Electrode for measuring plant leaf surface potential

Abstract

To provide an electrode for measuring the leaf surface potential of a plant, capable of closely adhering to the leaf surface even in the presence of hair-like projections. The present invention provides: SOLUTION: The electrode for measuring a plant leaf surface potential is composed of a conductive nano thin film including an elastomer layer and a carbon nanotube layer laminated on at least one surface of the elastomer layer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an electrode for measuring the surface electric potential of a plant leaf. [Background technology]

[0002] Plants respond to their surrounding environment and external stimuli, transmitting information throughout the plant as electrical signals to change their physiological activity. Measuring these signals as bioelectric potentials may enable continuous evaluation of the plant's health.

[0003] Needle electrodes, EEG dish electrodes, and gel electrodes have generally been used to measure bioelectric potentials in plants, but their invasiveness to plants during long-term measurements has not been fully investigated. Invasive electrodes such as needle electrodes cause significant damage to tissue. Wet electrodes such as EEG dish electrodes and ECG gel electrodes are known as non-invasive electrodes (surface electrodes), but the common EEG dish electrodes and hydrogel electrodes have the problem of being highly invasive to plant leaf tissue.

[0004] If we can develop a minimally invasive, flexible electrode that places minimal mechanical or chemical stress on plants, it will be possible to measure the health of plants over a long period of time in real time, which will contribute to improving the efficiency of agricultural production.

[0005] However, the numerous trichomes on the leaves of environmentally friendly crops reduce the contact area between the leaf surface and the thin-film electrode, thereby reducing the apparent adhesion of the electrode to the leaf surface. In other words, maintaining a stable contact state even in the presence of trichomes is important for an electrode that enables long-term measurements.

[0006] Ultra-thin polymer films (nanosheets) made of the conductive polymer PEDOT:PSS have been investigated for measuring surface potential, but they swell in water, making them difficult to use outdoors where they are exposed to wind and rain. Furthermore, they have problems such as poor adhesion to plant surfaces (such as leaves and stems) where trichomes are densely distributed, leading to breakage.

[0007] In Patent Document 1, the present inventors proposed a conductive nano-thin film intended for application to a dielectric elastomer actuator, which includes an elastomer layer and a carbon nanotube layer laminated on at least one of its surfaces. However, no study has been conducted on its application to electrodes for measuring the surface potential of plants. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2023 / 013558 Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention has been made in consideration of the above circumstances, and has as its object to provide an electrode for measuring the plant leaf surface potential that can be closely attached to the leaf surface even in the presence of trichomes. [Means for solving the problem]

[0010] As a result of extensive research to solve the above problems, the inventors discovered that a conductive nano-thin film made by laminating electronically conductive, hydrophobic carbon nanotubes onto an elastomer layer as an ultrathin polymer film electrode can maintain a stable adhesion even in the presence of trichomes, can self-adhere to the leaf surface, is lightweight, has high optical transparency, and enables long-term non-invasive measurement of leaf surface potential, leading to the completion of the present invention. Carbon nanotubes expand and contract due to their fiber network structure, which reduces the inhibition of the flexibility of the elastomer layer and maintains the conformability of the nanosheet, thereby maintaining a stable adhesion even in the presence of trichomes.

[0011] That is, the electrode for measuring the plant leaf surface potential of the present invention is characterized by comprising a conductive nano-thin film including an elastomer layer and a carbon nanotube layer laminated on at least one surface of the elastomer layer. [Effects of the Invention]

[0012] According to the present invention, there is provided a minimally invasive electrode for measuring the electric potential on a plant leaf surface, which can be brought into close contact with the leaf surface even in the presence of trichomes. [Brief explanation of the drawings]

[0013] [Figure 1A] 1 is a photograph of a SWCNT-SBS nanosheet prepared in an example. [Figure 1B] 1 shows the transmission spectra of the SWCNT-SBS nanosheet prepared in the example and a commercially available gel electrode in a wavelength region including the ultraviolet and visible regions. [Figure 2A] This shows the results of observing the adhesion state when an SWCNT-SBS nanosheet (film thickness 485 nm) was attached to the surface of a soybean leaf. (a) is a side photograph, and (b) is a bird's-eye view photograph. [Figure 2B] This shows the results of observing the adhesion state when an SWCNT-SBS nanosheet (320 nm thick) was attached to the surface of a soybean leaf. (a) is a side photograph, and (b) is a bird's-eye view photograph. [Figure 2C] The results of observing the adhesion state when a SWCNT-SBS nanosheet (70 nm thick) was attached to the surface of a soybean leaf are shown. (a) is a side photograph, and (b) is a bird's-eye view photograph. [Figure 2D] This is an overhead photograph showing the results of observing the adhesion state when an SBS nanosheet (film thickness 800 nm) was attached to the surface of a soybean leaf. [Figure 2E] This is an overhead photograph showing the results of observing the adhesion state when an SBS nanosheet (film thickness 30 nm) was attached to the surface of a soybean leaf. [Figure 2F] 1 is a side photograph showing the results of observing the state of adhesion when a polystyrene sheet is attached to the surface of a soybean leaf. [Figure 3A] These are overhead photographs of the leaves of a pothos plant after various electrodes were attached for 14 days. (a) is an EEG dish electrode, (b) is a commercially available gel electrode, and (c) is an SWCNT-SBS nanosheet electrode (film thickness 485 nm). [Figure 3B]These are overhead photographs of gel electrodes attached to the surface of tobacco leaves for 14 days. (a) is a commercially available gel electrode, (b) is a transparent gel electrode, and (c) is an SWCNT-SBS nanosheet electrode (film thickness 480 nm). [Figure 4] (a) shows the results of measuring LIB immediately after and after 14 days of application of a gel electrode, (b) shows the results of an EEG dish electrode, and (c) shows the results of measuring LIB immediately after application of a SWCNT-SBS nanosheet electrode to the leaf surface of a pothos plant. (d) shows the results of comparing the amplitude of LIB in these measurements. [Figure 5A] These are LIB spectra measured when a 485 nm thick SWCNT-SBS film was attached to the surface of a soybean leaf (the LED was turned on and off repeatedly every two hours). (a) shows the LIB immediately after the electrode was attached, and (b) shows the LIB 14 days after attachment. [Figure 5B] The LIB spectra are similar to those shown in Figure 5A, measured when a 320 nm thick SWCNT-SBS film was applied to the surface of soybean leaves (immediately after application and 14 days after application). [Figure 5C] The LIB spectra are similar to those shown in Figure 5A, measured when a 70 nm thick SWCNT-SBS was applied to the surface of soybean leaves (immediately after application and 14 days after application). DETAILED DESCRIPTION OF THE INVENTION

[0014] Hereinafter, embodiments of the present invention will be described in detail.

[0015] In the present invention, the thickness of the conductive nano-thin film, the carbon nanotube layer, and the elastomer layer are considered to be average values ​​determined by the difference in level between the support base and the film to be measured using a measuring device such as a cross-sectional profiler.

[0016] The electrode for measuring the plant leaf surface potential of the present invention comprises a conductive nano-thin film including an elastomer layer and a carbon nanotube layer laminated on at least one surface of the elastomer layer.

[0017] This conductive nano-thin film is lighter than commercially available wet electrodes such as gel electrodes for electrocardiograms and dish electrodes for electroencephalograms, and allows measurements to be made without placing a burden on the plant itself. Because it self-adheres to the leaf surface, no adhesives like the electrode paste used in wet electrodes are required, meaning no discoloration of the leaf surface is observed over long-term measurements, making it highly non-invasive. In other words, it is a minimally invasive, flexible electrode that places little mechanical or chemical burden on plants, making it possible to measure plant health in real time over long periods of time.

[0018] This conductive nano-thin film is gas-permeable and visible light-transmitting. In the visible light region of 400 to 800 nm, it exhibits a light transmittance of, for example, 80% or more at wavelengths of 400 to 500 nm and 600 to 700 nm, which are necessary for plant photosynthesis. Because it is extremely lightweight and has a film thickness on the order of nanometers, it exhibits high transmittance, thereby increasing the light intensity striking the leaf surface. Therefore, the electrode for measuring plant leaf surface potential of the present invention is capable of measuring with high signal intensity. In other words, it is a flexible electrode that does not block light in the wavelength range necessary for transpiration from the leaf surface or plant photosynthesis, and is unlikely to inhibit photosynthetic reactions. Furthermore, it has excellent water resistance and maintains adhesion without the use of adhesives, even on leaf surfaces with numerous trichomes.

[0019] The thickness of the conductive nano-thin film is not particularly limited, but in order of preference, it is less than 1000 nm, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, 100 nm or less, and 80 nm or less.

[0020] Among these, 500 nm or less is preferred, 400 nm or less is more preferred, 300 nm or less is even more preferred, 200 nm or less is even more preferred, 100 nm or less is particularly preferred, and 80 nm or less is most preferred. Numerous trichomes are present on the leaf surfaces of environmentally friendly crops, which reduce the contact area between the leaf surface and the thin-film electrode, thereby reducing the apparent adhesion of the electrode to the leaf surface. When the conductive nanofilm thickness is within the above range, it maintains stable adhesion even in the presence of trichomes, enabling long-term measurements. As the film thickness increases, some areas tend to lift off the leaf surface due to interference from the trichomes, and the conductive nanofilm tends to break when dried. Generally, the bending stiffness of the sheet is proportional to the Young's modulus and the cube of the film thickness. As the conductive nanofilm becomes thicker, gaps form between the electrode film and the trichomes, preventing it from adhering to the leaf surface due to the interference from the trichomes. On the other hand, when the film thickness is smaller, it tends to adhere to the leaf surface, even though some of the film floats. Some of the trichomes on the leaf surface break through the nanosheet, allowing the conductive nanofilm to come into contact with the leaf surface and exhibit adhesion. Furthermore, when the film thickness is reduced to, for example, 100 nm or less, it adheres to the leaf surface without being obstructed by the trichomes. In some cases, the trichomes break through the conductive nanofilm, allowing it to adhere stably to the trichomes for a long period of time. These tendencies affect the ease of potential measurements using conductive nanofilms as electrodes attached to leaves, such as LIB measurements, the accuracy of measurements over long periods of time, and the signal strength.

[0021] A small film thickness provides excellent flexibility, stretching in the thickness direction and surface direction, and good conformability to the leaf surface. On the other hand, a moderate film thickness provides self-supporting properties and ensures strength. Therefore, the conductive nano-thin film has no lower limit on its film thickness, but is preferably 20 nm or more, more preferably 30 nm or more, even more preferably 40 nm or more, and particularly preferably 50 nm or more.

[0022] The material of the elastomer layer in the conductive nano-thin film is not particularly limited, and may be a polymer having elasticity, such as a thermoplastic or thermosetting elastomer.

[0023] Specific examples include styrene-based elastomers, silicone-based elastomers, olefin-based elastomers, urethane-based elastomers, polyester-based elastomers, polyamide-based elastomers, acrylic-based elastomers, rubber-modified epoxy resins, etc. These may be used alone or in combination of two or more.

[0024] Examples of styrene-based elastomers include styrene-butadiene-styrene block copolymer (SBS), styrene-ethylene-butylene-styrene block copolymer (SEBS), styrene-isoprene-styrene block copolymer (SIS), styrene-ethylene-propylene-styrene block copolymer (SEPS, a hydrogenated product of SIS), styrene-ethylene-propylene block copolymer (SEP, a hydrogenated product of styrene-isoprene block copolymer), and styrene-isobutylene-styrene block copolymer (SIBS).

[0025] Silicone-based elastomers are primarily composed of organopolysiloxane, and examples thereof include polydimethylsiloxane, polymethylphenylsiloxane, and polydiphenylsiloxane. They may also be partially modified with vinyl groups, alkoxy groups, or the like. A thin film of organopolysiloxane can be obtained, for example, by treating a base material containing a siloxane compound with a curing agent to polymerize and / or crosslink the material. The curing agent used depends on the type of primary reactive group in the base material. For example, if the base material has a hydrosilyl group as the primary reactive group, a compound having an alkenyl group can be used; for example, if the base material has an alkenyl group as the primary reactive group, a compound having a hydrosilyl group can be used.

[0026] Examples of olefin-based elastomers include copolymers of α-olefins having 2 to 20 carbon atoms, such as ethylene, propylene, 1-butene, 1-hexene, and 4-methylpentene. Specific examples include ethylene-propylene copolymer (EPR), ethylene-propylene-diene copolymer (EPDM), copolymers of α-olefins with non-conjugated dienes having 2 to 20 carbon atoms, such as dicyclopentadiene, 1,4-hexadiene, cyclooctadiene, methylenenorbornene, ethylidenenorbornene, butadiene, and isoprene, and carboxy-modified NBR obtained by copolymerizing methacrylic acid with a butadiene-acrylonitrile copolymer.

[0027] Examples of urethane elastomers include those having structural units of a hard segment made of a low molecular weight glycol and a diisocyanate, and a soft segment made of a high molecular weight (long chain) diol and a diisocyanate.

[0028] Examples of polyester elastomers include those obtained by polycondensation of dicarboxylic acid or a derivative thereof and diol compound or a derivative thereof, and in particular those obtained by copolymerizing a polyester structure with a polyether structure.

[0029] Examples of polyamide elastomers include polyether block amides and polyether ester block amides, which use polyamide for the hard segment and polyether or polyester for the soft segment.

[0030] Acrylic elastomers are primarily composed of acrylic esters, and examples thereof include ethyl acrylate, butyl acrylate, methoxyethyl acrylate, and ethoxyethyl acrylate. Crosslinking monomers include glycidyl methacrylate and allyl glycidyl ether. Furthermore, acrylonitrile and ethylene can also be copolymerized. Specific examples include acrylonitrile-butyl acrylate copolymer, acrylonitrile-butyl acrylate-ethyl acrylate copolymer, and acrylonitrile-butyl acrylate-glycidyl methacrylate copolymer.

[0031] Examples of rubber-modified epoxy resins include those obtained by modifying some or all of the epoxy groups of bisphenol F epoxy resin, bisphenol A epoxy resin, salicylaldehyde epoxy resin, phenol novolac epoxy resin, or cresol novolac epoxy resin with both-end carboxylic acid-modified butadiene-acrylonitrile rubber, terminal amino-modified silicone rubber, etc. Among these, styrene-based elastomers and silicone-based elastomers are preferably used.

[0032] The elastomer layer may contain other components such as known additives within the scope of the present invention, provided that the effects of the present invention are not impaired. Examples of known additives include antioxidants, weather stabilizers, heat stabilizers, lubricants, crystal nucleating agents, ultraviolet absorbers, colorants, surfactants, and fillers. These may be used alone or in combination of two or more.

[0033] The thickness of the elastomer layer depends on the total thickness of the conductive nanothin film and the thickness of the carbon nanotube layer, but is preferably 750 nm or less, more preferably 500 nm or less, and even more preferably 200 nm or less. It is also preferably 30 nm or more. A small thickness provides excellent flexibility, good stretchability in the thickness direction and surface direction, and good conformability to the leaf surface. A moderate thickness provides self-supporting properties and ensures strength.

[0034] In the conductive nano thin film, the carbon nanotube layer is a layer having conductivity and functions as an electrode film, etc. Materials constituting the carbon nanotube layer include single-walled carbon nanotubes (SWCNTs) having a structure of one graphene sheet, multi-walled carbon nanotubes (MWCNTs) consisting of multiple graphene sheets, fullerene tubes, buckytubes, graphite fibrils, etc. These may be chemically modified to increase affinity for solvents, or may be a mixture of metallic carbon nanotubes and semiconducting carbon nanotubes, each of which is concentrated.

[0035] Among these, single-walled carbon nanotubes are preferably used because the conductive nano-thin film has excellent flexibility, stretches in the thickness direction and the surface direction, and has good conformability to the leaf surface.

[0036] The carbon nanotube layer may contain other components such as a dispersant for carbon nanotubes, within the range that does not impair the effects of the present invention.

[0037] The thickness of the carbon nanotube layer depends on the total thickness of the conductive nanothin film and the thickness of the elastomer layer, but is preferably 250 nm or less, more preferably 100 nm or less, and even more preferably 50 nm or less. It is also preferably 10 nm or more. When carbon nanotube layers are laminated on both sides of the elastomer layer, the thickness here refers to the total thickness of the carbon nanotube layers on both sides. A small thickness provides excellent flexibility, good stretchability in the thickness direction and surface direction, and good conformability to the leaf surface. A moderate thickness also ensures conductivity.

[0038] Carbon nanotubes, especially single-walled carbon nanotubes, have a smaller Young's modulus and a larger elongation at break than conductive polymers. Because single-walled carbon nanotubes stretch due to their fiber network structure, they minimize the inhibition of flexibility in the elastomer layer and maintain the conformability of the conductive nanofilm.

[0039] The Young's modulus of the conductive nano-thin film is not particularly limited, but is preferably 50 MPa to 200 MPa. A Young's modulus in this range provides excellent flexibility, good stretchability in the thickness direction and surface direction, and good conformability to the leaf surface.

[0040] In the conductive nano-thin film, the ratio of the thickness T2 of the carbon nanotube layer to the thickness T1 of the elastomer layer is preferably 0.01 or more and 1.85 or less, more preferably 0.05 or more and 1.85 or less. When carbon nanotube layers are laminated on both sides of the elastomer layer, the thickness of the carbon nanotube layer here is the total thickness of the carbon nanotube layers on both sides. A small ratio results in excellent flexibility, good stretchability in the thickness direction and in the surface direction, and good conformability to the leaf surface. A moderately large ratio also ensures conductivity.

[0041] The electrode for measuring plant leaf surface potential of the present invention is not particularly limited in its sheet shape or size, and may be selected according to the size of the leaf surface, etc. The carbon nanotube layer can be formed over the entire surface of the elastomer layer. Alternatively, it may be formed partially within the surface of the elastomer layer, for example, the carbon nanotube layer may be patterned within the surface of the elastomer layer. For example, since it has an area of ​​several to several tens of square centimeters for a thickness on the order of nanometers, it has flexibility and physical adhesiveness, and can adhere and conform to the leaf surface, which has fine irregularities such as leaf veins, at the nano level.

[0042] The conductive nanofilm can be produced by any known film-forming method, including, but not limited to, a roll-to-roll process using a gravure coater. For example, a gravure coater is used to coat a polyethylene terephthalate (PET) film with an aqueous solution of polyvinyl alcohol (PVA) for the first sacrificial layer, followed by drying to form a PVA layer. Next, a solution for the elastomer layer, such as a tetrahydrofuran solution of SBS, is coated on the PVA layer and dried to form the second layer. This results in a laminated film consisting of the first and second layers. A solution for the carbon nanotube layer, such as an aqueous dispersion of SWCNTs, is then coated and dried to form the third layer. This results in a laminated film consisting of the first, second, and third layers. Paper tape is then applied to the surface on which the third layer has been formed, framing the desired shape, and the tape is peeled off from the edge, allowing the three-layer film, including the first, second, and third layers, to be peeled off from the PET film while still held by the paper tape. The resulting three-layer film held by the paper tape is floated on pure water with the PVA side in contact, dissolving only the first PVA layer and obtaining a conductive nanothin film consisting of the elastomer layer and carbon nanotube layer held by the paper tape. The paper tape holding this conductive nanothin film can be cut into the desired shape and used, and can be attached to a separate substrate as needed.

[0043] The conductive nano-thin film is self-supporting, and by applying a carbon nanotube layer as a conductive layer to an elastomer layer, it has excellent flexibility while ensuring good conductivity.

[0044] The electrode for measuring plant leaf surface potential of the present invention is used to measure the potential on the leaf surface of a plant. The plant is not particularly limited, and can be used for environmental agricultural crops, ornamental plants, etc. Leaves, which are organs that grow laterally on stems, typically have a flat structure and a venation system made up of vascular bundles. The leaf is not particularly limited, but examples include ordinary leaves that have chloroplasts and perform photosynthesis. The leaves of angiosperms have a leaf blade as the main part that performs photosynthesis, and the tissue of the leaf blade consists of veins, mesophyll, and epidermis.

[0045] Trichomes are found on the leaf surface of plants. Trichomes are a collective term for appendages formed from the epidermis of vascular plants and are found in all organs of the plant. Trichomes vary in form, structure, and function, providing protection, secretion, and excreting substances from the body to the outside. They are classified as glands or non-secretory trichomes based on their function, and are also called hairs, scales, papillae, or root hairs based on their morphology and the organ in which they occur. Multiple types of trichomes are often found on a single plant. The plant leaf potential measurement electrode of the present invention can maintain stable contact with the leaf surface even in the presence of trichomes and can self-adhere to the leaf surface. In particular, carbon nanotubes, due to their fiber network structure, are able to expand and contract, thereby suppressing the inhibition of the flexibility of the elastomer layer and maintaining the conformability of the nanosheet, thereby maintaining stable contact with the leaf surface even in the presence of trichomes.

[0046] The electrode for measuring plant leaf potential of the present invention can be used to measure the light-induced biopotential (LIB) generated during photosynthesis. Biopotential is a type of biological signal and generally refers to the potential generated by the biological activity of animals and plants. In plants, its generation is due to the movement of ions through membrane transport and the like, and directly reflects the plant's activity. Previous research has already confirmed that the potential changes depending on the ambient temperature, light irradiation, and gas. Furthermore, plant biopotential is broadly classified into surface potential and cellular potential. Since the electrode for measuring plant leaf potential of the present invention measures surface potential, the surface potential is treated as the plant biopotential.

[0047] When measuring the potential of a plant leaf surface using the electrode for measuring plant leaf surface potential of the present invention, a conductive nano-thin film is attached to the leaf surface. The conductive nano-thin film is preferably attached so that the carbon nanotube layer, which is the conductive layer, is in close contact with the leaf surface. A collecting electrode may be attached to the conductive nano-thin film. When measuring light-induced bioelectric potential, the electrodes may be attached to two locations on the leaf surface, one on either side of the midvein, and one electrode may be covered with a light-shielding film and used as a reference electrode. The collecting electrode is connected to a measuring instrument, and the potential difference between the measuring electrode and the reference electrode is measured. Environmental conditions such as temperature and photon density are set according to the purpose.

[0048] The electrode for measuring plant leaf potential of the present invention can be applied to primary industries, including agriculture, for unmanned cultivation and artificial cultivation in plant factories, and is expected to contribute to resolving poverty, climate change, hunger, and other issues outlined in the SDGs. It is also expected to be applied as a simple potential measurement system to kits for junior and senior high school students to support their learning about bioelectric potentials and measurement systems. Plant factories, which manage and cultivate plants in completely artificial environments, have attracted attention in recent years because they enable stable cultivation unaffected by the external environment. However, fully unmanned management requires technology that can monitor and control plant growth status in real time. The electrode for measuring plant leaf potential of the present invention provides a method for measuring the bioelectric potential generated by individual plant activity as an indicator of the plant's live status. [Example]

[0049] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0050] We aimed to develop a minimally invasive nanosheet electrode that is lightweight, has a high visible light transmittance of over 80%, and is water-resistant. It maintains adhesion without adhesive even on leaf surfaces containing numerous trichomes. Specifically, we developed a conductive nanosheet (Patent Document 1) consisting of a polystyrene-polybutadiene-polystyrene copolymer (SBS) and single-walled carbon nanotubes (SWCNTs), which we have developed independently. This nanosheet electrode was designed to achieve stable adhesion to trichome-bearing plant leaves and enable bioelectric potential measurements for over a week. SWCNT-SBS nanosheets with different thicknesses were applied to soybean leaves containing numerous trichomes. The appropriate thickness for the electrode was determined by observing the contact state of the nanosheet and evaluating adhesion. We also measured the transmittance in the visible light region. Furthermore, SWCNT-SBS nanosheet electrodes were attached to the leaf tissue of pothos and soybean without adhesive, and the light-induced biopotential (LIB) generated during photosynthesis was measured over a long period of time by turning an LED light on and off.

[0051] 1. Preparation of SWCNT-SBS nanosheets Using a roll-to-role gravure coating system (Desktop Mini-Labo Test Coater, Yasui Seiki), a PVA (MW 22000, Kanto Chemical) aqueous solution was applied to a polyethylene terephthalate (PET) film roll at a microgravure roll (MG) rotation speed of 30 rpm and a substrate feed speed (LS) of 0.8 m / min. A 7 wt% PVA aqueous solution was used for the 1 wt% SBS sheet, and a 5 wt% PVA aqueous solution was used for the 2.5 wt% and 5 wt% SBS sheets. A thin PVA film was obtained on the PET by drying at 80°C using the system's internal heater. A 1 wt%, 2.5 wt%, or 5 wt% THF solution of SBS (Mw 280000, Sigma-Aldrich) was applied to the resulting PVA thin film at a rotation speed of 30 rpm and a linear speed of 1.3 m / min, followed by drying in a heater at 80°C. Furthermore, a 0.2 wt% aqueous dispersion of SWCNT (Meijo Nanocarbon) was applied to the SBS thin film at a rotation speed of 30 rpm and a linear speed of 0.8 m / min, followed by drying in a heater at 80°C. After film formation, the PVA / SBS / SWCNT sheet was peeled off using the tape frame method, and the PVA layer was removed in a deionized water bath to obtain a self-supporting SWCNT-SBS nanosheet.

[0052] 2. Film Thickness and Weight of SWCNT-SBS Nanosheets The SWCNT-SBS nanosheet was attached to a glass substrate, and the film thickness of the SWCNT-SBS nanosheet was measured using a cross-sectional profiler (DektakXT, Bruker). The film thickness of the SWCNT-SBS nanosheet was as follows: <SWCNT-SBSナノシート1> SBS: 379 nm SWCNT-SBS: 485 nm <SWCNT-SBSナノシート2> SBS: 150 nm SWCNT-SBS: 324nm (approx. 320nm) <SWCNT-SBSナノシート3> SBS: 33.6 nm SWCNT-SBS: 70.7nm (approx. 70nm)

[0053] The dry electrode, SWCNT-SBS nanosheet (film thickness: 485 nm, weight: 9.6 mg, Fig. 1A), weighed approximately 1 / 50 or less of the wet electrodes, commercially available gel electrodes for electrocardiograms (852 mg) and dish electrodes for electroencephalograms (439 mg).

[0054] 3. Visible light transmittance measurement of SWCNT-SBS nanosheets and commercial gel electrodes The SWCNT-SBS nanosheet was attached to a quartz glass substrate and its transmittance was measured in the 190-1100 nm range using a UV-visible spectrophotometer. The transmittance of a commercially available gel electrode was also measured using a similar method. The results are shown in Figure 1B. In the visible light range of 400-800 nm, the SWCNT-SBS nanosheet exhibited a transmittance of over 80% in the wavelengths of 400-500 nm and 600-700 nm (shown in gray in the figure), which are necessary for plant photosynthesis. In contrast, the transmittance of the commercially available gel electrode was nearly 0%. This is because the plastic substrate, which maintains the shape of the hydrogel layer of the gel electrode, absorbed and scattered light in the measured wavelength range. In other words, the nanosheet electrode is an electrode that is less likely to inhibit the photosynthetic reaction.

[0055] 4. Evaluation of adhesion of SWCNT-SBS nanosheets with different thicknesses to soybean leaf surfaces and LIB measurements As described above, SWCNT-SBS nanosheets with different thicknesses of 485 nm, 320 nm, and 70 nm were prepared using the gravure coating method. These nanosheets were peeled off from the PET roll substrate using the tape frame method, and the PVA layer was removed with deionized water. They were then placed in contact with soybean leaf surfaces, and the contact state between each nanosheet and the leaf surface was observed using a stereomicroscope.

[0056] The appearance of SWCNT-SBS nanosheets with thicknesses of 485 nm, 320 nm, and 70 nm attached to soybean leaves was observed using a stereomicroscope (Figures 2A, 2B, and 2C). The 485 nm thick nanosheet (Figure 2A) was observed to be obstructed by trichomes and to float away from the leaf surface, and when it dried further, the nanosheet broke. On the other hand, the 320 nm thick nanosheet (Figure 2B) was observed to adhere closely to the leaf surface, although some of the nanosheets were seen to be floating. It is thought that some of the trichomes on the leaf surface broke through the nanosheet, allowing the nanosheet to come into contact with the leaf surface, thereby exerting adhesion. Nanosheets with a thickness of 320 nm also adhere stably to the leaf surface, but are blocked by the trichomes. However, nanosheets with a thickness of 70 nm (Figure 2C) adhere to the leaf surface without being blocked by the trichomes, and in some cases, the trichomes were observed to break through the nanosheet. Nanosheets with a thickness of 70 nm can adhere stably to the trichomes for more than two weeks. The appearance of 800-nm-thick SBS nanosheets, 30-nm-thick SBS nanosheets, and polystyrene sheets attached to soybean leaves was observed under a stereomicroscope (Figures 2D, 2E, and 2F). The 800-nm-thick SBS nanosheet was inhibited by trichomes and did not adhere to the leaf surface, causing the edges of the nanosheet to shrink. The 30-nm-thick SBS nanosheet was too thin, so it broke and shrunk upon contact with trichomes. The polystyrene sheet was inhibited by trichomes and was observed to float off the leaf surface.

[0057] 5. Evaluation of leaf invasiveness when SWCNT-SBS nanosheets are applied to leaves for a long period of time Three types of surface electrodes were attached to the leaves of pothos (Epipremnumaureum), and the condition of the leaves was observed immediately after attachment and after 14 days. The electrodes used were a commercially available gel electrode, an EEG dish electrode (NE-118A, Nihon Kohden Corporation), and an SWCNT-SBS nanosheet. The EEG dish electrode was fixed in place using conductive paste for EEG and EMG (ZV-181E, Nihon Kohden Corporation). Furthermore, commercially available gel electrodes and transparent gel electrodes were attached to tobacco (Nicotianatabacum) leaves, which have lower mechanical strength than pothos, to evaluate the invasiveness of the electrodes to tobacco leaves.

[0058] When leaves with EEG plate electrodes, gel electrodes, and SWCNT-SBS nanosheet electrodes attached for 14 days or more were observed (Figure 3A(a), (b), (c)), blackening of the leaves was confirmed where the EEG plate electrodes were attached (Figure 3A(a)). When the EEG plate electrodes were attached for 14 days or more, the blackened tissue spread and the leaves withered and died. In contrast, no such discoloration was observed where the gel electrodes or nanosheet electrodes were attached (Figure 3A(b), (c)). The withering of the EEG plate electrode areas is thought to be due to the salt contained in the conductive paste used to secure the EEG plate electrodes. The paste contains NaCl as an ion-conductive material, and it is thought that if it is attached to the leaf surface for a long period of time, osmotic pressure will cause dehydration of the cells.

[0059] As with EEG dish electrodes, gel electrodes can also cause the area where they are applied to become dry. Figure 3B (a) shows the gel electrode, (b) shows the transparent gel electrode, and (c) shows the SWCNT-SBS nanosheet electrode applied to tobacco leaves for over 14 days. Even on tobacco leaves, no discoloration was observed at the applied area with the SWCNT-SBS nanosheet electrode, but the gel and transparent gel electrodes were highly invasive. Because the SWCNT-SBS nanosheet electrode is self-adhesive, the electrode paste required for wet electrodes is unnecessary, and measurements can be performed stably over a long period of time using the highly non-invasive dry SWCNT-SBS nanosheet electrode.

[0060] 6. LIB Measurement Using SWCNT-SBS Nanosheets and Comparison with Commercially Available Gel Electrodes The LIB was measured when a gel electrode, an EEG dish electrode, and an SWCNT-SBS nanosheet electrode were attached to the surface of a pothos leaf (Figure 4).

[0061] Electrodes were attached to the surface of the pothos leaves at two locations, one on each side of the midvein. The SWCNT-SBS nanosheet electrode consisted of a collector electrode made of copper foil and soldered gold wire attached with double-sided tape, and then a 20mm x 25mm SWCNT-SBS nanosheet was attached so that the conductive layer was in contact with the leaf surface. The pothos was placed in a dark box, and one electrode was covered with aluminum foil to serve as a reference electrode. The collector electrode was connected to a digital multimeter (Keysight 34461A) with a clip, and the potential difference between the measuring electrode and the reference electrode was measured. The leaf surface potential was measured at 1Hz in high impedance mode (>10GΩ). To measure LIB, a white LED in the dark room was turned on and off every two hours. Measurements were also taken when the photon density was 40-50μmol / m 2 The measurements were carried out in an environment with a temperature of 20-25°C. A photon meter (SE-MQ-500, Apogee) was used to measure the photon density. The LIB measured at each electrode was evaluated by the amplitude that appeared immediately after the LED was turned on. The amplitude was calculated by calculating the difference between the two peaks (P1, P2) that appeared immediately after the LED was turned on, and the average amplitude over several flashing cycles was found. The amplitude was analyzed by approximating the measured potential with a low-order polynomial and performing drift correction.

[0062] The LIB for each electrode was measured immediately after application and after 14 days (Figure 4(a), (b), (c)). Similar potential changes were measured for all electrodes even after 14 days, with a downward spike (P1) appearing 1-2 minutes after the LED was turned on, followed by an upward spike (P2) 10-20 minutes later. In all measurement results, there were differences in the LIB waveforms, such as a difference in the time elapsed from application to reaching P1 and P2, and the observation of two separate P2s. These differences in response may reflect differences in the plant's moisture status and the ambient temperature and humidity between immediately after application and during the experiment 14 days later.

[0063] Furthermore, when comparing the amplitude of LIB (Fig. 4(d)), the amplitude of LIB for the SWCNT-SBS nanosheet electrode and gel electrode remained close to that immediately after application even after long-term application. However, for the EEG dish electrode, the amplitude was found to decrease to about one-third after application for 14 days or more.

[0064] These results suggest that the paste used to secure the EEG dish electrodes is invasive to the pothos leaves and adversely affects LIB measurements. Furthermore, when comparing the amplitudes of each electrode, the LIB amplitude with the SWCNT-SBS nanosheet electrode was twice that of the gel electrode. This is thought to be because the SWCNT-SBS nanosheet electrode has higher transmittance than the gel electrode, resulting in a higher light intensity hitting the leaf surface and resulting in a larger amplitude. In other words, the SWCNT-SBS nanosheet electrode, with its high optical transparency, allows light to reach the area where it is attached, resulting in a larger LIB amplitude than the gel electrode.

[0065] In addition, the LIB was measured when SWCNT-SBS nanosheets with thicknesses of 485 nm, 320 nm, and 70 nm were attached to soybean leaves as shown in Figures 2A, 2B, and 2C, as shown in Figures 5A, 5B, and 5C.

[0066] When LIB measurements were performed on leaves with each nanosheet attached, the LIB waveform could be measured immediately after attachment in the case of soybean leaves with a 485 nm thick nanosheet attached (Figure 5A). However, two weeks after attachment, the nanosheets had dried and torn, and there was so much noise during measurement that it was difficult to measure LIB.

[0067] LIB measurements were also possible with a 70 nm thick nanosheet immediately after application, and continuous measurements were successful for over 70 hours (Figure 5C). Furthermore, the nanosheet remained in close contact with the leaf surface even one month after application, suggesting that long-term measurements over several months are possible. The results were best for nanosheets with thicknesses of 485 nm (Figure 5A), 320 nm (Figure 5B), and 70 nm (Figure 5C).

[0068] 7. Conclusion Compared to commercially available gel electrodes, SWCNT-SBS nanosheets are extremely lightweight and have a thickness on the order of nanometers, which has newly revealed a transmittance of over 80% in the visible light range. In other words, it is thought to be a flexible electrode that does not block light in the wavelength ranges (400-500nm, 600-700nm) necessary for transpiration from the leaf surface and plant photosynthesis, and is less likely to inhibit photosynthetic reactions.

[0069] When SWCNT-SBS nanosheets and SBS nanosheets of different thicknesses were applied to plant leaves, the 70 nm SWCNT-SBS nanosheets adhered closely to the plant surface and could be used for a long period of time. On the other hand, thicker nanosheets were lifted from the surface by the trichomes on the leaf surface, making them unsuitable for long-term use.

[0070] Furthermore, in a long-term electrode attachment experiment, the attachment area of ​​the plate electrode turned the leaf surface brown and caused the leaf to wither and die, whereas no discoloration was observed with the SWCNT-SBS nanosheet electrode. In an evaluation of abrasion resistance when wet, the SWCNT-SBS nanosheet showed high abrasion resistance, demonstrating that it is an electrode suitable for collecting the bioelectric potential of plants.

Claims

1. An electrode for measuring the surface potential of a plant leaf, comprising a conductive nano-thin film including an elastomer layer and a carbon nanotube layer laminated on at least one surface of the elastomer layer.

2. 2. The electrode for measuring plant leaf surface potential according to claim 1, wherein the carbon nanotubes are single-walled carbon nanotubes.

3. 3. The electrode for measuring plant leaf surface potential according to claim 1, wherein the conductive nano-thin film has a light transmittance of 80% or more in the visible light region of wavelengths of 400 to 800 nm.

4. 3. The electrode for measuring plant leaf surface potential according to claim 1, wherein the conductive nano-thin film has a thickness of 500 nm or less.

5. 3. The electrode for measuring plant leaf surface potential according to claim 1, wherein the conductive nano-thin film has a thickness of 100 nm or less.

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