CN106404864A - Method for detecting methyl jasmonate in plants on basis of microelectrode biosensing technique - Google Patents

Abstract

The invention provides a method for detecting methyl jasmonate in plants based on microelectrode biosensing technology, which realizes online detection of methyl jasmonate in plants through the microelectrode biosensor of the invention. This method has the characteristics of high accuracy, small sample damage, simple operation, and portability. It can realize the simultaneous acquisition of methyl jasmonate information in plants at the time and space levels, and provides technical support for plant research. Compared with traditional biological identification and chemical detection methods, the sample pre-treatment of this method is simple, the detection site does not need to be separated from the body, minimal damage, no essential damage, and will not cause substantial damage to plants. The detection result is reliable and the sensitivity is high.

Description

Detection method of methyl jasmonate in plants based on microelectrode biosensing technology

technical field

The invention relates to a microelectrode biosensing technology, in particular to a method for detecting methyl jasmonate in plants based on the microelectrode biosensing technology.

Background technique

Methyl jasmonate, a kind of growth regulating substance widely present in plants, has a variety of physiological effects, such as improving the resistance of seedlings to high and low temperature drought, and stimulating the synthesis of antifungal proteins to initiate or accelerate the defense response. Secondary metabolism, inhibition of growth and differentiation, promotion of rooting, maturation and senescence, etc. At present, due to the important physiological effects of methyl jasmonate and its relatively low content in plants, the detection of methyl jasmonate has attracted more and more attention.

At present, the methods used to detect methyl jasmonate in plants are mainly chromatography, including gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, high performance liquid chromatography, capillary liquid chromatography, etc., as well as enzyme-linked immunoassay technology. Most of the instruments used in these methods are complex in operation, high in cost, long in cycle, and highly destructive to samples, and cannot realize online detection of plant growth. Electrochemical analysis is most suitable for the online qualitative and quantitative detection of living plants due to its advantages of convenient operation, rapid response and high sensitivity.

Contents of the invention

The object of the present invention is to provide a method for detecting methyl jasmonate in plants based on microelectrode biosensing technology.

In order to realize the object of the present invention, the present invention at first provides a kind of microelectrode biosensor, and described microelectrode is triangular pyramid shape, long 1-10cm, and its tip part is long 2-20mm, and the side length of the other end cross-section triangle is 1-10mm. 15mm (preferably the cross-section of the other end is an equilateral triangle); the base electrode material of the microelectrode is silicon wafer, stainless steel or plexiglass etc. through insulating treatment; on each face of the base, one end is provided with a The component of the electrochemical workstation, the other end is the electrode material for detection, and the component is connected to the electrode material through a wire; the microelectrode has a three-electrode system, including an Ag/AgCl reference electrode, a platinum counter electrode and a platinum electrode. , graphene and perfluorosulfonic acid resin modified gold working electrode.

The microelectrode biosensor can be prepared as follows:

1) Prepare platinum counter electrode, Ag/AgCl reference electrode and gold working electrode on the substrate by micro-electromechanical technology (MEMS);

2) The gold working electrode is polished on metallographic sandpaper with alumina powder, ultrasonically cleaned with deionized water and ethanol respectively, and then the microelectrode is placed in a 0.5M dilute sulfuric acid solution at a potential of 0-1.5V. Cyclic voltammetry scan to obtain a typical cyclic voltammetry spectrum to ensure that the electrode surface is clean;

3) Prepare a mixed solution of 1 mg/ml graphene oxide and 1 mM chloroplatinic acid, immerse the microelectrode in the above solution, and perform electrochemical reduction deposition by chronoamperometry at a voltage of -0.8V for 0.5 to 2 hours (preferably 1 hour), Then wash with deionized water to obtain a modified graphene/platinum electrode;

4) drip-coat a perfluorosulfonic acid resin solution (preferably 0.02%) with a concentration of 0.005 to 0.1% on the graphene/platinum electrode, and dry it at 50°C to form a film (drying temperature is generally lower than 100°C, which meets the requirements of drying film-forming conditions), and then washed with deionized water to obtain a gold working electrode modified by platinum, graphene and perfluorosulfonic acid resin, thereby completing the preparation of the microelectrode biosensor.

The invention also provides the application of the microelectrode biosensor in online detection of plant methyl jasmonate. The method comprises inserting the microelectrode biosensor into the plant tissue to be tested, and connecting the electrochemical workstation to perform cyclic voltammetry scanning, so as to realize the online detection of methyl jasmonate in the plant tissue.

Before the detection, different concentrations of methyl jasmonate standard solutions were selected for differential pulse voltammetry scanning, and the working curve for microelectrode detection of methyl jasmonate was obtained according to the relationship between the concentration of methyl jasmonate and the current; the detection conditions were: potential 0.9-1.4V, The potential increment is 0.004V, the amplitude is 0.05V, the pulse width is 0.02s, the pulse period is 0.5s, and the rest time is 20s.

Wherein, the methyl jasmonate standard solutions of different concentrations are prepared using 10 mM phosphate buffer at pH 6.0.

The minimum detection limit of the microelectrode biosensor is 0.5 μmol/L, the linear range is 1-100 μM, the linear equation between the concentration of methyl jasmonate solution and the response peak current is: I=0.682+1.71C, and the correlation coefficient is 0.9847; Among them, I represents the current in μA, and C represents the concentration in μM.

The detection objects of the present invention include parts such as roots, stems, leaves or fruits of plants, and the insertion depth is about 0.5cm. The detection target materials are different stages of plant growth, such as the stems of wheat seedlings.

The aforementioned application also includes the step of calibrating the microelectrode biosensor before detection, the specific method is as follows: clean the microelectrode, and then use two parts of methyl jasmonate solutions with standard concentrations (such as 5 μM and 50 μM) respectively. Electrochemical calibration, if the slope deviation between the working curve and the standard curve is within 15%, it is determined that the electrode can work normally.

After correction, the sample was scanned by differential pulse voltammetry. The detection conditions were: potential 0.9-1.4V, potential increment 0.004V, amplitude 0.05V, pulse width 0.02s, pulse period 0.5s, rest time 20s.

Connect the electrochemical workstation, and after the system is activated for 20 minutes, run it stably for 1 minute to 10 minutes to collect the current data and substitute it into the linear equation to obtain the concentration of methyl jasmonate in the plant tissue.

The present invention provides an online method for detecting methyl jasmonate in plants based on microelectrode biosensing technology. The method has the characteristics of high accuracy, small sample damage, simple operation, and portability. The simultaneous acquisition of methyl jasmonate information in plants provides technical support for plant research. Compared with traditional biological identification and chemical detection methods, the sample pre-treatment of this method is simple, the detection site does not need to be separated from the body, minimal damage, no essential damage, and will not cause substantial damage to plants. The detection result is reliable and the sensitivity is high.

Description of drawings

Fig. 1 is a schematic diagram of the structure of the microelectrode biosensor prepared in Example 1 of the present invention; wherein, surface A-working electrode, surface B-reference electrode, surface C-counter electrode.

FIG. 2 is a schematic diagram of the modification of the working electrode of the microelectrode biosensor in Example 1 of the present invention.

detailed description

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

Example 1 Microelectrode biosensor and its preparation method

In this embodiment, micro-electromechanical technology is used to prepare micro-electrodes on an insulating stainless steel substrate. The micro-electrodes are in the shape of a triangular pyramid with a total length of 5 cm and a tip portion of 5 mm in length. The cross-section of the other end is an equilateral triangle with a side length of 3mm; on each surface of the base, one end is provided with an element for connecting to an electrochemical workstation, and the other end is an electrode material for detection, and the element and the electrode material are connected by wires; the microelectrode has Three-electrode system, including Ag/AgCl reference electrode, platinum counter electrode and gold working electrode modified by platinum, graphene and perfluorosulfonic acid resin (Figure 1 and Figure 2).

The gold working electrode modified by platinum, graphene and perfluorosulfonic acid resin can be prepared as follows:

1. Polish the gold working electrode with alumina powder on metallographic sandpaper, and ultrasonically clean it with deionized water and ethanol respectively, then place the microelectrode in 0.5M dilute sulfuric acid solution, and cycle at a potential of 0-1.5V Voltammetric scanning to obtain a typical cyclic voltammetric spectrum to ensure that the electrode surface is clean;

2. Prepare a mixed solution of 1mg/ml graphene oxide and 1mM chloroplatinic acid, immerse the microelectrode in the above solution, perform electrochemical reduction deposition by chronoamperometry at -0.8V for 1h, and then wash with deionized water Clean, obtain the modified graphene/platinum electrode;

3. Drop-coat the perfluorosulfonic acid resin solution with a concentration of 0.02% on the graphene/platinum electrode, dry it at 50°C to form a film, and then wash it with deionized water to obtain a solution composed of platinum, graphene and perfluorosulfonic acid A gold working electrode modified by sulfonic acid resin, thereby completing the preparation of the microelectrode biosensor.

Example 2 Real-time on-line detection of plant methyl jasmonate by microelectrode biosensor

1. Subjects

The plant to be tested was the wheat variety 'Nongda 6812' grown by the Beijing Academy of Agriculture and Forestry Sciences, and the stems of two wheat seedlings were selected as test samples.

After cleaning, insert the microelectrode prepared in Example 1 into the stem of the sample to be tested, and start electrochemical detection.

2. Experimental instruments and consumables

The experimental equipment was Shanghai Chenhua Electrochemical Workstation 760e.

3. Experimental method

(1) Drawing of standard curve:

Before the detection, different concentrations of methyl jasmonate standard solutions were selected for differential pulse voltammetry scanning, and the working curve for microelectrode detection of methyl jasmonate was obtained according to the relationship between the concentration of methyl jasmonate and the current; the detection conditions were: potential 0.9-1.4V, The potential increment is 0.004V, the amplitude is 0.05V, the pulse width is 0.02s, the pulse period is 0.5s, and the rest time is 20s.

Wherein, the methyl jasmonate standard solutions of different concentrations are prepared using 10 mM phosphate buffer at pH 6.0.

The minimum detection limit of the microelectrode biosensor is 0.5 μmol/L, the linear range is 1-100 μM, the linear equation between the concentration of methyl jasmonate solution and the response peak current is: I=0.682+1.71C, and the correlation coefficient is 0.9847; Among them, I represents the current in μA, and C represents the concentration in μM.

(2) Experimental steps:

The microelectrode biosensor is calibrated before detection, and the method is as follows: the microelectrode is cleaned, and then electrochemically calibrated with two parts of methyl jasmonate solutions of standard concentrations (5 μM and 50 μM), if the working curve is consistent with the standard curve If the slope deviation is within 15%, it is judged that the electrode can work normally.

After correction, the sample was scanned by differential pulse voltammetry. The detection conditions were: potential 0.9-1.4V, potential increment 0.004V, amplitude 0.05V, pulse width 0.02s, pulse period 0.5s, rest time 20s.

Connect the electrochemical workstation, and after the system is activated for 20 minutes, run it stably for 1 minute to 10 minutes to collect the current data and substitute it into the linear equation to obtain the concentration of methyl jasmonate in the plant tissue.

4. Experimental evaluation results

Adopt HPLC method and on-line detection analysis of the present invention to analyze the methyl jasmonate content comparison result of two wheat seedling stems in Table 1. It can be seen that the detection results of this method are reliable, and the online dynamic detection and analysis of methyl jasmonate can be realized through the electrochemical workstation, which provides a new method for online research on the content of methyl jasmonate in plants, and realizes the supplement to the existing detection methods.

Table 1 The test results of methyl jasmonate in the stems of wheat seedlings (unit, μg/g)

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. a kind of microelectrode biosensor it is characterised in that described microelectrode be in triangular pyramid, long 1-10cm, its point Divide long 2-20mm, the length of side of other end cross-sectional triangle is 1-15mm；The basal electrode material of described microelectrode is through exhausted Silicon chip, stainless steel or lucite that edge is processed；On each face of described substrate, one end is provided with for connecting electrochemistry work Make the element stood, the other end is the electrode material of detection, is connected by wire between described element and electrode material；Described micro- Electrode has three-electrode system, comprises Ag/AgCl reference electrode, and platinum is to electrode and by platinum, Graphene and perfluorinated sulfonic resin The metal working electrode modified.

2. microelectrode biosensor according to claim 1 is it is characterised in that the system of described microelectrode biosensor Preparation Method comprises the following steps：

1) platinum is prepared to electrode, Ag/AgCl reference electrode and metal working electrode in substrate using micro-electromechanical technology；

2) polish described metal working electrode, deionized water, ethanol are cleaned by ultrasonic respectively, subsequently microelectrode are placed in 0.5M In dilution heat of sulfuric acid, under 0-1.5V current potential, it is circulated voltammetric scan, obtain typical cyclic voltammetric spectrogram it is ensured that electrode table Face is clean；

3) prepare 1mg/ml graphene oxide and 1mM chloroplatinic acid mixed solution, described microelectrode is immersed in above-mentioned solution ,- Under 0.8V voltage, electrochemical reduction is carried out by timing Amperometric and deposit 0.5～2h, then wash, obtain the graphite modified Alkene/platinum electrode；

4) by the perfluor sulfoacid resin solution drop coating of concentration 0.005～0.1% on described graphene/platinum electrode, drying and forming-film, Then washing, obtaining the metal working electrode modified by platinum, Graphene and perfluorinated sulfonic resin, thus completing described microelectrode The preparation of biology sensor.

3. application in plant jasmonate acid methyl esters on-line checking for the microelectrode biosensor described in claim 1 or 2.

4. application according to claim 3 is it is characterised in that insert microelectrode biosensor described in claim 1 or 2 Enter in plant tissue to be measured, connect electrochemical workstation and be circulated voltammetric scan, realize carrying out online jasmine to plant tissue Sour methyl esters detection.

5. application according to claim 4 is it is characterised in that the methyl jasmonate standard choosing variable concentrations before detection is molten Liquid carries out differential pulse voltammetry scanning, obtains microelectrode detection methyl jasmonate according to methyl jasmonate concentration and current relationship Working curve；Testing conditions are：Current potential 0.9-1.4V, current potential increment 0.004V, amplitude 0.05V, pulse width 0.02s, pulse Cycle 0.5s, quiescent time 20s.

6. application according to claim 5 is it is characterised in that described microelectrode biosensor lowest detection is limited to 0.5 μ Mol/L, the range of linearity is 1-100 μM, and methyl jasmonate solution concentration with the linear equation of response peak current is：I=0.682+ 1.71C, relative coefficient is 0.9847；Wherein, I represents electric current, and unit is μ A, and C represents concentration, and unit is μM.

7. the application according to any one of claim 3-6 is it is characterised in that detection object includes the root of plant, stem, Ye Huo Fruit, insertion depth is 0.5cm；The stem of preferably wheat seedling.

8. the application according to any one of claim 3-7 it is characterised in that also include detection before to described microelectrode biology The step that sensor is corrected, concrete grammar is as follows：Microelectrode is carried out, then respectively with the jasmine of two parts of normal concentrations Jasmine acid methyl ester solution carries out electrochemistry calibration, if working curve and slope of standard curve deviation are within 15%, judges electrode Can normal work.

9. application according to claim 8, it is characterised in that carrying out differential pulse voltammetry scanning to sample after correction, is examined Survey condition is：Current potential 0.9-1.4V, current potential increment 0.004V, amplitude 0.05V, pulse width 0.02s, pulse period 0.5s, quiet Only time 20s.

10. application according to claim 9 is it is characterised in that connect electrochemical workstation, after system activation 20min, surely Surely run 1min-10min collection current data and substitute into linear equation, obtain the concentration of methyl jasmonate in plant tissue.