Ternary copolymer and surface-modified needle-like material including the same, and method for use thereof

Abstract

To provide new means for enhancing the adsorption efficiency, adsorption specificity, and release efficiency of substances on the adsorption surfaces of microneedles, nanoneedles, and the like.SOLUTION: A ternary copolymer has a structure represented by the formula (1), where each symbol is as defined in the specifications.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a terpolymer and a surface-modified needle-shaped object whose surface is modified with the terpolymer, as well as a microneedle array (hereinafter appropriately abbreviated as "MNA") using such a surface-modified needle-shaped object, a method for introducing a substance into a plant cell, and a probe and cantilever for an atomic force microscope (hereinafter appropriately abbreviated as "AFM"). [Background technology]

[0002] In various fields including the seed industry, methods such as hybridization, induction of mutations by radiation or DNA-damaging drugs, transduction by gene recombination using Agrobacterium or plant virus vectors, etc. are used to modify the functions of plants. In recent years, genome editing technology has been attracting attention as a method for modifying genetic properties, and research on genome editing technology is also being conducted in modifying the functions of plants. As a plant genome editing technology, a method of introducing DNA into cells using the above-mentioned Agrobacterium is known, but since this method relies on the insertion of the T-DNA of Agrobacterium into the genomic DNA, there is a risk that the gene at the insertion site will be destroyed. In addition, for example, depending on the tissue containing the target cell, there is a problem that the promoter of the protein to be introduced or the protein required for genome editing does not function, so the protein is not expressed and the desired editing operation cannot be performed.

[0003] On the other hand, genome editing can be performed regardless of the species, tissue, or cell by directly introducing proteins or RNA into cells. However, compared with animal cells and cultured cells, cells contained in plant tissues are covered with hard epidermal tissues and cell walls, making physical perforation difficult. To date, particle bombardment, electroporation, and microinjection are known as techniques for directly introducing target substances into such plant cells.

[0004] The particle bombardment method is a method in which a target substance (nucleic acid, protein, etc.) is attached to the surface of gold particles, which are then ejected with high pressure gas to perforate plant cells, thereby introducing the target substance into the plant cells. However, the particle bombardment method has problems such as low introduction efficiency and the inability to control the introduction site. The electroporation method is a method in which a target substance is introduced into plant cells by perforating them with electric pulses. However, the electroporation method requires the preparation of proplasts, and has a problem that the method cannot be directly applied to cells contained in plant tissue. The microinjection method is a method in which a solution of a target substance is injected into cells using a capillary, and can be directly applied to cells contained in plant tissue. However, the microinjection method has problems such as the shape and size of the capillary not being suitable for plant cells (especially cells deep in plant tissue), and the throughput is also low.

[0005] Patent Document 1 (JP Patent Publication No. 2015-181384) describes a method of introducing an artificial nuclease into a cell by puncturing a cell with a microneedle bound to the artificial nuclease, and Patent Document 2 (WO 2019 / 113396) describes a method of inserting a nanowire array into a plant cell to insert a biomolecule into the plant cell. According to such a method using a microneedle or nanowire array, the insertion operation into the cell is performed while performing positioning under microscope observation, so that it is possible to introduce a target substance into a target cell in a plant tissue. However, it is difficult to insert these microneedles and nanowire arrays into plant cells, particularly plant cells contained in plant tissues such as tissue culture cells and cells of plant species that are difficult to regenerate plants, and there are cases where the target substance cannot be introduced.

[0006] The present inventors have developed a technology that reduces damage to cells by using a nanoscale ultrafine needle-shaped material (Patent Document 3: Japanese Patent No. 4625925), and a technology that improves the efficiency of substance introduction into cells by inserting a nanoneedle into a cell and then vibrating the nanoneedle array at a high frequency (Patent Document 4: Japanese Patent No. 6449057). In addition, a technology that can introduce a target substance into plant cells contained in plant tissue with high efficiency by using a microneedle array (MNA) in which microneedles having a specific high aspect ratio, which are convex structures, are aligned on a support at specific intervals (Patent Document 5: Japanese Patent Publication No. 2021-151201). Although these are excellent technologies, when introducing a target substance using such microneedles / nanoneedles, a solution of the target substance is dropped onto the surface of the microneedle / nanoneedle and adsorbed onto the surface of the microneedle / nanoneedle through electrostatic interaction, but there is a limit to the efficiency of introducing the target substance, and there is room for further improvement. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2015-181384 A [Patent Document 2] International Publication No. 2019 / 113396 [Patent Document 3] Patent No. 4625925 [Patent Document 4] Patent No. 6449057 [Patent Document 5] Patent Publication No. 2021-151201 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been made in consideration of the above problems, and aims to provide a new means for improving the adsorption efficiency, adsorption specificity, release efficiency, etc. of substances on the substance adsorption surfaces of microneedles, nanoneedles, etc. [Means for solving the problem]

[0009] As a result of extensive research to achieve the above-mentioned object, the inventors have developed a terpolymer with a novel structure and discovered that by modifying the surface of a needle-shaped object with such a terpolymer, it is possible to improve the adsorption efficiency, adsorption specificity, release efficiency, etc. of a substance. In turn, the use of such surface-modified needle-shaped objects can provide a variety of advantages when applied to a variety of applications, such as improving the introduction efficiency of target substances using microneedles and nanoneedles, and improving the adsorption efficiency and adsorption specificity of target substances using AFM cantilevers, and have thus completed the present invention.

[0010] That is, the present invention includes the following aspects. [1] A terpolymer having a structure represented by formula (1). [ka] (However, in formula (1), R 11 represents a linear or branched divalent aliphatic hydrocarbon group having 1 to 6 carbon atoms, R 12 represents hydrogen or methyl; R 21 represents a linear or branched monovalent aliphatic hydrocarbon group having 1 to 6 carbon atoms substituted with 1 to 3 hydroxyl groups, R 22 represents hydrogen or methyl; R 3 represents hydrogen or methyl; Ring A represents an aromatic hydrocarbon group having 1 to 3 hydroxyl groups; Ring B represents a nitrogen-containing cyclic group having 1 to 3 oxo groups; T 1 and T 2 each independently represents a terminal group; x represents a natural number between 1 and 5000. y is a natural number between 1 and 8000, z is a natural number between 1 and 5000. Here, the x, y, and z monomer units may exist as blocks or may coexist randomly. [2] The terpolymer according to item 1, wherein Ring A is a dihydroxyphenyl group. [3] The terpolymer according to item 1, wherein Ring B is an N-succinimide group. [4] A surface-modified needle-shaped object, at least a part of whose surface is modified with the terpolymer according to any one of items 1 to 3. [5] A microneedle array comprising a support and a plurality of needle parts arranged on the support, Item 5. A microneedle array, wherein some or all of the needles are the surface-modified needle-shaped objects according to Item 4. [6] The diameter of the base surface of the needle portion is 1 to 20 μm, The diameter of the tip surface of the needle portion is 0.5 to 3 μm, The length of the needle portion is 30 to 100 μm, The diameters of the tip surface and cross section of the needle portion are equal to or smaller than the diameter of the base surface, and the rate of increase in the diameter of the cross section from the tip surface to the length of the needle portion minus 10 μm is 0 to 5%, Item 6. The microneedle array according to item 5, wherein the distance between the tip surfaces of the needle parts is 20 to 1000 μm. [7] The microneedle array according to item 6, wherein the aspect ratio of the needle portion (length / (diameter of 1 / 2 length face)) is 5 to 200. [8] The microneedle array according to item 6, wherein the support and the needle portion are integral and made of single crystal silicon. [9] The microneedle array according to item 6, which is used for introducing a target substance into a plant cell.

[10] The microneedle array according to item 9, wherein the plant cells are cells contained in plant tissue.

[11] The microneedle array according to item 10, wherein the plant tissue is a shoot apical meristem.

[12] A method for introducing a target substance into a plant cell, an insertion step of inserting the surface-modified needle-shaped object according to Item 4 or the needle part of the microneedle array according to Item 5, having the target substance attached to the surface, into the plant cell; A method for introducing a substance into a plant cell, comprising the steps of:

[13] The method for introducing a substance into a plant cell according to Item 12, wherein the insertion speed in the insertion step is 0.01 to 50 μm / sec.

[14] The method for introducing a substance into a plant cell according to Item 12, wherein in the inserting step, the needle part is vibrated in the insertion direction at an amplitude of 0.01 to 50 μm and a frequency of 0.1 to 2000 Hz.

[15] The method for introducing a substance into a plant cell according to Item 12, wherein the plant cell is a cell contained in a plant tissue, and the method is a method for introducing a substance into a plant tissue.

[16] A probe having a needle-shaped tip that is attached to a cantilever of an atomic force microscope (AFM) or an intermolecular force measuring device, Item 5. A probe, wherein the needle-shaped tip is the surface-modified needle-shaped material according to Item 4.

[17] A cantilever for an atomic force microscope (AFM) or an intermolecular force measuring device, comprising the probe according to item 16. Effect of the Invention

[0011] The terpolymer of the present invention can be used for surface modification of needle-shaped objects such as microneedles, nanoneedles, and cantilevers of atomic force microscopes (AFMs) to improve the adsorption efficiency, adsorption specificity, release efficiency, etc. of substances. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic longitudinal sectional view showing one embodiment of a microneedle array of the present invention. [Diagram 2] 1 is a SEM photograph showing the appearance of a microneedle array used in an example. [Diagram 3] 1 is a graph showing the fluorescence intensity of GFP protein immobilized on the surface of modified MNA in each Example and Comparative Example, using a plant cytoplasm-mimicking buffer solution, before and after washing. [Figure 4]FIG. 1 is a schematic diagram for explaining the structure of the GUS reporter gene used in the Examples. [Diagram 5] (a) and (b) are optical microscope photographs of GUS-stained leaf tissue after the introduction of modified MNA in Example 1. The dotted line in (a) indicates the MNA insertion site. (b) is an enlarged photograph of the part of (a) where GUS staining was observed. [Figure 6] FIG. 2 is a schematic diagram for explaining the configuration of a nestin tail-immobilized AFM probe used in the examples. [Figure 7] FIG. 2 is a schematic diagram for explaining the configuration of an actin-immobilized glass substrate used in the Examples. [Figure 8] FIG. 2 is a schematic diagram for explaining the configuration of the nestin tail tensile test carried out in the examples. [Figure 9] 1(a) and (b) are schematic diagrams of force curves obtained when stretching the nestin tail used in the nestin tail tensile test in the Examples, where (a) shows a characteristic curve obtained by polymer stretching, and (b) shows a curve at a stretch distance of 0, which indicates the bond breaking force of nonspecific interactions between the substrate and the probe. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention will be described below based on specific embodiments, but the present invention is not limited to these embodiments. All documents cited in this specification, including patent publications, patent application publications, and non-patent publications, are incorporated herein by reference in their entirety for all purposes.

[0014] [Ternary copolymer] One aspect of the present invention relates to a terpolymer having a structure represented by formula (1) (hereinafter, appropriately abbreviated as "the terpolymer of the present invention").

[0015] [ka]

[0016] In formula (1), the definitions of each symbol are as follows:

[0017] R 11 represents a straight or branched divalent aliphatic hydrocarbon group having 1 to 6 carbon atoms. Specific examples include a methylene group, an ethylene group, a propylene group (n-propylene group, isopropylene group), a butylene group (n-butylene group, isobutylene group, sec-butylene group), a pentylene group, a hexylene group, etc. Among these, a methylene group, an ethylene group, a propylene group, etc. are preferred, and an ethylene group is particularly preferred.

[0018] R 12 represents hydrogen or a methyl group.

[0019] R 21 represents a linear or branched monovalent aliphatic hydrocarbon group having 1 to 6 carbon atoms substituted with 1 to 3 hydroxyl groups. Specific examples include groups in which a methyl group, an ethyl group, a propyl group (n-propyl group, isopropyl group), a butyl group (n-butyl group, isobutyl group, sec-butyl group), a pentyl group, a hexyl group, or the like is substituted with 1 to 3 hydroxyl groups. Among these, a hydroxymethyl group, a hydroxyethyl group, a hydroxypropyl group, or the like is preferred, with a hydroxypropyl group being particularly preferred.

[0020] R 21 represents hydrogen or a methyl group.

[0021] R 3 represents hydrogen or a methyl group.

[0022] Ring A represents an aromatic hydrocarbon group having 1 to 3 hydroxyl groups. Specific examples include hydroxyphenyl, dihydroxyphenyl, trihydroxyphenyl, etc. Among them, a dihydroxyphenyl group is preferable.

[0023] Ring B represents a nitrogen-containing cyclic group having 1 to 3 oxo groups. Specific examples include N-succinimide and N-phthalimide. Among them, the N-succinimide group is preferable.

[0024] T 1 and T 2 each independently represents an end group. Examples include a linear or branched monovalent aliphatic hydrocarbon group having 1 to 3 carbon atoms. Specific examples include a methyl group, an ethyl group, a propyl group (n-propyl group, isopropyl group), and the like. Among these, each independently represents a methyl group, an ethyl group, and the like, and a methyl group is particularly preferred.

[0025] x represents a natural number of 1 or more and 5000 or less. More specifically, x can be, for example, 1 or more, or 3 or more, or 10 or more, or 20 or more, and can be, for example, 5000 or less, or 3000 or less, or 2000 or less, or 1000 or less. y represents a natural number of 1 or more and 8000 or less. More specifically, y can be, for example, 1 or more, or 5 or more, or 20 or more, or 50 or more, and can be, for example, 8000 or less, or 6000 or less, or 5000 or less, or 4000 or less. z represents a natural number of 1 or more and 5000 or less. More specifically, z can be, for example, 1 or more, or 3 or more, or 10 or more, or 20 or more, and can be, for example, 5000 or less, or 3000 or less, or 2000 or less, or 1000 or less.

[0026] The ratio of x:y:z is not particularly limited, but when x is 10, y can be in the range of 10 to 200, and z can be in the range of 1 to 50.

[0027] The x monomer units (which are appropriately referred to as "monomer units X") may be the same units, or two or more different units may be mixed. A particularly preferred example of the monomer unit X is a 3,4-dihydroxyphenylalanine (DOPA) unit.

[0028] The y monomer units (which are appropriately referred to as "monomer units Y") may be the same units, or may be a mixture of two or more different units. A particularly preferred example of the monomer unit Y is a 2-hydroxypropylacrylamide (HPA) unit.

[0029] The z monomer units (which are appropriately referred to as "monomer units Z") may be the same units, or two or more different units may be mixed. A particularly preferred example of the monomer unit Z is an N-hydroxysuccinimide (NHS) unit.

[0030] The x number of monomer units X, the y number of monomer units Y, and the z number of monomer units Z may be present together as a block, or may be present randomly.

[0031] The degree of polymerization (the sum of x+y+z) of the terpolymer of the present invention is not particularly limited, and can be, for example, 20 or more, or 50 or more, or 100 or more, and can be, for example, 18,000 or less, or 15,000 or less, or 12,000 or less, or 10,000 or less, or 7,000 or less, or 5,000 or less.

[0032] A specific example of the terpolymer of the present invention is, but is not limited to, a DOPA / HPA / NHS copolymer represented by the following formula (2).

[0033] [ka]

[0034] In formula (2), the definitions of x, y, and y are the same as those in formula (1).

[0035] The method for producing the terpolymer of the present invention is not particularly limited, but includes a method in which monomer compounds corresponding to the monomer unit X, the monomer unit Y, and the monomer unit Z, which are individually prepared, are used so as to have a desired ratio of x:y:z, and polymerized by any conventionally known polymerization method. As the monomer compounds corresponding to the monomer unit X, the monomer unit Y, and the monomer unit Z, various known commercially available compounds may be used, or they may be synthesized by any conventionally known synthesis method. Various synthesis and polymerization methods for such monomer compounds are known, and those skilled in the art can appropriately select and use them. As a specific example, when synthesizing the DOPA / HPA / NHS copolymer represented by the above formula (2), the method adopted in the "Synthesis Example" described below can be used.

[0036] The ternary copolymer of the present invention can be used for surface modification of various structures including needle-shaped objects described below. In particular, the ternary copolymer of the present invention has the effect of improving the adsorption efficiency and / or release efficiency of the target substance by using it for surface treatment of the adsorption surface in various physical, chemical, or biological treatment or analysis devices used in the operation of adsorbing a target substance or the operation of releasing the target substance after adsorption. For this reason, the ternary copolymer of the present invention is preferably used on the surface of the adsorption surface of such treatment or analysis devices. Examples of structures to be subjected to such surface treatment include, but are not limited to, various needle-shaped objects such as microneedle arrays (MNA), nanoneedle arrays, and cantilevers of atomic force microscopes (AFMs) and molecular force measuring devices. Note that a surface modification material containing the ternary copolymer of the present invention is also included in one aspect of the present invention.

[0037] [Surface modified needles] One aspect of the present invention relates to a needle-shaped object whose surface is modified by the terpolymer of the present invention. In the present invention, the needle-shaped object means a structure having a needle shape. The surface-treated needle-shaped object of the present invention is preferably used for adsorbing a substance. In the present invention, the needle-shaped object for adsorbing a substance means a needle-shaped object used in an operation of adsorbing a target substance or an operation of releasing the target substance after adsorption in various physical, chemical, or biological processing or analysis devices. Examples of such needle-shaped objects include, but are not limited to, the needle-shaped portion of a microneedle array (MNA) or a nanoneedle array, and the cantilever probe of an atomic force microscope (AFM) or an intermolecular force measuring device.

[0038] The material of the needle-shaped object is not particularly limited, and any material can be used. Specific examples include, but are not limited to, inorganic materials such as glass, metals such as silicon (single crystal silicon, etc.), silicon nitride, gold, chromium, indium arsenide, and silica, resins such as polyethylene terephthalate, polyethylene, polypropylene, polystyrene, polycarbonate, and acrylic resin, fibrous materials such as carbon nanotubes and cellulose nanofibers, and whisker materials made of zinc oxide, silicon carbide, etc.

[0039] The size of the needle-shaped object is not limited and can be any size depending on the application, but it is preferable that at least the cross-sectional diameter is on the microscale or nanoscale (i.e., it is a microneedle or nanoneedle).

[0040] At least a part of the surface of such a needle-shaped object is modified with the terpolymer of the present invention to produce the surface-treated needle-shaped object of the present invention. The method of surface modification with the terpolymer of the present invention is not particularly limited, and any conventionally known modification method can be appropriately selected and used. As a specific example, the method employed in the examples described below can be used.

[0041] The needle-shaped material of the present invention, which has been surface-modified with the terpolymer of the present invention, has superior substance adsorption efficiency, adsorption specificity, release efficiency, etc., compared to conventional surface-unmodified needle-shaped materials and needle-shaped materials surface-modified with conventional copolymers.

[0042] In particular, the ternary copolymer of the present invention is preferably used for surface treatment of various microneedles or nanoneedles, and for surface treatment of the probe of the cantilever of an atomic force microscope (AFM) or an intermolecular force measuring device. Examples of nanoneedles include nanoneedles described in Patent Document 3 (Japanese Patent No. 4625925) and Patent Document 4 (Japanese Patent No. 6449057) developed by the present inventors. Examples of microneedles include microneedles possessed by a microneedle array (MNA) described in Patent Document 5 (Japanese Patent Publication No. 2021-151201) developed by the present inventors. Note that an example in which the ternary copolymer of the present invention is applied to a microneedle array (MNA) having a plurality of microneedles, and an example in which the ternary copolymer of the present invention is applied to the surface treatment of a probe of a cantilever of an atomic force microscope (AFM) or an intermolecular force measuring device will be described below in a separate chapter.

[0043] [Microneedle Array (MNA)] One aspect of the present invention relates to a microneedle array (MNA) having needle-shaped objects whose surfaces are modified with the ternary copolymer of the present invention. In the present invention, the term "microneedle array" or "MNA" refers to a support on which a plurality of convex structures (microneedles, needle parts) of the order of micrometers are dispersed. The configuration of such an MNA is not particularly limited, but examples thereof include an MNA having a support and a plurality of needle parts arranged on the support, in which some or all of the needle parts are surface-treated needle-shaped objects of the present invention that have been surface-modified with the ternary copolymer of the present invention.

[0044] In particular, the ternary copolymer of the present invention is preferably used for surface treatment of the needle portion of a specific MNA described in Patent Document 5 (JP Patent Publication No. 2021-151201) developed by the present inventors. In particular, the MNA described in Patent Document 3 has a structure in which needle portions having a specific high aspect ratio, which are convex structures, are aligned on a support at specific intervals. In this way, by precisely controlling the size and arrangement of the needle portions, it is possible to introduce a target substance into plant cells contained in plant tissue with high efficiency. Hereinafter, an embodiment in which a specific MNA described in Patent Document 3 is subjected to surface treatment with the ternary copolymer of the present invention (hereinafter referred to as the "microneedle array of the present invention" or "MNA of the present invention") will be described in detail with examples, sometimes with reference to FIG. 1, but the MNA to which the ternary copolymer of the present invention can be applied is not limited to this, and can be applied to any MNA.

[0045] The MNA of the present invention is an MNA comprising a support and a plurality of needle portions arranged on the support, wherein the diameter of the base surface of the needle portions is 1 to 20 μm, the diameter of the tip surface of the needle portions is 0.5 to 3 μm, the length of the needle portions is 30 to 100 μm, the diameters of the tip surface and cross section of the needle portions are equal to or less than the diameter of the base surface, and the rate of increase in the diameter of the cross section from the tip surface to a point 10 μm below the length of the needle portions is 0 to 5%, the distance between the tip surfaces of the needle portions is 20 to 1000 μm, and at least a portion of the surface of the needle portions is modified with the terpolymer of the present invention.

[0046] Fig. 1 is a schematic longitudinal sectional view of one embodiment (MNA10) of the MNA of the present invention, which comprises a support 1 and multiple needles 2. However, the size ratio of the MNA shown in Fig. 1 does not correspond to the size ratio of the MNA of the present invention. In the following explanation and drawings, the same or corresponding elements are given the same reference numerals, and duplicate explanations are omitted.

[0047] In the present invention, the "support" has the function of supporting the needle part described below, and the surface on which the needle part is arranged is preferably flat (support 1 in FIG. 1). Examples of the shape of the support include a linear shape and a substrate shape (the needle part may be arranged on the upper or lower surface, or on the side surface).

[0048] The size of the support according to the present invention is not particularly limited, but for example, when the needles are arranged in a single row, the width is preferably 0.3 to 20 mm (more preferably 0.5 to 2 mm) and the length (in the row direction) is preferably 1 to 50 mm (more preferably 3 to 10 mm). When the needles are arranged in two or more rows, the width is preferably 1 to 100 mm (more preferably 1 to 50 mm) and the length is preferably 1 to 50 mm (more preferably 3 to 10 mm). The thickness of the support is not particularly limited, and the support may be integrated with a structure that further supports the support.

[0049] The material of the support according to the present invention is not particularly limited as long as it is not toxic to cells, and examples thereof include metals such as silicon (single crystal silicon, etc.), silicon nitride, gold, chromium, indium arsenide, and silica; resins such as polyethylene terephthalate, polyethylene, polypropylene, polystyrene, polycarbonate, and acrylic resin; fibrous materials such as carbon nanotubes and cellulose nanofibers; and Wesker materials made of zinc oxide, silicon carbide, etc., and among these, single crystal silicon is preferred from the viewpoint of the tendency to easily control the strength and microstructure of the support. Furthermore, from the viewpoint of the strength of the MNA, the support according to the present invention is preferably integrated with the needle part described below.

[0050] In the present invention, the "needle" means a convex structure. In other words, the "needle" is not limited to a needle shape having a sharp tip. The needle (needle 2 in FIG. 1) according to the present invention has a base surface (the lower surface of needle 2 in FIG. 1) which is the boundary surface with the support, and a tip surface (the upper surface of needle 2 in FIG. 1) opposite thereto. Note that, when the support and the needle are integral, the boundary surface refers to the surface on which the needle is arranged on a surface that is an extension of the surface on which the needle of the support is arranged.

[0051] In the present invention, the diameter of the basal surface of the needle portion must be 1 to 20 μm, preferably 1 to 10 μm, and more preferably 1 to 5 μm. If the diameter of the basal surface is less than the lower limit, it tends to be difficult to introduce the target substance into the plant cell. On the other hand, if the diameter of the basal surface exceeds the upper limit, it tends to be difficult to insert the target substance into the plant cell contained in the plant tissue. In the present invention, the "diameter of the basal surface" refers to the maximum diameter of the basal surface, and if the basal surface is not a circle, it refers to the diameter of the circumscribed circle of the basal surface (d1 in FIG. 1).

[0052] In the present invention, the diameter of the tip surface of the needle portion must be 0.5 to 3 μm. If the diameter of the tip surface is less than the lower limit, it tends to be difficult to introduce the target substance into the plant cell. On the other hand, if the diameter of the tip surface exceeds the upper limit, it tends to be difficult to insert the target substance into the plant cell contained in the plant tissue. In the present invention, the "diameter of the tip surface" refers to the maximum diameter of the tip surface, and when the tip surface is not a circle, it refers to the outer diameter of the tip surface. This indicates the diameter of the tangent circle (d2 in Figure 1).

[0053] The shape of the needle part according to the present invention may be a columnar shape in which the diameter of the tip surface and cross section of the needle part is equal to or less than the diameter of the base surface (for example, FIG. 1(a)), a frustum shape in which the cross sections from the tip surface to the base surface are continuously homologous, or a trapezoidal plate shape in which a pair of opposite sides of the cross section from the tip surface to the base surface changes continuously (for example, FIG. 1(b)). The needle part may have a structure in which the inclination (angle) of the side surface of the needle part changes continuously, or a multi-layer structure of two or more layers in which the inclination (angle) of the side surface of the needle part changes discontinuously, but is preferably a columnar shape, trapezoidal plate shape, or frustum shape. In the present invention, the "cross section of the needle part" refers to a transverse section parallel to the tip surface and the base surface, and the "diameter of the cross section" refers to the maximum diameter of the cross section, and when the cross section is not a circle, refers to the diameter of the circumscribed circle of the cross section.

[0054] The shapes of the base surface, tip surface, and cross-section of the needle portion are not particularly limited, and include circles, ellipses, triangles, squares, rectangles, trapezoids, and polygons with pentagons or more sides. From the viewpoint of the efficiency of introducing the target substance, however, a circle or a regular polygon is preferable.

[0055] In addition, in the needle according to the present invention, when the diameters of the tip and cross section of the needle are less than the diameter of the base, the cross-sectional diameters at any length from the tip to the length of the needle minus 10 μm (h' in FIG. 1) in the direction of the needle are the same, or the cross-sectional diameter of the base is larger than the cross-sectional diameter of the tip, and the increase rate of the diameter between them is 5% or less, that is, the increase rate of the cross-sectional diameter from the tip to the length of the needle minus 10 μm is 0 to 5%. If the increase rate of the cross-sectional diameter exceeds the upper limit, it tends to be difficult to introduce the target substance into the plant cell. In the present invention, the "increase rate of the cross-sectional diameter" refers to the increase rate (%) of the cross-sectional diameter of the base to the cross-sectional diameter of the tip between any two points on the length axis within the range of minus 10 μm (h': h-10 μm) from the tip to the length of the needle.

[0056] In the present invention, the length of the needle portion must be 30 to 100 μm, and is preferably 50 to 100 μm. If the length is less than the lower limit, it tends to be difficult to insert the needle portion into a plant cell contained in the plant tissue. On the other hand, if the length exceeds the upper limit, it tends to be difficult to insert the needle portion into a plant cell due to buckling. In the present invention, the "length of the needle portion" refers to the length (height) of the perpendicular line drawn from the apical surface to the basal surface (h in FIG. 1).

[0057] The needle part according to the present invention has a higher aspect ratio than conventional microneedles used for introducing a substance into a cell. The aspect ratio is the ratio (length / (diameter of 1 / 2 length face)) of the length of the needle part to the diameter of the cross section at 1 / 2 the length of the needle part, and is preferably 5 to 200, more preferably 10 to 100, and even more preferably 25 to 67. If the aspect ratio is less than the lower limit, it tends to be difficult to insert the needle into a plant cell contained in a plant tissue. On the other hand, if the aspect ratio exceeds the upper limit, it tends to be difficult to insert the needle into a plant cell due to buckling of the needle part. In the present invention, the "diameter of 1 / 2 length face" refers to the maximum diameter of the cross section at 1 / 2 the length of the needle part (2 / h in FIG. 1), and if the cross section is not a circle, it refers to the diameter of the circumscribed circle of the cross section (d3 in FIG. 1).

[0058] In the MNA of the present invention, the multiple needle parts must be arranged with a distance between the tip surfaces of 20 to 1000 μm. The distance between the tip surfaces is preferably 20 to 100 μm. If the distance between the tip surfaces is less than the lower limit, insertion into plant cells contained in plant tissues tends to be difficult. On the other hand, if the distance between the tip surfaces exceeds the upper limit, the number of needle parts per cell tends to decrease, and the introduction efficiency of the target substance tends to decrease. In the present invention, the "distance between the tip surfaces" refers to the distance between the tip surfaces of one needle part and the tip surface of the adjacent needle part, at which the horizontal distance between the points on the outer periphery is the smallest (w in FIG. 1).

[0059] In the MNA of the present invention, the shapes of the multiple needles may be the same or different as long as the above conditions are met. Furthermore, the needles of the present invention may be arranged in any manner on the support as long as the above conditions are met, but it is preferable that they are arranged in one row or multiple rows of two or more rows. The row length of the MNA is preferably 0.5 to 50 mm, more preferably 3 to 10 mm. If the row length is less than the lower limit, handling such as visual alignment tends to become difficult. On the other hand, if the row length exceeds the upper limit, handling as a material tends to become difficult.

[0060] The material of the needle part according to the present invention is not particularly limited as long as it is not toxic to cells. Examples include the various materials mentioned above. Among them, single crystal silicon is preferable from the viewpoint of the tendency to easily control the strength and microstructure of the needle part. Moreover, the needle part according to the present invention is preferably integrated with the support from the viewpoint of the strength of the MNA.

[0061] The MNA of the present invention may have other components in addition to the support and the needle, so long as the effects of the present invention are not impaired. Examples of such other components include a biocompatible polymer bound to the surface of the needle, mainly for the purpose of binding the target substance described below.

[0062] The method for producing the MNA of the present invention is not particularly limited as long as it is a method capable of producing an MNA that satisfies the above conditions, and known methods or methods based thereon can be appropriately adopted. Examples of such methods for producing MNA include a method for producing a needle shape using photolithography and / or etching techniques, a method for producing a resin MNA by injection molding using a mold in which a recess corresponding to the shape of the MNA is formed, a method for stacking a columnar structure by catalytic reaction, and a method for producing a columnar structure by deposition.

[0063] For example, when arranging the needle portions in a single row on the support, a method can be used in which the edge of a wafer that is the material for the support and the needle portions is etched to cut out the needle portions, and the support and the needle portions are molded as a single unit.

[0064] Furthermore, for example, when arranging the needle portions in one or more rows on the support, a method can be used in which a dot-shaped resist mask is applied to a wafer such as a silicon wafer and then etching is performed to form the needle portions, as described in JP 2013-183706 A.

[0065] The MNA of the present invention is characterized in that some or all of its multiple needles are needle-shaped objects whose surfaces have been modified with the above-mentioned terpolymer of the present invention (surface-treated needle-shaped objects of the present invention).The MNA of the present invention has the effect of improving the efficiency of substance introduction compared to MNAs using conventional needle-shaped objects whose surfaces have not been modified or needle-shaped objects whose surfaces have been modified with conventional copolymers.

[0066] [Methods for introducing substances into plant cells] According to the MNA of the present invention, it is possible to introduce a target substance into not only cultured cells but also plant cells contained in plant tissues. Therefore, the present invention also provides an MNA for introducing a target substance into a plant cell, which is used for introducing a target substance into a plant cell.

[0067] The "plant cells" into which the target substance is introduced are not particularly limited, and include, for example, cells of cereals, oil crops, forage crops, fruits, and vegetables. Examples of the plants are also not particularly limited, and include, for example, rice, barley, wheat, rye, barnyard millet, sorghum, corn, banana, peanut, sunflower, tomato, rapeseed, tobacco, potato, soybean, cotton, and carnation. The plant cells include, for example, cells contained in plant tissue in a plant individual or organ, cells contained in plant tissue separated from a plant individual or organ, and cultured cells derived from these plant tissues. Although not particularly limited, the MNA of the present invention can also be applied to cells contained in plant tissue. In the present invention, "cells contained in a plant cell (plant cell)" refers to cells (plant cells) that constitute plant tissue and are not separated from the plant tissue. The tissue may be separated from a plant individual or organ, or may be tissue cultured.

[0068] Examples of the plant organs include leaves, stems, shoot apex, roots, tubers, tuberous roots, seeds, hypocotyls, pollen, and ovaries. Examples of the plant tissues include shoot apical meristems, root apical meristems, shoot apical meristems, and calluses, and among these, shoot apical meristems are preferred.

[0069] In the present invention, the "target substance" to be introduced into the plant cell includes a substance that is expected to have an effect when introduced into the plant cell, and includes, but is not limited to, for example, a protein, a nucleic acid (DNA, RNA), a vector, a peptide, a lipid, a sugar, a low molecular weight compound (coenzyme, toxin, antibiotic, antibacterial drug, antiviral drug, etc.), a metal ion, a metal complex, and a composite molecule containing two or more of these molecules, and one type may be used alone or two or more types may be used in combination. In addition, if necessary, it may be combined with a labeling substance to confirm the introduction.

[0070] For example, the target substance can be introduced into a plant cell using a site-specific nuclease protein, thereby enabling genome editing of the plant cell, but the object of the present invention is not limited to genome editing. Examples of the site-specific nuclease protein include Cas proteins (Cas9, Cpf1 (Cas12), Cas12b, CasX (Cas12e), Cas13, Cas14, etc.), meganucleases, zinc finger nucleases (ZFNs), TALENs, PPR-ND1, and PPR-ND2, and one of these may be used alone or two or more may be used in combination. These may be combined with a guide RNA for a Cas protein, etc., as necessary.

[0071] The present invention also provides a method for introducing a target substance into a plant cell using the above-mentioned MNA of the present invention, comprising an insertion step of inserting into the plant cell the needle part of an MNA of the present invention having the target substance attached to its surface (hereinafter appropriately referred to as the "substance introduction method of the present invention").

[0072] In the substance introduction method of the present invention, the needle of the MNA of the present invention having the target substance attached to its surface is inserted into the plant cell (insertion step). The MNA of the present invention used in the insertion step may be used alone or in a stack of two or more sheets. When two or more sheets are used in a stack, one type may be used alone or a combination of two or more types may be used, and they may be inserted simultaneously or sequentially.

[0073] Examples of methods for attaching the target substance to the needle surface of the MNA include a method in which a solution of the target substance is dropped onto the surface of a target plant cell, or a plant tissue or organ containing the target plant cell, and the needle of the MNA is inserted therein. Alternatively, a method in which the target substance is preliminarily adsorbed onto the needle surface of the MNA, and this is then inserted into the target plant cell, or a plant tissue or organ containing the target plant cell. This allows the target substance to be introduced into the target plant cell (preferably the plant cell contained in the plant tissue). The solvent for the target substance solution is not particularly limited as long as it is not toxic to cells, and examples include buffer solutions and culture media.

[0074] In the above-mentioned insertion, the plant cell or plant tissue or organ is preferably fixed or held on a substrate, and the needle of the MNA is preferably inserted while aligning the insertion site under microscopic observation. The above-mentioned insertion may be performed manually or by an automated method controlled by a computer. When a labeling substance is combined with the target substance, the insertion of the needle of the MNA can be confirmed by detecting the labeling substance by a method appropriate for the labeling substance.

[0075] In the insertion step, the speed at which the needle part is inserted into the plant cell is preferably 0.01 to 50 μm / sec.

[0076] In the insertion step, it is preferable to vibrate the needle, and the condition of such vibration is preferably an amplitude of 0.01 to 50 μm in the insertion direction (parallel to the length direction of the needle). In addition, the frequency of the vibration at this time is preferably 0.1 to 2000 Hz. The combination of the amplitude and frequency is not particularly limited, but for example, an amplitude of 0.1 to 5 μm and a frequency of 1 to 200 Hz. Since plant tissue is hard, buckling of the microneedle (needle) is likely to occur, making insertion difficult, but such vibration makes it difficult for the needle to buckle, allowing easier and more accurate insertion. As a device for applying vibration to the MNA to vibrate in this way, for example, a selective cell separation device described in JP 2011-182761 A can be used as an MNA operation device.

[0077] In the inserting step, when the plant cells are cells contained in a plant tissue, the insertion depth of the needle is preferably 20 to 100 μm, more preferably 20 to 60 μm, from the surface of the plant tissue or an organ containing the plant tissue, thereby enabling the target substance to be introduced into cells in a deep layer of the plant tissue or organ.

[0078] In the substance introduction method of the present invention, after the insertion step, an extraction step of extracting the needle of the MNA from the plant cell allows only the target substance to remain in the target plant cell. The conditions of the extraction step are not particularly limited. The insertion time of the needle is preferably 0.1 to 120 minutes from the start of insertion to the completion of extraction. During the insertion time, it is preferable that the needle is maintained at the insertion depth at which it reaches the target plant cell for 0.1 to 120 minutes. Furthermore, when the needle is maintained at the insertion depth at which it reaches the target plant cell, it is preferable to vibrate the needle, and the conditions for such vibration include the same conditions as those described above.

[0079] In the substance introduction method of the present invention, after the extraction step of the present invention, if necessary, a step of incubating the plant cells and the plant tissue containing the plant cells may be further included. The incubation conditions can be appropriately set depending on the target substance, the plant cells, and the plant tissue.

[0080] According to the substance introduction method of the present invention, a target substance can be introduced into plant cells contained in plant tissue. Therefore, the substance introduction method of the present invention is not particularly limited and can be used for various purposes depending on the target substance. For example, according to the substance introduction method of the present invention, it is possible to introduce a target substance into plant cells in a deep layer of a plant tissue (for example, a layer 20 to 100 μm or a layer 20 to 60 μm from the tissue surface), so that it is possible to introduce the site-specific nuclease protein into plant tissue containing undifferentiated cells such as the shoot apical meristem by an in planta method to perform genome editing, thereby modifying the function of the plant.

[0081] [Probes and cantilevers] One aspect of the present invention relates to a probe having a needle-like tip, which is attached to the cantilever of an atomic force microscope (AFM) or an intermolecular force measuring device for use, and a cantilever having such a probe at its tip. The needle-like tip may be formed as a separate member from the probe and fixed to the probe, or the probe itself may be sharpened to form a needle shape.

[0082] The probe and cantilever of the present invention are characterized in that their needle-shaped tip is a needle-shaped object whose surface is modified with the above-mentioned terpolymer of the present invention (surface-treated needle-shaped object of the present invention). The probe and cantilever of the present invention having a needle-shaped tip whose surface is modified with the terpolymer of the present invention has the effect of reducing non-specific interactions, compared with a conventional needle-shaped object whose surface is unmodified or a probe and cantilever using a needle-shaped object whose surface is modified with a conventional copolymer. EXAMPLES

[0083] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative and are not intended to limit the present invention in any way.

[0084] [Synthesis example: synthesis of DOPA / HPA / NHS copolymer] (1) Synthesis of HPA (2-hydroxypropylacrylamide) monomer 41.04g of potassium carbonate was dissolved in 60mL of water and stirred in a round-bottom flask in an ice bath. 18.58g of 1-amino-2-propanol was dissolved in 200mL of ethyl acetate and added to the flask. 20mL of acryloyl chloride was added dropwise, stirred in ice for 30 minutes, then returned to room temperature and further stirred overnight to react. After the reaction, the target compound was extracted from the aqueous phase with ethyl acetate (oil phase). The oil phase was dehydrated with sodium sulfate and then concentrated with an evaporator to remove as much ethyl acetate as possible. Air bubbling was further performed for 18 hours to completely remove the ethyl acetate, and the target compound was obtained as a transparent liquid. 1H NMR (D2O): δ (ppm): 1.06 (d, 3H), 3.14 (m, 1H), 3.21 (br , 1H), 3.84 (m, 1H), 5.66 (dd, 1H), 6.08 (m, 1H), 6.17 (dd , 1H)..

[0085] (2) Synthesis of DOPA (N-(3,4-dihydroxyphenethyl)methacrylamide) monomer 8.0g of dopamine hydrochloride and 4.309g of triethylamine were dissolved in 80mL of ultra-dehydrated methanol. A tetrahydrofuran solution containing 5.29g of methacryloyl chloride and a methanol solution containing 6.278g of triethylamine were alternately dropped into the resulting solution in an ice bath. After stirring at room temperature for 2 hours, the solution was washed with 1M hydrochloric acid. After dehydration with sodium sulfate, the solution was concentrated in an evaporator. The resulting powdery compound was recrystallized and purified using ethyl acetate to obtain the target compound as a brownish-white powder. 1H NMR (DMSO-d6): δ (ppm): 1.80 (s, 3H, CH2CCH3), 2.47 (t, 2H, C6H3CH2CH2), 3.13-3.25 (m, 2H, C6H3CH2CH2), 5.26, 5.58 (s, 2H, CH2CCH3), 6.33-6.66 (m, 3H, C6H3), 7.89 (s, 1H, CONH), 8.59, 8.69 (s, 2H, OH).

[0086] (3) Synthesis of DOPA / HPA / NHS terpolymer HPA / NHS / DOPA / RAFT / AIBN were mixed in a molar ratio of 170 / 10 / 20 / 1 / 0.1 or 160 / 20 / 20 / 1 / 0.1, respectively, and polymerized in DMF under an argon atmosphere in an oil bath at 70°C for 2 hours to synthesize a DOPA / HPA / NHS terpolymer. After the reaction, the molecular weight and molecular weight distribution of the resulting polymer were determined by DMF-based GPC measurement. The resulting terpolymer was purified by reprecipitation recovery using acetone. 1 It was confirmed by 1 H NMR that the unreacted monomers had been removed and that the contents of HPA, BPA, and NHS in the polymer chain were almost consistent with the feed ratios.

[0087] [Table 1] [ka]

[0088] [Example group A: Study on MNA needle surface modification] (1) Preparation of MNA A silicon single crystal was machined to produce a microneedle array 10 integral with the support, in which 167 needle parts, all of which are regular square prisms (length of one side of square cross section: 1 μm, length: 100 μm) with the same cross section from the tip surface to the base surface, were aligned in a row with a distance of 30 μm between the tip surfaces. An SEM photograph of the appearance of the obtained microneedle array 10 is shown in Figure 2.

[0089] (2) Surface modification of the MNA needle Example A1: MNA needle surface modification with DOPA / HPA / NHS copolymer The surface of the MNA (MNA) was cleaned by immersing it in isopropanol for 1 minute and 1% hydrofluoric acid for 1 minute. The cleaned MNA was treated with UV-ozone for 30 minutes, and then cleaned by immersing it in an SPM solution prepared with a 1:1 ratio of sulfuric acid and hydrogen peroxide for 10 minutes at room temperature. The treated MNA was washed with ultrapure water and ethanol, and then immersed in a hydrochloric acid-hydrogen peroxide mixture (HPM) made by mixing ultrapure water, hydrochloric acid, and hydrogen peroxide in a ratio of 6:1:1, and reacted at 50°C for 30 minutes to hydroxylate the silicon surface of the MNA. The MNA surface was then washed with ultrapure water, ethanol, and 10 mM borate buffer (pH 9.0). The DOPA / HPA / NHS=10 / 85 / 5 copolymer obtained in the synthesis example was dissolved in a borate buffer solution containing 10% ethanol at 0.5 wt% to prepare a solution. The washed MNA was immersed in this DOPA / HPA / NHS polymer solution and left to stand overnight at 18°C. Then, EDS / NHS reaction was carried out in MES buffer solution (pH 5.0) for 15 minutes to reintroduce NHS groups into the polymer modified with MNA. After washing the polymer modified MNA with MES buffer solution and borate buffer solution, it was reacted in a 10 mM N-(5-amino-1-carboxypentyl)iminodiacetic acid free form (AB-NTA) solution dissolved in borate buffer solution at 37°C for 2 to 3 hours. After washing the polymer modified MNA after the reaction with borate buffer solution, it was reacted with an ethanolamine solution with a final concentration of 20 mM dissolved in borate buffer solution at room temperature for 1 hour to block the unreacted NHS groups. After blocking, the polymer-modified MNA was washed with borate buffer and distilled water, and then reacted at room temperature for 1 hour in a nickel (II) chloride (NiCl2) solution with a final concentration of 100 mM dissolved in ultrapure water. The polymer-modified MNA after the reaction was immersed in a His-tag fusion protein solution and modified by reacting overnight at 4°C to produce MNA whose surface was modified with DOPA / HPA / NHS copolymer.

[0090] Comparative Example A1: Modification of MNA surface with silane-PEG-NHS copolymer The surface of the MNA was washed with 1% hydrofluoric acid, and then treated with a sulfuric acid-hydrogen peroxide mixture (SPM) and a hydrochloric acid-hydrogen peroxide mixture (HPM) to hydroxylate the surface. The resulting hydroxylated MNA was immersed in a solution of silane-PEG-NHS (final concentration 10 mg / mL) dissolved in dimethyl sulfoxide (DMSO) and reacted at room temperature for 1 hour. After the reaction, the MNA was immersed in a free form of N-(5-amino-1-carboxypentyl)iminodiacetic acid (AB-NTA; final concentration 10 mM) dissolved in 0.1 M HEPES buffer (pH 8.0) and reacted at 37°C for 1 hour, and then blocked with a solution of ethanolamine (final concentration 20 mM) dissolved in HEPES buffer for 1 hour. After blocking, the modified MNA was modified by reacting it overnight at 4°C in a GFP solution diluted with PBS (pH 7.5) to a final concentration of 40 μg / mL, thereby producing MNA whose surface was modified with silane-PEG-NHS copolymer.

[0091] Comparative Example A2: Modification of MNA surface with APTES-GTA copolymer The surface of the MNA was washed with 1% hydrofluoric acid, and then SPM and HPM treatments were performed to hydroxylate the surface. The resulting surface-hydroxylated MNA was washed in turn with ultrapure water, ethanol, and toluene. The washed MNA was immersed in a 1 (v / v)% APTES solution dissolved in toluene and reacted at 70°C for 1 hour. Thereafter, it was washed with toluene and ethanol, and reacted at 37°C for 1 hour in 10% glutaraldehyde dissolved in ethanol. The modified MNA after the reaction was modified by reacting it overnight at 4°C in a GFP solution with a final concentration of 40 μg / mL diluted with PBS (pH 7.5) to prepare MNA whose surface was modified with APTES-GTA copolymer.

[0092] Measurement of the amount of GFP immobilized and released by MNA Green fluorescent protein (GFP) was immobilized by a conventional method on the DOPA / HPA / NHS polymer-modified MNA of Example 1, the silane-PEG-NHS-modified MNA of Comparative Example 1, and the APTES-GTA-modified MNA of Comparative Example 2 obtained by the above procedure. Each of the obtained GFP-immobilized MNAs was immersed in a buffer solution simulating plant cytoplasm (100 mM potassium chloride, 30 mM sodium chloride, 500 mM mannitol, 1.2 M sucrose, 25 mM MES, 25 mM HEPES, 0.4 mM magnesium chloride (MgCl2), pH 8.0) and allowed to stand for 90 seconds to release GFP. The GFP fluorescence intensity per needle was calculated from the fluorescent microscope images of the MNA before and after immersion, and the amount of GFP immobilized and released was compared.

[0093] The results are shown in the graph in Figure 3. Approximately 76% of the protein immobilized on the DOPA / HPA / NHS-modified MNA in Example 1 was released in the plant cytoplasm-mimicking buffer, whereas the amount of protein released on the silane-PEG-NHS-modified MNA in Comparative Example 1 was 45%, and the amount of protein released on the APTES-glutaraldehyde-modified MNA in Comparative Example 2 was only 47%.

[0094] Direct delivery of Cas9-gRNA complexes into Arabidopsis leaves using MNA 2.5 pmol of Cas9 and 7.5 pmol of forward sgRNA and reverse sgRNA were added to Cas9 buffer (20 mM HEPES, 500 mM NaCl, 10% glycerol, pH 7.4) and the solution volume was adjusted to 10 μL. The Cas9-gRNA complex was formed by reacting on ice for 10 minutes. MNA modified with DOPA / HPA / NHS copolymer of Example 1 and reacted with Ni-NTA was immersed in this Cas9-gRNA complex solution and reacted overnight at 4 ° C. This MNA was placed on the stage of the array operation device, and the xGxGUS reporter Arabidopsis thaliana leaf was fixed to the sample stage with adhesive double-sided tape. The β-glucuronidase (GUS) reporter gene integrated into the genome of this reporter Arabidopsis thaliana contains a partial sequence of the phytoene desaturase (PDS) gene that is the target of the gRNA. A sequence identical to the part of the GUS gene preceding the PDS sequence also exists following the PDS sequence. When the Cas9 / sgRNA complex cleaves within the PDS sequence, homologous recombination occurs between the repeated sequences, and the GUS gene is modified into a normal GUS gene (Figure 4).

[0095] 5 mM magnesium chloride (MgCl2), 0.4 U / μL RNase inhibitor, and 0.05% Silwet (登録商標)Nuclease-free water containing 100% ethanol was dropped into the gap between the MNA and the leaf tissue. The MNA was inserted into the leaf tissue at a speed of 10 μm / sec, left at rest for 1 minute, and then removed at a speed of 10 μm / sec. The leaf tissue was incubated for 48 hours on a B5 medium plate (0.5% agar, 20 g / L glucose, 0.6 g / L MES, 1x Gamborg B5 medium mixed salts, 1x Murashige and Skoog vitamin solution (MS vitamins, pH 5.7). The leaf tissue was then incubated for 5 hours at 37°C in a staining solution (20% (w / v) methanol, 0.1% TritonX-100, 50 mM sodium dihydrogen phosphate (NaH2PO4, pH 7.0) containing 0.5 mg / mL of X-glucuronide. The leaves were then placed in a 6:1 mixture of ethanol and acetic acid and allowed to stand overnight to decolorize. After washing with 99.5% ethanol and sterile distilled water, the tissue was placed on a slide and the blue color was observed under an inverted microscope (Figure 5).

[0096] The results are shown in Table 3 below. It was confirmed that the genome editing efficiency of the MNA of Example 1 modified with DOPA / HPA / NHS copolymer was improved by about 20% compared to the case where untreated MNA was used.

[0097] [Table 2]

[0098] [Example Group B: Study on AFM probe surface modification] (1) Surface modification of AFM tips Example B1: AFM tip surface modification with DOPA / HPA / NHS copolymer An unmodified AFM probe was treated with a plasma asher at 200 W for 10 minutes, and then immersed in ultrapure water. It was then immersed in an SPM solution of 0.5 mL of hydrogen peroxide and 2 mL of sulfuric acid, and left at 80°C for 30 minutes. It was then washed with ultrapure water, immersed in an APM solution of 0.4 mL of hydrogen peroxide, 0.4 mL of ammonia water, and 2 mL of ultrapure water, left at 65°C for 30 minutes, and then washed with ultrapure water. Next, the AFM probe was immersed in an SPM solution of 1 mL of hydrogen peroxide and 1 mL of sulfuric acid, and left at room temperature for 10 minutes to generate surface hydroxyl groups on the silicon surface. The treated AFM probe was washed with ultrapure water and then washed with dehydrated ethanol to remove water molecules adsorbed on the surface. Furthermore, the substrate was immersed in an HPM solution prepared by mixing 0.4 mL of hydrogen peroxide, 0.4 mL of hydrochloric acid, and 2 mL of ultrapure water, and left to stand at 80° C. for 10 minutes, and then washed with ultrapure water and dehydrated ethanol.

[0099] DOPA / HPA / NHS polymer powder was added to a dimethyl sulfoxide solution to a final concentration of 5 wt% and dissolved. The solution was further diluted with a 10 mM borate buffer (pH 9.0) to a final concentration of 0.5 wt% to obtain a polymer solution. The AFM probe with its surface cleaned was immersed in this polymer solution and reacted for 12 hours. N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC) were dissolved in a 10 mM MES buffer (pH 4.7) to a final concentration of 50 mM, and the AFM probe was left in 100 μL of this solution for 30 minutes, and then washed with MES buffer. The AFM probe was left in AB-NTA dissolved in a borate buffer to a final concentration of 10 mM for 30 minutes to introduce NTA groups, and the unreacted NHS groups were blocked by leaving the AFM probe in a solution of ethanolamine (final concentration 10 mM) dissolved in borate buffer for 30 minutes. The membrane was immersed in 100 mM nickel chloride (II) (NiCl2) dissolved in ultrapure water for 10 minutes, and then left to stand in a 20 nM nestin tail protein solution at 4°C for more than 8 hours for modification (Figure 6).

[0100] Comparative Example B1: AFM tip surface modification with silane-PEG-NHS copolymer The surface of the AFM probe was washed with 1% hydrofluoric acid, then treated with a sulfuric acid-hydrogen peroxide mixture (SPM) and a hydrochloric acid-hydrogen peroxide mixture (HPM) to hydroxylate the surface. The resulting hydroxylated AFM probe was immersed in a solution of silane-PEG-NHS (final concentration 10 mg / mL) dissolved in dimethyl sulfoxide (DMSO) and reacted at room temperature for 1 hour. N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC) were dissolved in 10 mM MES buffer (pH 4.7) to a final concentration of 50 mM, and the AFM probe was left in 100 μL of this solution for 30 minutes, and then washed with MES buffer. The AFM probe was left for 30 minutes in AB-NTA dissolved in borate buffer to a final concentration of 10 mM to introduce NTA groups, and then left for 30 minutes in a solution of ethanolamine (final concentration 10 mM) dissolved in borate buffer to block unreacted NHS groups. After immersion in 100 mM nickel (II) chloride (NiCl2) dissolved in ultrapure water for 10 minutes, the blocked modified AFM probe was modified by leaving it in a 20 nM nestin tail protein solution at 4°C for more than 8 hours.

[0101] (2) Preparation of actin-immobilized substrate A skeletal muscle actin protein solution with a final concentration of 4 μM was prepared using actin polymerization buffer (10 mM HEPES (pH 7.4), 0.15 M potassium chloride, 2 mM magnesium chloride, 0.2 mM ATP, 1 mM DTT) and allowed to stand at room temperature for 30 minutes to polymerize actin fibers. After that, rhodamine phalloidin (final concentration 0.4 μM) was added and the dish was allowed to stand on ice for more than 1 hour. Next, 0.4 mL of 1 mM (3-aminopropyl) triethoxysilane (APTES) solution dissolved in ethanol was dropped onto a 27φ glass base dish and allowed to stand for 1 hour. After washing three times with ethanol, 0.4 mL of 10% glutaraldehyde solution dissolved in ethanol was dropped and allowed to stand for 1 hour. After washing three times with ethanol, the dish was washed once with 1 mL of actin polymerization buffer. 20 μL of actin filament solution diluted with actin polymerization buffer to a final concentration of 400 nM was dropped onto the plate, and the plate was immobilized by leaving the plate at room temperature for 30 minutes (Figure 7). The plate was then washed three times with actin polymerization buffer at 1 mL, 5 minutes, and 100 rpm. Finally, 2 μM BSA solution dissolved in actin polymerization buffer was dropped onto the plate, and the plate was left at room temperature for 30 minutes to block aldehyde groups not bound to actin. The plate was then washed three times with BSA-containing actin polymerization buffer at 1 mL, 5 minutes, and 100 rpm.

[0102] (3) Tensile test of nestin tail using AFM A nestin tail tensile test was performed at 64 points (8 × 8) within a square of 10 μm on a side of an actin filament-immobilized glass substrate by moving the nestin tail-modified probe up and down at a set point of 1 nN and a speed of 1 μm / s using an AFM, and the force curves obtained were analyzed. A characteristic curve obtained by polymer stretching is shown in FIG. 9(a). FIG. 9(b) shows a curve of stretch distance 0, which indicates the bond breaking force of nonspecific interaction between the substrate and the probe. The number of force curves in which a peak of stretch distance 0 in FIG. 9(b), which indicates nonspecific interaction, was observed among the obtained force curves was counted. The number of curves was 23 out of 64 curves for the AFM probe of Example B1, in which the nestin tail was modified with DOPA / HPA / NHS copolymer, and 40 out of 64 curves for the AFM probe of Comparative Example B1, in which the nestin tail was modified with silane-PEG-NHS copolymer. That is, the occurrence frequency of nonspecific interactions was 35.9% for the AFM probe of Example B1 modified with the DOPA / HPA / NHS copolymer, and 62.5% for the AFM probe of Comparative Example B1 modified with the silane-PEG-NHS copolymer. That is, it was confirmed that nonspecific interactions between the substrate and the probe were significantly suppressed by modifying the nestin tail protein with the DOPA / HPA / NHS copolymer of the present invention. [Industrial Applicability]

[0103] The terpolymer of the present invention has the effect of improving the adsorption efficiency, adsorption specificity, release efficiency, etc. of substances when used for surface modification of substance-adsorbing needle-shaped objects such as microneedles, nanoneedles, and cantilevers of atomic force microscopes (AFMs), and thus has extremely high applicability in the fields of experiments and analyses in physics, chemistry, biology, etc. [Explanation of symbols]

[0104] 1: Support 2: Needle part 10: Microneedle array

Claims

1. A terpolymer having a structure represented by formula (1): 【Chemistry 1】 (However, in formula (1), R １１ represents a linear or branched divalent aliphatic hydrocarbon group having 1 to 6 carbon atoms, R １２ represents hydrogen or methyl, R ２１ represents a linear or branched monovalent aliphatic hydrocarbon group having 1 to 6 carbon atoms substituted with 1 to 3 hydroxyl groups, R ２２ represents hydrogen or methyl, R ３ represents hydrogen or methyl, Ring A represents an aromatic hydrocarbon group having 1 to 3 hydroxyl groups; Ring B represents a nitrogen-containing cyclic group having 1 to 3 oxo groups; T １ and T ２ each independently represents a terminal group, x represents a natural number between 1 and 5000, y represents a natural number between 1 and 8000, z is a natural number between 1 and 5000, Here, the x, y, and z monomer units may exist as blocks or may coexist randomly.

2. 2. The terpolymer of claim 1, wherein Ring A is a dihydroxyphenyl group.

3. 2. The terpolymer of claim 1, wherein Ring B is an N-succinimide group.

4. A surface-modified needle-shaped object, at least a portion of whose surface is modified with the terpolymer according to claim 1.

5. A microneedle array comprising a support and a plurality of needle portions arranged on the support, A microneedle array, wherein some or all of the plurality of needle portions are the surface-modified needle-shaped objects described in claim 4.

6. The diameter of the base surface of the needle portion is 1 to 20 μm, The diameter of the tip surface of the needle portion is 0.5 to 3 μm, The length of the needle portion is 30 to 100 μm, The diameters of the tip surface and cross section of the needle portion are equal to or smaller than the diameter of the base surface, and the rate of increase in the diameter of the cross section from the tip surface to the length of the needle portion minus 10 μm is 0 to 5%, The microneedle array according to claim 5, wherein the distance between the tip surfaces of the needle portions is 20 to 1000 μm.

7. The microneedle array according to claim 6, wherein the aspect ratio of the needle portion (length / (diameter of 1 / 2 length face)) is 5 to 200.

8. The microneedle array according to claim 6 , wherein the support and the needle portion are integral and made of single crystal silicon.

9. The microneedle array according to claim 6, which is used for introducing a target substance into a plant cell.

10. The microneedle array according to claim 9 , wherein the plant cells are cells contained in plant tissue.

11. The microneedle array according to claim 10 , wherein the plant tissue is a shoot apical meristem.

12. A method for introducing a target substance into a plant cell, an insertion step of inserting the needle portion of the surface-modified needle-shaped article according to claim 4 or the microneedle array according to claim 5, having the target substance attached to the surface of the plant cell; A method for introducing a substance into a plant cell, comprising:

13. The method for introducing a substance into a plant cell according to claim 12, wherein the insertion speed in the insertion step is 0.01 to 50 μm / sec.

14. 13. The method for introducing a substance into a plant cell according to claim 12, wherein in the inserting step, the needle is vibrated in the insertion direction at an amplitude of 0.01 to 50 μm and a frequency of 0.1 to 2000 Hz.

15. The method for introducing a substance into a plant cell according to claim 12, wherein the plant cell is a cell contained in a plant tissue, and the method is a method for introducing a substance into a plant tissue.

16. A probe having a needle-like tip that is attached to a cantilever of an atomic force microscope (AFM) or an intermolecular force measurement device, A probe, wherein the needle-shaped tip is the surface-modified needle-shaped object according to claim 4 .

17. A cantilever for an atomic force microscope (AFM) or molecular force measuring device, comprising the probe of claim 16.

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