the solution A and the solution B according to different proportions to obtain the phosphoric acid buffer solution with the required pH value.
Examples 1 to 3
A method of detecting salicylic acid in plant tissue comprising the steps of:
1) preparing 7 first salicylic acid mixed solutions with different pH values, wherein the pH values of the 7 first salicylic acid mixed solutions are 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, and the concentration of the salicylic acid in the 7 first salicylic acid mixed solutions is 0.1 mmol/L. And connecting a working electrode, a reference electrode and a counter electrode with an electrochemical workstation, respectively inserting the working electrode, the reference electrode and the counter electrode into each first salicylic acid mixed solution, testing by adopting a differential pulse voltammetry method to obtain 7 voltage-current curves of the first salicylic acid mixed solution, and taking the pH value corresponding to the maximum intensity current in the 7 voltage-current curves as X, wherein the X is shown in table 1. The preparation method of each first salicylic acid mixed solution comprises the following steps: mixing the phosphoric acid buffer solution after the pH is adjusted with salicylic acid; setting the potential range of the differential pulse voltammetry to be 0.4-1.4V; the potential increment was set to 0.004V, the amplitude was set to 0.05V, the pulse width was set to 0.06s, and the pulse period was set to 0.5 s.
2) M second salicylic acid mixed solutions with the pH value of X are prepared, and the preparation method of the second salicylic acid mixed solutions comprises the following steps: mixing phosphoric acid buffer solution with pH of X with salicylic acid, wherein the concentration of salicylic acid in each second salicylic acid mixed solution is CiAnd i is 1 … … M. Detecting the plant tissue as tomato and the concentration of salicylic acid C in the plant tissueTo be treatedAbout 2 to 9. mu. mol/L, for Cmin≤CTo be treated≤CmaxAnd is
(CmaxIs the maximum value of the concentration of salicylic acid in M second salicylic acid mixed solutions, CminThe minimum value of the salicylic acid concentration in the M second salicylic acid mixed solutions), therefore, the salicylic acid concentrations in the M second salicylic acid mixed solutions are sequentially shown in table 1; and connecting a working electrode, a reference electrode and a counter electrode with an electrochemical workstation, respectively inserting the working electrode, the reference electrode and the counter electrode into each second salicylic acid mixed solution according to the sequence of salicylic acid concentration from low to high, testing the M second salicylic acid mixed solutions by adopting a differential pulse voltammetry method to obtain current-voltage curves of the M second salicylic acid mixed solutions, and setting the potential range of the differential pulse voltammetry method to be 0.4-1.4V; the potential increment was set to 0.004V, the amplitude was set to 0.05V, the pulse width was set to 0.06s, and the pulse period was set to 0.5 s. Obtaining the concentration C of each salicylic acid through the current-voltage curves of M second salicylic acid mixed solutionsiCorresponding salicylic acid response characteristic peak, and the current I corresponding to the salicylic acid response characteristic peakiWith salicylic acid concentration CiDrawing curves for horizontal and vertical coordinates, fitting to obtain working curve and at least 1 linear regression equation I of the working curvei＝a+bCi(the number of linear regression equations is 1 if the working curve is a straight line; the number of linear regression equations is more than 1 if the working curve is a broken line), wherein a and b are both constants, and the value of M is shown in Table 1.
3) Obtaining a coefficient K, and linearly regressing equation I with the coefficient K through formula (1)i＝a+bCiCorrection is performed (i.e. linear regression equation I)i＝a+bCiCorrected to equation (1)):
Ii'＝K(a+bCi) Formula (1) wherein Ii' for the corrected current, the coefficient K is obtained by: preparing a substance of the same kind as the detected plant tissue as a correction plant tissue, and obtaining 2 resistances of the correction substance by using the squeezed liquid of the correction plant tissue and the correction plant tissue as the correction substance respectively, wherein the resistance of each correction substance is obtained by the following method: connecting a working electrode, a reference electrode and a counter electrode with an electrochemical workstation and respectively inserting the working electrode, the reference electrode and the counter electrode into the correction substance, obtaining the open-circuit voltage of the correction substance by adopting an open-circuit potential-time curve method, and obtaining the resistance by adopting alternating current impedance measurement based on the open-circuit voltage; dividing the resistance of the liquid obtained after the correction of the plant tissue juicing by the resistance of the correction of the plant tissue to obtain a coefficient K;
4) connecting the working electrode, the reference electrode and the counter electrode with an electrochemical workstation, inserting the electrochemical workstation into the detected plant tissue to obtain a DPV curve of the detected plant tissue, wherein the current value corresponding to the peak of the DPV curve is IDetection ofIs shown byDetection ofSubstituting into I in formula (1)i', obtaining CiTo measure the salicylic acid concentration of plant tissues.
In the technical scheme, the reference electrode is a saturated calomel electrode, and the counter electrode is a platinum sheet electrode. The working electrode is a manganese oxide/graphene modified electrode, and the preparation method of the manganese oxide/graphene modified electrode comprises the following steps:
① preparing glassy carbon electrode as solid electrode, and cleaning the surface of the solid electrode by polishing the solid electrode with alumina powder to mirror surface, and sequentially ultrasonic treating with 50 wt% nitric acid aqueous solution, ultrapure water, anhydrous alcohol and ultrapure water for 10min to remove organic and inorganic dirt.
② graphite is prepared by electrochemical stripping method by connecting a graphite rod with diameter of 3mm to the positive and negative electrodes of a voltage-stabilized DC power supply, immersing 2 graphite rods in dilute sulfuric acid at 10V and 300mA for 15min until the positive electrode has a large amount of graphite solid, vacuum filtering with circulating water, and oven drying to obtain graphite with dilute sulfuric acid concentration of 5 mol/L.
The potassium permanganate, the potassium chloride, the graphite and the ultrapure water are uniformly mixed to obtain a mixed solution, the concentration of the potassium permanganate in the mixed solution is H mol/L, the concentration of the potassium chloride in the mixed solution is 0.1mol/L, the concentration of the graphite in the mixed solution is 10mg/mL, and H is shown in Table 1. And inserting the solid electrode, the reference electrode and the counter electrode into the mixed solution, performing electrodeposition treatment by adopting a cyclic voltammetry, setting the potential range of the cyclic voltammetry to be-1.5V, setting the voltage scanning speed to be 0.2V/s, continuously scanning for 50 circles, taking out the solid electrode, washing the solid electrode with ultrapure water, and drying the solid electrode with nitrogen to obtain the manganese oxide/graphene modified electrode.
TABLE 1
As shown in fig. 1, fig. 1 is a scanning electron microscope photograph of the manganese oxide/graphene modified electrode obtained in example 1, and it can be seen that manganese oxide particles and graphene sheets are tightly combined together, and the strips dispersed to 200nm uniformly cover the surface of the manganese oxide/graphene modified electrode.
Example 1 the current-voltage curve of M second salicylic acid mixed solutions in step 2) is shown in FIG. 2b, the working curve is shown in FIG. 2a, and the linear regression equation based on the working curve is Ii(μA)＝4.54+0.18Ci(μmol/L),(Ci: 8-400 μmol/L, R ═ 0.976), the lower limit of detection is 8 μmol/L. Example 1, the calibration plant tissue in step 3) was tomato, the open circuit voltage of the calibration plant tissue in example 1 was 0.7V, and the resistance measured by ac impedance based on the open circuit voltage was 988.5 Ω. The open circuit voltage of the liquid after the correction of the plant tissue juicing in example 1 was 0.6V, and the resistance measured by ac impedance based on the open circuit voltage was 63.98 Ω. Therefore, the coefficient K was 0.065. Corrected Ii'＝K(a+bCi) Is Ii'(μA)＝0.293+0.0116Ci(μmol/L),(Ci: 8-400 mu mol/L), the lower limit of detection is 8 mu mol/L. Example 1 detection of plant tissue as tomato in step 4), IDetection of0.39 muA, and substitution IiAfter neutralization to give CiThe concentration was 8.5. mu. mol/L.
As shown in fig. 3, fig. 3 is a scanning electron microscope photograph of the manganese oxide/graphene modified electrode obtained in example 2, and it can be seen that manganese oxide particles and graphene sheets are tightly combined together, and the manganese oxide particles are dispersed into 200nm small spheres and uniformly cover the surface of the manganese oxide/graphene modified electrode.
Example 2, in step 2), the current-voltage curves of M second salicylic acid mixed solutions are shown in fig. 4b, the working curves are shown in fig. 4a, and the linear regression equations based on the working curves are 2, which are: i isi(μA)＝106.0+0.13Ci(μmol/L),(Ci：90～1000μmol/L，R＝0.998)；Ii(μA)＝9.23+1.11Ci(μmol/L),(Ci: 2 to 90. mu. mol/L, R0.991), the lower limit of detection is 2. mu. mol/L. Example 2, the corrected plant tissue in step 3) was tomato, the open circuit voltage of the corrected plant tissue in example 2 was 0.05V, and the resistance measured by ac impedance based on the open circuit voltage was 405 Ω. Correction in example 2The open circuit voltage of the liquid after the plant tissue was extracted was 0.07V, and the resistance measured by ac impedance based on the open circuit voltage was 85.5 Ω. Therefore, the coefficient K is 0.211. Corrected Ii'＝K(a+bCi) Is Ii'(μA)＝22.3+0.027Ci(μmol/L),(Ci：90～1000μmol/L)；Ii'(μA)＝1.94+0.233Ci(μmol/L),(Ci: 2-90 mu mol/L), the lower limit of detection is 2 mu mol/L. Example 2 detection of plant tissue as tomato in step 4), IDetection ofIs 2.78 muA, and is substituted into IiAfter neutralization to give CiIt was 3.62. mu. mol/L.
As shown in fig. 5, fig. 5 is a scanning electron microscope photograph of the manganese oxide/graphene modified electrode obtained in example 3, and it can be seen that manganese oxide particles and graphene sheets are tightly combined together, and the sheets dispersed to 100nm uniformly cover the surface of the manganese oxide/graphene modified electrode.
Example 3, the current-voltage curves of M second salicylic acid mixed solutions in step 2) are shown in fig. 6b, the working curves are shown in fig. 6a, and the linear regression equations based on the working curves are 2, which are: i isi(μA)＝59.64+0.16Ci(μmol/L),(Ci：100～600μmol/L，R＝0.996)；Ii(μA)＝7.14+0.68Ci(μmol/L),(Ci: 2 to 100. mu. mol/L, R0.993), the lower limit of detection is 2. mu. mol/L. Example 3 the calibration plant tissue in step 3) was tomato, the open circuit voltage of the calibration plant tissue in example 3 was 0.8V, and the resistance measured by ac impedance based on the open circuit voltage was 798.6 Ω. The open circuit voltage of the liquid after the correction of the plant tissue juicing in example 3 was 0.62V, and the resistance measured by ac impedance based on the open circuit voltage was 32.15 Ω. Therefore, the coefficient K is 0.04. Corrected Ii'＝K(a+bCi) Is Ii'(μA)＝2.4+0.0064Ci(μmol/L),(Ci：100～600μmol/L)；Ii'(μA)＝0.287+0.027Ci(μmol/L),(Ci: 2 to 100. mu. mol/L) and the lower limit of detection is 2. mu. mol/L. Example 3 detection of plant tissue as tomato in step 4), IDetection of0.35 μ A, substituted into IiAfter neutralization to give CiIs 2.3 mu mol/L。
Interference test of manganese oxide/graphene modified electrode
① preparing 6 groups of solutions with pH of 7.0 and salicylic acid concentration of 0.1mmol/L with phosphoric acid buffer solution as base solution, adding glucose into the solutions respectively, and stirring to obtain solutions with glucose concentrations of 0.001, 0.01,0.05,0.1,0.5, and 1 mmol/L;
② the manganese oxide/graphene modified electrode, the saturated calomel electrode and the platinum sheet electrode are connected with the electrochemical workstation and are respectively inserted into 6 groups of prepared solutions, and the current-voltage curve is tested by adopting a differential pulse voltammetry method, so that the salicylic acid oxidation peak of each group is at the same position, the salicylic acid peak current has no great change, the anti-interference capability is proved, and the manganese oxide/graphene modified electrode is not interfered by other biological ions when detecting salicylic acid.
The same test plant tissue (tomato) is repeatedly tested for 2 times according to the method in the embodiment 1, and the difference of the salicylic acid concentration of the test plant tissue obtained in 2 times is not more than 0.1 mu mol/L, so that the manganese oxide/graphene modified electrode and the method based on the manganese oxide/graphene modified electrode have good reproducibility, repeatability and stability.
The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.
Claims (10)
1. A preparation method of a manganese oxide/graphene modified electrode is characterized by comprising the following steps:
① cleaning the surface of the solid electrode;
② potassium permanganate, potassium chloride, graphite and ultrapure water are uniformly mixed to obtain a mixed solution, a solid electrode, a reference electrode and a counter electrode are inserted into the mixed solution, electrodeposition treatment is carried out by adopting a cyclic voltammetry, the potential range of the cyclic voltammetry is set to-1.5V, the voltage scanning speed is 0.05-0.2V/s, continuous scanning is carried out for 30-100 circles, the solid electrode is taken out and washed by the ultrapure water and dried to obtain the manganese oxide/graphene modified electrode, wherein the concentration of the potassium permanganate in the mixed solution is 0.1-0.5 mol/L, the concentration of the potassium chloride in the mixed solution is 0.1-0.5 mol/L, and the concentration of the graphite in the mixed solution is 5-10 mg/mL.
2. The method according to claim 1, wherein in the step ①, the cleaning method comprises polishing the solid electrode with alumina powder to a mirror surface, and then sequentially subjecting the mirror surface to ultrasonic treatment with 30-80 wt% nitric acid aqueous solution, ultrapure water, absolute ethanol and ultrapure water for 5-15 min to remove organic and inorganic dirt;
in the step ①, the solid electrode is a glassy carbon electrode;
in the step ②, the drying is blowing dry with nitrogen;
in the step ②, the graphite is graphite powder, graphene, carbon nanotubes or graphite prepared by an electrochemical stripping method, and the method for preparing graphite by the electrochemical stripping method comprises the steps of connecting a graphite rod to each of the positive electrode and the negative electrode of a voltage-stabilized direct-current power supply, immersing 2 graphite rods in dilute sulfuric acid, setting the voltage to be 5-15V and the current to be 100-400 mA, keeping for 5-20 minutes, carrying out vacuum filtration, and drying to obtain the graphite, wherein the concentration of the dilute sulfuric acid is 3-5 mol/L, and the diameter of each graphite rod is 0.5-3 mm.
3. A method for detecting salicylic acid in plant tissue, comprising the steps of:
1) preparing N first salicylic acid mixed solutions with different pH values, connecting a working electrode, a reference electrode and a counter electrode with an electrochemical workstation, respectively inserting the working electrode, the reference electrode and the counter electrode into each first salicylic acid mixed solution, testing by adopting a differential pulse voltammetry to obtain voltage-current curves of the N first salicylic acid mixed solutions, and taking a pH value corresponding to the maximum intensity current in the N voltage-current curves as X, wherein N is 6-10, and the preparation method of each first salicylic acid mixed solution comprises the following steps: mixing a phosphoric acid buffer solution with salicylic acid;
2) preparing M second salicylic acid mixed solutions with the pH value of X, wherein M is 10-30, and the concentration of salicylic acid in each second salicylic acid mixed solution is Ci，i＝1……M，CmaxIs the maximum value of the concentration of salicylic acid in M second salicylic acid mixed solutions, CminIs the minimum value of the concentration of the salicylic acid in the M second salicylic acid mixed solutions,
connecting a working electrode, a reference electrode and a counter electrode with an electrochemical workstation and respectively inserting the working electrode, the reference electrode and the counter electrode into each second salicylic acid mixed solution, testing the M second salicylic acid mixed solutions by adopting a differential pulse voltammetry method to obtain current-voltage curves of the M second salicylic acid mixed solutions, and obtaining the concentration C of each salicylic acid through the current-voltage curves of the M second salicylic acid mixed solutionsiCorresponding salicylic acid response characteristic peak, and the current I corresponding to the salicylic acid response characteristic peakiWith salicylic acid concentration CiDrawing curves for horizontal and vertical coordinates, fitting to obtain a working curve and a linear regression equation I of the working curvei＝a+bCiWherein a and b are constants, and the preparation method of the second salicylic acid mixed solution comprises the following steps: mixing a phosphoric acid buffer solution with salicylic acid;
3) obtaining a coefficient K, and linearly regressing equation I with the coefficient K through formula (1)i＝a+bCiAnd (3) correcting:
Ii'＝K(a+bCi) In the formula (1),
wherein, Ii' for the corrected current, the coefficient K is obtained by: preparing a substance of the same kind as the detected plant tissue as a correction plant tissue, and obtaining 2 resistances of the correction substance by using the squeezed liquid of the correction plant tissue and the correction plant tissue as the correction substance respectively, wherein the resistance of each correction substance is obtained by the following method: connecting a working electrode, a reference electrode and a counter electrode with an electrochemical workstation and respectively inserting the working electrode, the reference electrode and the counter electrode into the correction substance, obtaining the open-circuit voltage of the correction substance by adopting an open-circuit potential-time curve method, and obtaining the resistance by adopting alternating current impedance measurement based on the open-circuit voltage; will be provided withCorrecting the resistance of the liquid after the plant tissue is juiced to be divided by the resistance of the corrected plant tissue to obtain the coefficient K;
4) connecting the working electrode, the reference electrode and the counter electrode with an electrochemical workstation, and inserting the working electrode, the reference electrode and the counter electrode into the detected plant tissue to obtain a DPV curve of the detected plant tissue, wherein the current value corresponding to the peak of the DPV curve is IDetection ofIs shown byDetection ofSubstituting into I in formula (1)i', obtaining CiTo measure the salicylic acid concentration of plant tissues.
4. The method as claimed in claim 3, wherein in the step 1), the pH of the N first salicylic acid mixed solutions comprises neutrality, acidity and alkalinity.
5. The method according to claim 4, wherein in step 1), N is 7, and the pH of the N first salicylic acid mixed solutions is 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0.
6. The method according to claim 5, wherein in the step 1), the concentration of salicylic acid in the first salicylic acid mixed solution is 0.1-1 mmol/l.
7. The method of claim 6, wherein in step 2), Cmin≤CTo be treated≤CmaxWherein, CTo be treatedTo detect the concentration of salicylic acid in plant tissue;
in the step 2), testing M second salicylic acid mixed solutions by adopting a differential pulse voltammetry method according to the sequence of salicylic acid concentration from low to high;
in the step 2), the number of the linear regression equations is at least 1.
8. The method according to claim 7, wherein the potential range of the differential pulse voltammetry is set to 0-1.4V; the potential increment is set to be 0.001-0.05V, the amplitude is set to be 0.01-0.05V, the pulse width is set to be 0.005-0.25 s, and the pulse period is set to be 0.01-0.5 s.
9. The method of claim 8, wherein the working electrode is the manganese oxide/graphene modified electrode, a glassy carbon electrode, a gold electrode, or a conductive glass.
10. The method of claim 9, wherein the reference electrode is a saturated calomel electrode, an Ag/AgCl electrode, or a mercury/mercurous sulfate electrode and the counter electrode is a platinum sheet electrode.
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