Electrochemical and spectroscopic analysis methods for internal components of plants, hydrogels using aqueous solvents or aqueous electrolytes as a medium, and electrochemical and spectroscopic analysis kits

Abstract

To provide a method for non-destructively extracting or detecting plant internal substances, using a phenomenon (leaching) that the plant internal substances leaches in a transepidermal manner from wet leaves.SOLUTION: An electrochemical analysis method for internal constituents of a plant comprises: extracting internal constituents of a plant into an aqueous electrolyte solution by immersing a diagnosis target portion of a growing plant in the aqueous electrolyte solution; and performing electrochemical measurement of the aqueous electrolyte solution containing extracted constituents. A spectroscopic analysis method for internal constituents comprises: extracting internal constituents of a plant into an aqueous solvent by immersing a diagnosis target portion of a growing plant in the aqueous solvent; and measuring an aqueous solution containing extracted constituents by ultraviolet / visible spectroscopy or fluorescence spectroscopy.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for electrochemical and spectroscopic analysis of internal components of plants. [Background technology]

[0002] When agricultural crops such as vegetables, fruit trees, and rice are infected or damaged by pests, the impact on farmers is severe, with reduced yields and reduced quality. One survey reported that yields could be reduced by 20 to 40%.

[0003] Smart agriculture, which uses information and communication technology (ICT), is expected to solve these problems. In smart agriculture, crops are managed based on environmental monitoring data obtained using various sensors. However, while this method can identify areas of damage or infection to a certain extent using data measured at several locations within a facility, there are also areas where damage cannot be detected, which can result in pesticide spraying over a wider area than necessary.

[0004] When a plant is infected or damaged by a pest, signal substances produced in the affected area transmit information about the infection or damage throughout the plant, providing resistance to the entire plant, including healthy leaves, and suppressing secondary infection by pests. Among these signal substances produced by plants, salicylic acid is produced nonspecifically within the plant from the early stages of infection, regardless of the type of pathogen or the location of infection. It also possesses autofluorescence, which is advantageous for spectroscopic analysis. Detection of salicylic acid could potentially enable early detection of plant pathogen infection. Furthermore, spraying fungicides during the early stages of plant pathogen infection could potentially provide significant therapeutic benefits without the need for widespread fungicide spraying.

[0005] As a method for eluting metabolites from within plants, there have been reports of a method for evaluating the allelopathic effects of components eluted from fallen leaves, utilizing the phenomenon of transepidermal elution of plant substances from leaves wetted by natural rain or fog (leaching) (Non-Patent Documents 1 and 2). For example, in the sandwich method described in Non-Patent Document 2, fallen leaves are sandwiched between two layers of agarose sheets, and plants are grown on the surface of the agarose, allowing the evaluation of how eluted components from fallen leaves affect plant growth. 2 It has been reported that by sandwiching fallen leaves (50 mg) of various plants in 0.5-1.0% agarose gel per cell, fallen leaves containing components that promote the growth of lettuce roots and hypocotyls could be identified. However, no method has been reported to detect signal substances such as salicylic acid that are produced in plants at the early stage of plant pathogen infection. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Weed Research, Vol.43, p.258-266 (1998) [Non-patent document 2] Weed Biology and Management Vol.4, p.19-23 (2004) Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a method for non-destructively extracting or detecting substances in plants using leaching. [Means for solving the problem]

[0008] The present invention comprises the following items. [1] A method for electrochemically analyzing the internal components of a plant body, comprising the steps of: immersing a target part of a growing plant body in an aqueous electrolyte to extract the internal components of the plant body into the aqueous electrolyte; and performing electrochemical measurements of the aqueous electrolyte containing the extracted components. [2] A method for electrochemically analyzing internal components of a plant body, comprising the steps of: immersing a target part of a growing plant body in an aqueous solvent to extract the internal components of the plant body into the aqueous solvent; mixing an aqueous electrolyte with the aqueous solvent containing the extracted components; and performing electrochemical measurements of the mixed solution of the aqueous solvent containing the extracted components and the aqueous electrolyte. [3] A method for electrochemically analyzing the internal components of a plant body, comprising the steps of attaching a hydrogel containing an aqueous electrolyte solution as a medium to the target area of ​​a growing plant body, extracting the internal components of the plant body into the hydrogel, and contacting an electrode with the hydrogel to perform electrochemical measurement of the hydrogel containing the extracted components.

[0009] [4] A method for spectroscopic analysis of internal components of a plant body, comprising the steps of extracting the internal components of the plant body into an aqueous solvent by immersing the part of the plant body to be diagnosed during growth in the aqueous solvent, and measuring the aqueous solvent containing the extracted components using ultraviolet-visible spectroscopy or fluorescence spectroscopy. [5] A method for spectroscopic analysis of internal components of a plant body, comprising the steps of attaching a hydrogel containing an aqueous solvent to the target area of ​​a growing plant body, extracting the internal components of the plant body into the hydrogel, and measuring the hydrogel containing the extracted components using ultraviolet-visible spectroscopy or fluorescence spectroscopy.

[0010] [6] A hydrogel using an aqueous solvent or aqueous electrolyte as a medium, which is used for electrochemical or spectroscopic analysis of target areas of a growing plant body. [7] An electrochemical analysis kit consisting of a hydrogel with an aqueous electrolyte as a medium and electrodes, used for electrochemical analysis of target areas of growing plants. [8] A spectroscopic analysis kit consisting of a hydrogel with an aqueous solvent as a medium, and a light detection sensor, used for spectroscopic analysis of target areas of growing plants. [Effects of the Invention]

[0011] According to the present invention, by immersing the part of a growing plant body to be diagnosed in an aqueous solvent or aqueous electrolyte, substances within the plant body can be detected or measured non-destructively using electrochemical measurement, ultraviolet-visible spectroscopy, or fluorescence spectroscopy.

[0012] According to the present invention, a hydrogel containing an aqueous solvent or aqueous electrolyte as a medium is attached to the diagnostic target area of ​​a growing plant body, and substances within the plant body are extracted into the hydrogel.Substances within the plant body can then be detected or measured non-destructively by performing electrochemical measurement, ultraviolet-visible spectroscopic analysis, or fluorescent spectroscopic analysis of the hydrogel.

[0013] When a plant is infected with a pathogen, salicylic acid accumulates in the plant body at the early stage of infection. Detection of this by electrochemical or spectroscopic analysis allows for early detection of infection. Therefore, the present invention can prevent widespread contamination by pathogens and the spread of disease in agricultural crops. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows the experimental procedure (Figure 1a) and the amount of potassium ions (K+) (ppm) (Figure 1b) and sodium ions (Na+) (ppm) (Figure 1c) when tomato leaves were immersed in an aqueous electrolyte solution prepared by mixing various surfactants in phosphate-buffered saline (PBS) at a final concentration of 0.3 mg / mL for 18 hours to extract the material, and the amount of potassium ions (K+) (ppm) (Figure 1b) and sodium ions (Na+) (ppm) was quantified using an electrochemical ion sensor. [Figure 2] Figure 2 shows the experimental procedure (Figure 2a) when a potassium ion sensor coated with agarose gel using PBS as a medium was attached to the surface of a tomato leaf and the eluted potassium ions were monitored over time, as well as the potential response of the potassium ion sensor, which was investigated by adding known concentrations of potassium ions to PBS as a preliminary study (Figure 2b), and the change over time (Figure 2c). [Figure 3]Figure 3 shows the experimental procedure (Figure 3a) and graphs showing the amount of glucose (μM) (Figure 3b) and lactic acid (μM) (Figure 3c) when tomato leaves were immersed in an aqueous electrolyte solution prepared by mixing various surfactants in PBS at a final concentration of 0.3 mg / mL for 18 hours for extraction, and then a colorimetric reagent was added and quantified using visible light spectroscopy. [Figure 4] Figure 4 shows the fluorescence spectrum of salicylic acid extracted into an agarose gel sheet using ultrapure water as a solvent, attached to the surface of a tomato leaf. DETAILED DESCRIPTION OF THE INVENTION

[0015] The electrochemical analysis method and spectroscopic analysis method for the internal components of a plant body according to the present invention will be described in detail below. [Electrochemical analysis method] One embodiment of the method for electrochemically analyzing internal components of a plant of the present invention comprises the steps of: immersing a target portion of a growing plant in an aqueous electrolyte to extract the internal components of the plant into the aqueous electrolyte; and performing electrochemical measurement of the aqueous electrolyte containing the extracted components. Alternatively, the method comprises the steps of immersing a target portion of a growing plant in an aqueous solvent to extract the internal components of the plant into the aqueous solvent; mixing the aqueous solvent containing the extracted components with the aqueous electrolyte; and performing electrochemical measurement of the mixed solution of the aqueous solvent containing the extracted components and the aqueous electrolyte.

[0016] The present invention aims to determine whether or not a plant is responding to infection by a pathogen or damage by a pest by immersing a target portion of the plant in an aqueous electrolyte or an aqueous solvent, without destroying the plant, and detecting or measuring metabolites dissolved from the target portion into the aqueous electrolyte or the aqueous solvent.

[0017] The diagnostic target area is preferably a part of the plant that excretes many internal components, such as a leaf, which is flat, soft, and easy to handle when immersed in an aqueous electrolyte or aqueous solvent. The veins that form the veins between the network of veins in leaves supply water and nutrients to mesophyll cells and also transport metabolites. Therefore, many metabolites are eluted from the leaves. Alternatively, the roots and stems of plants, which have active cell division at their growing points and are involved in internal metabolism, are also suitable diagnostic target areas.

[0018] Plants that can be used in electrochemical analytical methods include cultivated crops, such as root vegetables (radish, carrot, potato, taro, turnip, burdock, lotus root, yam), leafy vegetables (Chinese cabbage, cabbage, spinach, lettuce, leeks, onions, komatsuna, bok choy, butterbur, mitsuba, chrysanthemum, mizuna, celery, asparagus, cauliflower, broccoli, chives, garlic), fruit vegetables (cucumber, eggplant, tomato, bell pepper, pumpkin, sweet corn, green beans, snow peas, green peas, broad beans, edamame), spicy vegetables (ginger), and fruit vegetables (strawberries, melon, watermelon).

[0019] Components inside the plant include metabolites such as sucrose, glucose, lactic acid, hydrogen peroxide, ethylene, jasmonic acid, abscisic acid, salicylic acid, and methyl salicylate, as well as ionic species such as sodium ions, potassium ions, and nitrate ions. Of these, from the perspective of early detection of disease infection, the present invention focuses on salicylic acid, which has autofluorescence and is produced inside the plant at the early stage of pathogen infection.

[0020] The aqueous electrolyte solution is an aqueous solution containing an electrolyte such as sodium chloride or potassium chloride.

[0021] The surfactant may be any of anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants.

[0022] Examples of anionic surfactants include carboxylates (such as soap), sulfonates (linear alkylbenzenesulfonates, α-olefin sulfonates, α-sulfofatty acid methyl ester salts, and sulfosuccinates), and sulfates (alkyl sulfates and polyoxyethylene alkyl sulfates). Of these, sodium dodecyl sulfate (SDS) is preferred. Cationic surfactants include amine salt types and quaternary ammonium salt types.

[0023] Amphoteric surfactants are surfactants that, when dissolved in water, exhibit the properties of an anionic surfactant in the alkaline range and the properties of a cationic surfactant in the acidic range. Specific examples include alkyldiaminoethylglycine hydrochloride and alkylpolyaminoethylglycine hydrochloride.

[0024] Nonionic surfactants include esters (glycerin fatty acid esters, sorbitan fatty acid esters, sucrose fatty acid esters) and ethers (polyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers). Among these, Tween® 20 (polyoxyethylene sorbitan monolaurate (Tris-buffered saline)) and Triton® X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (emulsifier)) are preferred.

[0025] Among the surfactants, nonionic surfactants such as Tween 20 and Triton X-100, which are spreaders for agricultural chemicals, are particularly suitable.

[0026] Buffer solutions typically include phosphate-buffered saline (PBS) and Hepes buffer (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid). Buffer solutions adjust the pH or salt conditions in aqueous solvents and aqueous electrolytes, and are primarily added for electrochemical measurements.

[0027] The buffer in the aqueous electrolyte serves as an electrolyte. The concentration of the electrolyte in the aqueous electrolyte is usually 1 μM to 1 M, and preferably 1 μM to 10 mM. The pH of the aqueous electrolyte is 6 to 8.

[0028] Instead of the aqueous electrolyte, a specific aqueous solvent may be used. Examples of the aqueous solvent include those that do not contain ions, such as pure water (distilled water, salt water, purified water) and ultrapure water. However, since electrochemical measurements cannot be performed without an electrolyte, aqueous solvents for electrochemical measurements should contain sodium ions (Na + ), magnesium ions (Mg 2+ ), potassium ions (K + ), calcium ions (Ca 2+ ), nitrate ions (NO3 - ), sulfate ions (SO4 2- ), or chloride ions (Cl - In order to improve the extraction efficiency, an electrolyte such as ethanol must be added to the electrolyte and aqueous solvent. In addition to the surfactant and buffer solution, alcohols may be added to the electrolyte and aqueous solvent. Ethanol is particularly preferred as the alcohol.

[0029] Another embodiment of the electrochemical analysis method for internal components of a plant body of the present invention comprises the steps of attaching a hydrogel containing an aqueous electrolyte solution as a medium to the diagnostic target area of ​​a growing plant body and extracting the internal components of the plant body into the hydrogel, and contacting an electrode with the hydrogel and performing electrochemical measurement of the hydrogel containing the extracted components.

[0030] A hydrogel has a three-dimensional structure and is a water-insoluble polymeric substance that encapsulates water. The hydrogel of the present invention may have a water content of about 70 to 99% and may be used in electrochemical or spectroscopic analysis. As long as the hydrogel uses an aqueous electrolyte as a medium, for example, paper containing a moderate amount of water can be used to achieve the same effect. Specific examples of the hydrogel include agarose, gelatin, xanthan gum, gellan gum, sclerotium gum, gum arabic, tragacanth gum, karaya gum, cellulose gum, tamarind gum, guar gum, locust bean gum, glucomannan, chitosan, carrageenan, quince seed, galactan, mannan, starch, dextrin, curdlan, casein, pectin, collagen, fibrin, peptides, chondroitin sulfates such as sodium chondroitin sulfate, hyaluronic acid, hyaluronates such as sodium hyaluronate, alginic acid, alginates, and natural polymers such as derivatives thereof; methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, ... Examples of suitable polymers include cellulose derivatives such as cellulose and their salts; poly(meth)acrylic acids such as polyacrylic acid, polymethacrylic acid, and acrylic acid-alkyl methacrylate copolymers and their salts; and synthetic polymers such as polyvinyl alcohol, polyethylene glycol di(meth)acrylate polymers (PPEGDA, PPEGDM), polyhydroxyethyl methacrylate, polyacrylamide, poly(N,N-dimethylacrylamide), poly2-acrylamido-2-methylpropanesulfonic acid, poly(N-isopropylacrylamide), polyvinylpyrrolidone, polystyrene sulfonic acid, polyethylene glycol, carboxyvinyl polymers, alkyl-modified carboxyvinyl polymers, maleic anhydride copolymers, polyalkylene oxide resins, crosslinked poly(methyl vinyl ether-alt-maleic anhydride) and polyethylene glycol, crosslinked polyethylene glycol, N-vinylacetamide crosslinked acrylamide crosslinked acrylamide crosslinked acrylamide, and crosslinked starch-acrylate graft copolymer.Among these hydrogels, agarose gel is preferred because it has a pore size of 0.1 to 1 μm, which is large enough not to affect the physical diffusion of the extract, is electrostatically neutral, and is non-toxic.

[0031] The electrochemical analysis kit of the present invention comprises a hydrogel containing an aqueous electrolyte solution as a medium, and electrodes, which are used for electrochemical analysis of a target site of a growing plant.

[0032] Electrochemical measurements typically involve measuring the current flowing due to an electrochemical reaction that occurs when a voltage is applied to an electrode immersed in a test solution, or the electrode potential when the electrochemical reaction is in equilibrium at the electrode / test solution interface. In the present invention, internal plant components dissolved in an aqueous electrolyte solution, specifically metabolites containing signal substances (e.g., salicylic acid) and ionic species, are detected or measured from the electrochemical reaction at an electrode in contact with a hydrogel using an aqueous electrolyte solution as a medium. In the present invention, a two-electrode system is used to measure ionic species, and the potential difference between the working electrode and the reference electrode is measured to detect changes in the working electrode potential. Alternatively, when measuring the target based on the current response, a three-electrode system (using three electrodes: a working electrode, a counter electrode, and a reference electrode) is used, and the current flowing between the working electrode and the counter electrode is measured while controlling the potential of the working electrode relative to the reference electrode. This allows electrochemical analysis of internal plant components.

[0033] In the case of a two-electrode type, it consists of a working electrode for detecting the analyte and a reference electrode that indicates the reference potential. In the case of a three-electrode type, it consists of a working electrode for detecting the analyte, a counter electrode that forms a circuit while preventing current from passing through the reference electrode, and a reference electrode that indicates the reference potential. The reference electrode is an electrode that takes the reference potential, such as an Ag / AgCl electrode. In the present invention, since electrochemical measurements are performed in an aqueous system, electrode materials that are less likely to cause water electrolysis (wide potential window) are used for the working electrode and counter electrode. Specific examples include carbon, gold, platinum, diamond, indium tin oxide (ITO), nickel, and conductive polymers (poly(3,4-ethylenedioxythiophene), polyacetylene, polypyrrole, polythiophene, polybithiophene, polyisothiophene, polydodecylthiophene, polyisonite thiophene, poly-3-hexylthiophene, polyanions, polyisothianaphthene, polythiazyl, polyphenylene, polyfluorene, polydiacetylene, polyacene, polyparaphenylene, polythienylenevinylene, and polyphenylene sulfide). To selectively measure plant substances, an extract reactant may be immobilized on the surface of the working electrode. Extract reactants react with plant extracts, such as enzymes, antibodies, ionophores, nucleic acids (DNA, RNA), artificial receptors, cells, microorganisms, tissues, and organs. Enzymes, antibodies, ionophores, nucleic acids (DNA, RNA), and artificial receptors are preferred because they react selectively with the extracted components. These extract reactants may be used alone or in combination. Here, the term "artificial receptor" refers to a compound that forms chemical interactions (e.g., oxidation-reduction reactions, coordinate bonds, hydrogen bonds, van der Waals forces) with the extract components.

[0034] In the electrochemical analysis kit of the present invention, electrodes are placed in contact with the hydrogel, and the extract is detected or measured electrochemically. In this case, the electrochemical analysis kit may have an integrated structure in which the surface of the electrochemical sensor is coated with the hydrogel, or the electrochemical sensor may be prepared separately and brought into contact with the hydrogel attached to the target site of diagnosis, such as a leaf, for electrochemical measurement.

[0035] [Spectroscopic analysis method] Analysis of internal components of a plant of the present invention can also be carried out by a spectroscopic method. One embodiment of the method for spectroscopic analysis of internal components of a plant of the present invention comprises the steps of extracting the internal components of the plant into an aqueous solvent by immersing a target site of the plant during growth in the aqueous solvent, and measuring the aqueous solution containing the extracted components by ultraviolet-visible spectroscopy or fluorescence spectroscopy.

[0036] The plant and aqueous solvent used in the spectroscopic analysis method are the same as those used in the electrochemical analysis method. Unlike the electrochemical analysis, the spectroscopic analysis does not require the addition of an electrolyte to the aqueous solvent.

[0037] In UV-visible spectroscopy, a sample is irradiated with light in the UV to visible range (200-800 nm), and the transmitted, reflected, or absorbed light is detected to obtain a spectrum. The peak intensity of the spectrum obtained indicates the concentration of the sample, and the peak wavelength allows the sample to be identified.

[0038] If the extracted component itself does not have distinctive UV-visible spectral characteristics, there is a method that enables UV-visible spectroscopic measurement by using a chemical reaction with a colorimetric reagent. Colorimetric reagents consist of a pigment, or a pigment and a catalyst. If the UV-visible spectral characteristics change due to a direct reaction between the extracted component and the pigment, only the pigment is used. If the extracted component and the pigment do not react directly, a catalyst is used to induce a change in the UV-visible spectral characteristics.

[0039] In fluorescence spectroscopy, a sample is irradiated with excitation light and the light emitted when the excited electrons return to their ground state is detected. When detecting or measuring extracts that have autofluorescence, such as salicylic acid, it is effective to irradiate the hydrogel with excitation light and measure the fluorescence.

[0040] In the present invention, UV-visible spectroscopy or fluorescence spectroscopy can be used to detect or quantify internal plant components dissolved in an aqueous solvent, specifically metabolites such as salicylic acid and ionic species. As mentioned above, salicylic acid is a component produced in plants from the early stages of disease infection. However, salicylic acid can also be converted to derivatives such as methyl salicylate within the plant. Therefore, the detection of salicylic acid or salicylic acid derivatives indicates that the plant has recently been infected with a disease, but that action is required.

[0041] Another embodiment of the spectroscopic analysis method for internal components of a plant body of the present invention comprises the steps of attaching a hydrogel containing an aqueous solvent as a medium to the diagnostic target area of ​​a growing plant body, extracting the internal components of the plant body into the hydrogel, and measuring the hydrogel containing the extracted components using ultraviolet-visible spectroscopy or fluorescence spectroscopy.

[0042] The same hydrogels as those used in electrochemical analysis can be used. However, unlike electrochemical measurements, the medium can also be ion-free, such as pure water (distilled water, purified water), ultrapure water, or alcohol-containing water.

[0043] In the spectroscopic analysis of the present invention, a spectroscopic analysis kit having an integrated structure in which the surface of a light detection sensor is coated with a hydrogel may be used, or a photodetector may be prepared separately and brought close to a hydrogel attached to the diagnostic site such as a leaf to measure fluorescence. [Example]

[0044] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. [Electrochemical analysis] The instruments used for the electrochemical analysis are as follows: Compact sodium ion meter (LAQUAtwin Na-11, manufactured by Horiba Advanced Techno Co., Ltd.) Compact potassium ion meter (LAQUAtwin K-11, manufactured by Horiba Advanced Techno Co., Ltd.)

[0045] [Spectroscopic analysis] The instruments and measurement conditions used for the spectroscopic analysis are as follows: (1) Ultraviolet-visible (UV-vis) spectrophotometer Shimadzu UV-3150 Measurement conditions: Scan speed: Medium, Measurement range: 200-800 nm, Sampling pitch: 0.5 nm, Slit width: 0.5 nm Thermo Scientific plate reader (Multiskan Sky T)

[0046] (2) Spectrofluorometer JASCO Corporation FP8600 Measurement conditions: Excitation wavelength 310 nm, measurement range 350-550 nm, excitation bandwidth 10 nm, fluorescence bandwidth 5 nm, scanning speed 100 nm / min

[0047] [Example 1] Extraction test using various solutions 1 mL of ultrapure water or PBS containing or not containing various surfactants at a final concentration of 0.3 mg / mL was prepared (liquids (1) to (8)). Tomato leaves were immersed in these for 18 hours. The extract was then collected and measured for potassium ions (K) using an electrochemical ion sensor. + ) and sodium ions (Na + The amount of extractables (ppm) was measured.

[0048] The surfactants used are shown below. Triton® X-100 (non-ionic surfactant) Tween® 20 (Tris-buffered saline) SDS (sodium dodecyl sulfate)

[0049] The compositions of liquids (1) to (8) are shown below. The concentrations of surfactants added to ultrapure water and PBS were all 0.3 mg / mL. (1) Ultrapure water (2) PBS (phosphate-buffered saline) (3) A mixture of Triton X-100 and ultrapure water (4) Mixture of Triton X-100 and PBS (5) Mixture of Tween 20 and ultrapure water (6) Mixture of Tween 20 and PBS (7) Mixture of SDS and ultrapure water (8) Mixture of SDS and PBS

[0050] The results are shown in Figure 1(b). A positive value for the extraction concentration indicates an increase in the concentration of the ionic species in the extraction solution, while a negative value indicates a decrease. + The amounts of Na extracted were highest in PBS containing Triton X-100 and PBS containing SDS, at 110.0 μM and 116.7 μM, respectively. + The amount of extraction was the highest in the case of SDS-containing PBS, at 166.7 μM.

[0051] [Example 2] Hydrogel-coated K + Electrochemical measurements using sensors Figure 2(a) shows a photograph of the sensor. + A sensor (working electrode) and an Ag / AgCl reference electrode were prepared. + The sensor was prepared by dropping a solution of extract reactant onto the surface of a carbon electrode and drying it. The extract reactant solution was prepared by dissolving 2 mg of valinomycin (Fujifilm Wako Pure Chemical Industries, Ltd.), 0.5 mg of potassium tetrakis(4-chlorophenyl)borate (Sigma-Aldrich), 32.7 mg of polyvinyl chloride (Sigma-Aldrich), and 71 μL of bis(2-ethylhexyl) sebacate (Tokyo Chemical Industry Co., Ltd.) in 350 μL of tetrahydrofuran (Tokyo Chemical Industry Co., Ltd.). Figure 2(b) shows the K values ​​measured at various concentrations in PBS containing 0.3 mg / mL Triton X-100. +This shows the potential response of the sensor to ions. A silicone rubber sheet frame (15 mm x 15 mm x 2 mm through-holes, manufactured by AS ONE Corporation) was attached to surround these two electrodes. 0.8 g of agarose powder (gelation temperature 30-31°C, manufactured by Nacalai Tesque, Inc.) was added to 19.2 mL of ultrapure water and dissolved by boiling. This agarose solution was filled into the silicone rubber sheet frame and cooled to room temperature to obtain an agarose gel sheet. Figure 2(c) shows the results of continuous measurement of the potential response of this sensor attached to the surface of a tomato leaf for 48 hours.

[0052] [Example 3] Spectroscopic analysis (visible spectroscopic analysis using colorimetric reagents) (1) Preparation of colorimetric reagents Preparation of each reagent An Oxired probe (a dye for colorimetric measurement, manufactured by Thermo Scientific) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM DMSO solution of the Oxired probe. A 100 U / mL glycerol diluted solution of horseradish peroxidase (Fujifilm Wako Pure Chemical Industries, Ltd.) was prepared. Two types of enzyme solutions were prepared: a PBS solution containing 200 U / mL glucose oxidase (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and a PBS solution containing 200 U / mL lactate oxidase (manufactured by Toyobo Corporation).

[0053] Preparation of colorimetric reagents A colorimetric reagent was prepared by mixing 50 μL of the Oxired probe in DMSO, 10 μL of a glycerol-diluted solution of 100 U / mL horseradish peroxidase, 100 μL of a PBS-diluted solution of 200 U / mL glucose oxidase, or 100 μL of a PBS-diluted solution of 200 U / mL lactate oxidase, with 4.84 mL of PBS.

[0054] (2) Quantitation of glucose and lactic acid by visible spectroscopy Metabolites were extracted from tomato leaves into liquids (1) to (8) using the procedure described in Example 1. The amounts of glucose and lactic acid dissolved in the aqueous solvent were measured by visible spectroscopy (Figure 3(b)). Glucose (Fujifilm Wako Pure Chemical Industries, Ltd.) and lactic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) were diluted with PBS to prepare standard glucose and lactic acid solutions (0, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 1 mM, and 10 mM solutions). 50 μL of each of these standard solutions and the tomato leaf extract solution was dispensed into a 96-well plate. The colorimetric reagent was dispensed into a 96-well plate containing 50 μL of standard solution or extract (100 μL in total), and the extract components and colorimetric reagent were allowed to react at room temperature for 30 minutes. The absorbance at 570 nm was measured using a plate reader (Multiskan Sky T, Thermo Scientific). The glucose and lactate concentrations in the extract were quantified using a calibration curve prepared using the standard solution. The results are shown in Figure 3(b)(c). Glucose was highest in the ultrapure water containing Triton X-100, at 80.7 μM. Lactic acid was highest in the ultrapure water containing Triton X-100, at 163.3 μM.

[0055] [Example 4] Spectroscopic analysis (fluorescence) 0.8 g of agarose powder (gelling temperature 30-31°C, manufactured by Nacalai Tesque, Inc.) was added to 19.2 mL of ultrapure water and dissolved by boiling. This aqueous agarose solution was poured into the gap between two glass plates sandwiching a 1 mm thick Teflon (registered trademark) plate as a spacer. After cooling to room temperature, the glass plates were removed to obtain a 1 mm thick agarose gel sheet. An agarose gel sheet was attached to the surface of a tomato leaf and left to stand for 24 hours. The tomato leaf was then peeled off from the agarose gel sheet, and the fluorescence of salicylic acid and its derivatives extracted into the agarose gel sheet was measured using a spectrofluorometer. Fluorescence was observed in the fluorescence wavelength range of salicylic acid and salicylic acid esters when irradiated with 310 nm excitation light. The resulting fluorescence spectrum is shown in Figure 4. Quantitation using a calibration curve revealed that the concentration of salicylic acid and salicylic acid esters in the aqueous solution contained in the agarose gel was 0.01 mmol / L, calculated as a salicylic acid concentration.

Claims

1. A method for electrochemically analyzing the internal components of a plant, comprising the steps of: immersing a target area of ​​a growing plant in an aqueous electrolyte to extract the internal components of the plant into the aqueous electrolyte; and performing electrochemical measurements of the aqueous electrolyte containing the extracted components.

2. A method for electrochemically analyzing internal components of a plant body, comprising the steps of: immersing a target portion of a growing plant body in an aqueous solvent to extract the internal components of the plant into the aqueous solvent; mixing an aqueous electrolyte with the aqueous solvent containing the extracted components; and performing electrochemical measurements on the mixed solution of the aqueous solvent containing the extracted components and the aqueous electrolyte.

3. A method for electrochemically analyzing the internal components of a plant, comprising the steps of: attaching a hydrogel containing an aqueous electrolyte solution as a medium to the target area of ​​a growing plant body, extracting the internal components of the plant body into the hydrogel; and contacting an electrode with the hydrogel and performing electrochemical measurements of the hydrogel containing the extracted components.

4. A method for spectroscopic analysis of internal components of a plant body, comprising the steps of attaching a hydrogel containing an aqueous solvent to the target area of ​​a growing plant body, extracting the internal components of the plant body into the hydrogel, and measuring the hydrogel containing the extracted components using ultraviolet-visible spectroscopy or fluorescence spectroscopy.

5. A spectroscopic analysis kit consisting of a hydrogel with an aqueous solvent as a medium, used for spectroscopic analysis of target areas of growing plants, and a light detection sensor.

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