JP5311456B2 - Fabrication method of two-dimensional fine thin film - Google Patents

Description

  The present invention relates to a technique for producing a two-dimensional fine thin film on a substrate from an organic molecular material having a peptide chain.

  A monomolecular film (two-dimensional fine thin film) made of an organic molecular material having a peptide chain (hereinafter sometimes referred to as “peptide material”) is expected to be applied as a functional material (Non-patent Document 1). . As one of general methods for producing a monomolecular film on a substrate, a Langmuir-Blodgett method (Langmuir-Blodgett method, LB method) is known. In the LB method, a monomolecular film is produced by transferring a film of amphiphilic molecules compressed to an interface between two different phases (typically an air / water interface) onto a solid substrate. Non-patent documents 2 and 3 are cited as prior art documents related to the preparation of monomolecular films of peptide materials by the LB method.

Journal of the American Chemical Society Vol. 121, pp. 7266-7267, 1999. Chemistry Letters Vol. 36, No.4, pp. 562-563, 2007. Journal of the American Chemical Society Vol. 122, pp. 12523-12529, 2000.

However, the LB method is not simple because it involves precise control of the compression pressure and requires a special device. Moreover, since a highly water-soluble peptide material cannot form a thin film at the air / water interface, it is difficult to produce a monomolecular film of such a peptide material by the LB method.
Accordingly, an object of the present invention is to provide a method for easily producing a monomolecular film of a peptide material on the surface of a substrate using a solution in which the peptide material is in a free state.

  When the present inventors supply a solution in which an organic molecular material having a predetermined peptide chain (peptide material) is free to a predetermined substrate surface, the organic molecular material is adsorbed on the substrate surface. Then, the present inventors have found that a plurality of the peptide chain portions exhibiting an α-helix or a random coil structure are adjacent to each other on the substrate to form a β-sheet structure, thereby completing the present invention.

According to the present invention, a method for producing a two-dimensional fine thin film on a substrate using a predetermined organic molecular material (peptide material) is provided. That is, the two-dimensional fine thin film production method according to the present invention is a method of producing a two-dimensional fine thin film on a substrate,
As an organic molecular material for forming the thin film, it has at least a peptide chain,
The peptide chain is, have at least a hydrophilic amino acid residues and hydrophobic amino acid residues, can be adsorbed on the base material, when it is intake wearing an amino acid sequence capable of forming a β- sheet structure ,
Formula _{(X 1 -X 2 -X 3 -X} 4) n ( where, n is an integer of 2 or _more, X _1, X 2, at least one of _{X 3} and _{X 4} are the same to each other or A step of preparing an organic molecular material characterized by having an amino acid sequence structure represented by different hydrophilic amino acid residues and the rest being the same or different hydrophobic amino acid residues. When,
Preparing a solution containing the organic molecular material at a concentration at which the peptide chain can maintain an α-helix or a random coil structure and in which the organic molecular material is individually released and dispersed;
The solution is supplied to the surface of the substrate, the organic molecular material in the solution is adsorbed on the substrate, and the peptide chains of the organic molecular material when adsorbed on the substrate form β-sheets, respectively. Providing a monomolecular film formed on the substrate;
This is a method for producing a two-dimensional fine thin film containing.

  As described above, the organic molecular material used in the method for producing a two-dimensional fine thin film of the present invention has a peptide chain composed of 4 to 50 amino acid residues as its constituent elements. In addition, the organic molecular material used in the two-dimensional fine thin film manufacturing method of the present invention has a property that can be adsorbed to the base material used in the present method, and forms a β-sheet structure when adsorbed. It has the resulting amino acid sequence. That is, the organic molecular material used in the present method has an amphiphilic peptide chain (that is, having both a hydrophilic amino acid residue and a hydrophobic amino acid residue) capable of forming a β-sheet structure. In the method according to the present invention, the width of the β-sheet can be controlled to be nano-sized (typically about 20 nm or less) according to the number of amino acid residues constituting the peptide chain. A monomolecular film composed of fine peptide fibers (peptide fibers) having a certain nanosize width (that is, a predetermined peptide chain length defined by the number of amino acid residues) can be produced.

Further, in a preferred embodiment of the two-dimensional fine thin film manufacturing method disclosed herein, the peptide chain may contain any amino acid residue in addition to the predetermined hydrophilic amino acid residue and hydrophobic amino acid residue. Often, the β-sheets formed by the peptide chains can be parallel or antiparallel.
Further, in a preferred embodiment of the two-dimensional fine thin film manufacturing method disclosed herein, the organic molecular material includes a chemical structure other than the peptide chain (for example, a protective group, a spacer, a functional group, a functional group, etc.). obtain. Therefore, in the present specification, the “organic molecular material (peptide material)” means an organic molecular material that has at least the above-described peptide chain and can contain a chemical structure other than the peptide chain. In a particularly preferred embodiment, the organic molecular material is characterized in that a spacer is bonded to a part of the peptide chain. Suitable examples of such spacers include polyethylene glycol (PEG) having various molecular weights.

In addition, the peptide chain contained in the organic molecular material used in the two-dimensional fine thin film manufacturing method of the present invention has a predetermined length (or a fine thin film to be formed) defined by the number of amino acid residues when adsorbed on the base material. Depending on the formation pattern, the β-sheet may have a composition (amino acid sequence) that can form a β-sheet structure having a line width), the types of hydrophilic and hydrophobic amino acid residues that are constituent elements thereof, There is no particular restriction on the order. However, from the viewpoint of ease of synthesis and the like, an amino acid sequence structure in which a predetermined amino acid sequence is repeated can be suitably employed.
In other words, the general formula _{I: (X 1 -X 2 -X} 3 -X 4) can be preferably used a peptide chain having a structure represented by n. Here, n is an integer of 2 or more (for example, 2 to 12, preferably 2 to 8, particularly preferably 3 to 5), and at least one of X ₁ , X ₂ , X ₃ and X ₄ is mutually The same or different hydrophilic amino acid residues, and the rest are the same or different hydrophobic amino acid residues. In the present specification and claims, the above for the general formula X in I _{1 (X 2} be about to X ₄ the same.) "Mutually same or different" is represented by the general formula The position of X _{1 in} the formula which is repeatedly present in the amino acid sequence according to the number of n, such as the first X ₁ counted from the N-terminal, the next X ₁ , and the next X ₁ among the repeated amino acid sequences This means that all the amino acid residues may be the same type of hydrophilic (or hydrophobic) amino acid residues, or may be different types of hydrophilic (or hydrophobic) amino acid residues. . That is, the above general formula represents the presence pattern of hydrophilic amino acid residues and hydrophobic amino acid residues in the repetitive amino acid sequence structure represented by the formula. From the viewpoint of ease of synthesis, ease of formation of β-sheet, etc., an amino acid sequence in which a predetermined amino acid sequence consisting of a specific amino acid residue (for example, the amino acid sequence shown in parentheses in the above general formula) is repeated. A structure can be suitably employed.

The composition of the above general formula I (amino acid sequence) is obtained when the peptide chain of the organic molecular material has an α-helix or random coil structure in a state dispersed in the solvent, and is adsorbed on the substrate (monomolecular film). As long as it has a β-sheet structure, there are no particular restrictions on the proportion and order of hydrophilic amino acid residues and hydrophobic amino acid residues. Typically, X ₁ and X ₃ in the formula may each be the same or different hydrophilic amino acid residues, and X ₂ and X ₄ may each be the same or different hydrophobic amino acid residues. . X ₁ and X ₃ may be the same or different hydrophobic amino acid residues, and X ₂ and X ₄ may be the same or different hydrophilic amino acid residues.

The hydrophilic amino acid residues constituting the peptide chain can be, for example, independently any of aspartic acid, glutamic acid, arginine, lysine, histidine, serine, threonine, asparagine, glutamine, and tyrosine. Preferably, the hydrophilic amino acid residues constituting the peptide chain are each independently selected from the group consisting of aspartic acid, glutamic acid, arginine and lysine.
Further, the hydrophobic amino acid residues constituting the peptide chain can be independently any of valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, glycine, and alanine. Preferably, the hydrophobic amino acid residues constituting the peptide chain are each independently selected from the group consisting of valine, leucine, alanine and isoleucine.

Further, in the two-dimensional fine thin film manufacturing method of the present invention, one kind of solvent or a mixture of two or more kinds of mutually soluble solvents is used for preparing the solution containing the organic molecular material in a dispersed state. . Preferably, when any solvent selected from the group consisting of at least water and a lower alcohol solvent (for example, methanol, ethanol, butanol, isopropanol) is used, the peptide material is easily dissolved and in a free state (dispersed state). Can keep stable. Moreover, the said solution may contain substances other than the said peptide material, such as an acid and a base.
The concentration of the peptide material contained in this solution is such that the peptide material can remain free, and when the peptide material is adsorbed on the substrate surface, β-sheets can be efficiently formed in a two-dimensional direction. The degree (preferably about 1 × 10 ⁻⁶ to 1 × 10 ⁻⁴ mol / L).

Moreover, the base material used in the two-dimensional fine thin film manufacturing method disclosed herein is not particularly limited in material (composition), shape, size, and the like as long as the peptide material can be adsorbed. Although various metal substrates or ceramic substrates can be used, a substrate having a highly hydrophilic surface is preferred. For example, a suitable example includes a base material in which at least one surface (side on which a film is provided) is made of any material selected from the group consisting of silicon carbide (SiC), silica glass, alumina, and mica.
Therefore, as another aspect of the present invention, a monomolecular film is formed on a substrate having a relatively high hydrophilicity (for example, a substrate made of the above-described substance) using a highly hydrophobic peptide material. By making it, the surface of the base material can be modified. That is, as another aspect of the present invention, a method for producing a monomolecular film made of a peptide material (organic molecular material) having water repellency on the surface of a hydrophilic substrate is provided. Moreover, the article | item provided with the highly water-repellent two-dimensional fine thin film (monomolecular film) on the base material produced by the two-dimensional fine thin film production method disclosed here is provided.
In addition, since the hydrophobic surface has adhesiveness to cells, a substrate having such a surface can be used for cell culture or the like.
Accordingly, as yet another aspect of the present invention, there is provided a substrate having on its surface a peptide thin film produced by any of the two-dimensional fine thin film production methods disclosed herein. As a result of having such a peptide thin film on the surface, such a substrate has high living tissue adaptability. Here, “living tissue adaptability” refers to easiness of survival, proliferation, etc. of living tissue such as cells and tissues (organs) on a substrate. For example, the improvement of the adhesion of the adherent cells to the substrate surface means the improvement of the adaptability of the living tissue here. By applying the two-dimensional fine thin film preparation method provided by the present invention to produce a peptide thin film on the surface of the substrate, it is possible to improve the adaptability of the substrate to the living tissue, such as improvement of the adhesion of adhesive cells. .

  In the two-dimensional fine thin film manufacturing method of the present invention, the method for supplying the solution to the substrate surface is not particularly limited. For example, the solution can be supplied to the surface of the substrate without using a special apparatus by a method of immersing the substrate in a solution or a method utilizing capillary action. Thus, according to the method of the present invention, since it is not necessary to form a thin film of the peptide material at the gas phase / liquid phase interface, a simple method can be used on a predetermined substrate without using a special apparatus. A monomolecular film of the organic molecular material (peptide material) can be efficiently produced.

Hereinafter, preferred embodiments of the present invention will be described. Note that matters other than matters specifically mentioned in the present specification and necessary for the implementation of the present invention can be grasped as design matters of those skilled in the art based on the prior art in this field. The present invention can be carried out based on the contents disclosed in this specification and common technical knowledge in the field.
Moreover, the peptide material to be used can be produced by generally known chemical synthesis or genetic engineering biosynthesis. Since such a synthesis method itself does not characterize the present invention in any way, a detailed description is omitted.

  In the two-dimensional thin film manufacturing method disclosed herein, an organic molecular material having an amphiphilic peptide chain is used. The total number of amino acid residues in the peptide chain is preferably 4 or more and 50 or less, more preferably 8 or more and 32 or less. 12 or more and 20 or less are particularly preferable. As a result, a preferable β-sheet is formed at the time of adsorption to the base material, and a fine β-sheet (peptide) having a line width corresponding to the chain length of the peptide chain of about 20 nm or less (more preferably 10.5 nm or less). Monolayers composed of fibers) can be produced.

The hydrophilic amino acid residue and hydrophobic amino acid residue constituting the amphiphilic peptide chain are preferably those in which the peptide chain can form a linear structure (that is, a structure that can be a component of β-sheet). use. Although not particularly limited, preferred examples of the hydrophilic amino acid residue include aspartic acid, glutamic acid, arginine, lysine, histidine, serine, threonine, asparagine, glutamine, and tyrosine. In particular, aspartic acid, glutamic acid, arginine, and lysine are preferable.
Similarly, although not particularly limited, preferred examples of the hydrophobic amino acid residue include valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, glycine and alanine. In particular, valine, leucine, alanine, and isoleucine are preferable.

As long as the peptide chain can form a β-sheet on the substrate, the composition (type, ratio, order, etc. of hydrophilic and hydrophobic amino acid residues contained) is not particularly limited. However, from the viewpoint of ease of synthesis and the like, a structure in which a predetermined amino acid sequence is repeated is preferable. For example, a structure represented by the general formula I: (X ₁ -X ₂ -X ₃ -X ₄ ) n can be preferably adopted. Here, preferably, n is an integer of 1 or more, at least one of X ₁ , X ₂ , X ₃ and X ₄ is a hydrophilic amino acid residue which is the same or different from each other, and the rest are the same as each other Or a different hydrophobic amino acid residue.

As a preferred composition of the above general formula I, for example, any one of X ₁ , X ₂ , X ₃ and X ₄ is a hydrophilic amino acid residue, and the rest are the same or different hydrophobic amino acid residues from each other Composition, composition in which any one of X ₁ , X ₂ , X ₃ and X ₄ is a hydrophobic amino acid residue and the rest are the same or different hydrophilic amino acid residues, and X ₁ , X ₂ , X Examples include a composition in which any two of ₃ and X ₄ are the same or different hydrophilic amino acid residues, and the remaining two are the same or different hydrophobic amino acid residues. Especially, the composition of the aspect which the hydrophilic amino acid residue and the hydrophobic amino acid residue couple | bonded alternately is especially preferable. That is, a composition in which X ₁ and X ₃ in the formula are the same or different hydrophilic amino acid residues, and X ₂ and X ₄ are the same or different hydrophobic amino acid residues, or X ₁ and X 3 _A composition in which ₃ is the same or different hydrophobic amino acid residues and X ₂ and X ₄ are the same or different hydrophilic amino acid residues can be particularly preferably used. Since the peptide chain having such a composition is linearly extended, only a hydrophilic amino acid residue is arranged on one side and only a hydrophobic amino acid residue is arranged on the other side, so that the β-sheet is particularly efficient. Can be formed.
As one of the preferable specific examples included in the general formula I, a peptide chain (LELE) having a total of 16 residues consisting of a repeating sequence of leucine (L) and glutamic acid (E) from the N-terminus described in SEQ ID NO: 1 ₄ is mentioned.
As another specific example, a peptide chain of 16 residues in total consisting of a repetitive sequence of arginine (R), alanine (A), aspartic acid (D) and alanine (A) from the N-terminus described in SEQ ID NO: 2 ( RADA) ₄ .
As another specific example, a peptide chain of 16 residues in total (LELL) consisting of a repeating sequence of leucine (L), glutamic acid (E), leucine (L) and leucine (L) from the N-terminus described in SEQ ID NO: 3 ₄ ).

In addition to the general formula I, the peptide chain having a structure that repeats the predetermined amino acid sequence may preferably have a structure represented by, for example, the general formula II: (Y ₁ -Y ₂ -Y ₃ ) n. Here, n is preferably an integer of 1 or more, at least one of Y ₁ , Y ₂ and Y ₃ is the same or different hydrophilic amino acid residue, and the rest are the same or different from each other It is a hydrophobic amino acid residue. That is, a composition in which any one of Y ₁ , Y ₂ and Y ₃ is a hydrophilic amino acid residue, and the rest are the same or different hydrophobic amino acid residues, and Y ₁ , Y ₂ and Y ₃ Any one of the compositions in which any one of them is a hydrophobic amino acid residue and the rest are the same or different hydrophilic amino acid residues can be preferably used.

  As long as β-sheet formation is not inhibited, the peptide chain may contain one or more arbitrary amino acid residues in addition to the predetermined hydrophilic amino acid residue and hydrophobic amino acid residue. The arbitrary amino acid residue may be an amino acid sequence having a predetermined function such as adhesion activity. Moreover, the side chain of the amino acid residues constituting the peptide chain may be altered or modified. Furthermore, the N-terminus and / or C-terminus of the main chain may be altered or modified (such as amidation).

  In addition to the peptide chain, a preferred embodiment of the organic molecular material used in the two-dimensional fine thin film production method of the present invention is, for example, other chemical structures such as a protective group, a spacer, a functional group, a functional group, etc. Organic molecular material containing For example, when an organic molecular material in which the N-terminus of a peptide chain that is a main chain is modified with a protecting group and a spacer (PEG or the like) is bonded to the C-terminus of the main chain is used, each peptide fiber is bound by the spacer. Since it can arrange | position on a board | substrate in the state arranged at substantially equal intervals, the monomolecular film excellent in the homogeneity can be produced preferably. Further, by using such an organic molecular material with a spacer, a fine wiring (in other words, a pattern in which a predetermined pattern is formed, in which a space is provided by the spacer and β-sheets are arranged adjacent to each other) Can be formed on a substrate.

  The solvent used for the preparation of the solution containing the peptide material is not particularly limited as long as the peptide material is soluble at a concentration at which the peptide material can be kept free. For example, typically, one solvent selected from the group consisting of water and a lower alcohol, or a mixture of two or more mutually soluble solvents can be preferably used. Preferable examples of the lower alcohol include methanol, ethanol, 2,2,2-trifluoroethanol (TFE) and the like. Depending on the solubility of the organic molecular material used, a mixture of a lower alcohol and an organic solvent soluble in the alcohol (such as benzene) can be preferably employed. Moreover, you may employ | adopt the acidic solvent or alkaline solvent which added the acid or the base.

The upper limit of the concentration of the peptide material contained in the solution is preferably such that the peptide material can be dispersed in the solution in a free state. That is, it is preferable that the upper limit concentration of the peptide material does not exceed the critical aggregation concentration (CAC) of the peptide material. The lower limit concentration is preferably such that when supplied to the substrate surface, a β-sheet can be efficiently formed on the substrate. For example, a concentration of about 1 × 10 ⁻⁶ mol / L to 1 × 10 ⁻⁴ mol / L can be preferably used. If the concentration is too higher than the concentration range, the peptide material aggregates in the solution to form a β-sheet. When such a β-sheet is transferred to the surface of the substrate, the β-sheet is likely to be multilayered (lamination in the three-dimensional direction) on the substrate, making it difficult to produce a thin film having a uniform thickness. On the other hand, if the concentration is too lower than the concentration range, the efficiency of adsorption of the peptide material onto the substrate surface decreases, and it becomes difficult to form a monomolecular film.

  In the present invention, a two-dimensional fine thin film made of the peptide material is formed on the surface of the substrate using a solution in which the above-described peptide materials are dispersed in a state of being released from each other. Here, the material, shape, and size of the substrate are not particularly limited, and a substrate made of a substance capable of adsorbing the peptide material at least on the surface on which the thin film is disposed may be used. Suitable examples of such materials include silicon carbide (SiC), silica glass, alumina, mica and the like.

There is no restriction | limiting in particular in the method of supplying the said solution to the surface of a base material. For example, a method of immersing the substrate in the solution for a predetermined time can be preferably employed. When this method is employed, the embodiment is not particularly limited, and may be allowed to stand for a predetermined time while the substrate is immersed in the solution. There is no restriction | limiting in particular in the way (direction, position) of the said base material in making it immerse. For example, the substrate may be placed perpendicular to or parallel to the liquid level of the solution. According to this solution supply method, there is no particular limitation on the container in which the solution is placed.
In another solution supply method, the peptide material solution can be suitably supplied to the surface of the substrate using capillary action. A preferred embodiment of this method is shown in the schematic diagram of FIG. In this embodiment, at least a gap 30 having a desired thickness (typically about 100 μm to 250 μm) is formed from the inner wall surface 12 of a container 10 (such as a container made of Teflon (registered trademark)) whose inner wall surface is hydrophobic, and thus hydrophilic. The substrate 20 is raised. This is not particularly limited, but is preferably performed by placing a sheet piece (such as a tape made of Teflon (registered trademark)) having the same thickness as the gap 30 between the inner wall surface 12 and the substrate surface 22. can do. When the peptide material solution is supplied to the bottom portion 24 of the substrate 20 in such an embodiment by a pipette or the like, the solution rises through the gap 30 formed by the inner wall surface 12 and the substrate surface 22 by capillary action. By maintaining this state for a predetermined time, a monomolecular film in which a plurality of peptide fibers are associated with the substrate surface 22 can be produced.
Here, the peptide material solution may be disposed directly on the bottom 24 as described above, or a predetermined amount of a predetermined solvent (typically, the solvent used for preparing the peptide material solution) may be placed in the bottom 24. The peptide material solution may be further supplied to the bottom 24 and diffused into the solvent after the solvent has risen through the gap 30 by capillary action.

Moreover, the schematic diagram of another suitable one aspect | mode of the method of supplying a peptide material solution to the base-material surface using a capillary phenomenon is shown in FIG. In this embodiment, the peptide material solution 40 is supplied along at least one bottom side of the container 10 whose inner wall surface is hydrophobic (FIG. 2A), and the lower side of the substrate 20 tilted obliquely is the one bottom side of the container 10 described above. The substrate 20 is immersed in the peptide material solution 40 so as to be parallel to the substrate 20 (FIG. 2B), and the upper side of the substrate 20 is gradually pulled up (FIG. 2C), so that the substrate 20 is surfaced on the inner wall surface. 12 upright (FIG. 2D). In this embodiment, the rate at which the peptide material solution 40 ascends the substrate surface 22 by capillary action can be controlled more preferably.
Preferably, as shown in FIG. 3, in any of the above-described methods utilizing the capillary phenomenon, a single molecule is obtained by providing a β-sheet growth starting point 26 on the substrate surface 2A (FIG. 3 (a)). The peptide material fibers constituting the membrane can be oriented (FIGS. 3B and 3C).

The time for immersing the substrate surface in the peptide material solution is not particularly limited, and may be set as appropriate according to the desired formation state of the monomolecular film. Typically, it can be set preferably between several minutes (about 10 minutes) to several days (about 3 days). It is possible to control the length of the peptide fiber by the immersion time. For example, if the immersion time is short, the peptide fibers constituting the monomolecular film can be arranged in a dot shape. In addition, when the immersion time is increased, the peptide fibers can be linear.
Moreover, it is preferable that the solution temperature at the time of immersing a base material in a peptide material solution is 10 to 40 degreeC, and it is suitable that it is 20 to 35 degreeC. If the temperature is too lower than the temperature range, the secondary structure transition of the peptide chain may be difficult to occur. If the temperature is too high, precipitation of the peptide material on the substrate may be inhibited.
After the substrate having the monomolecular film formed on the surface is pulled out of the above solution, it is allowed to stand in a predetermined solvent (typically the same solvent used for the preparation of the peptide material solution. For example, pure water). As a result, the surface of the substrate can be washed. Moreover, typically, the produced film can be preferably dried at room temperature and normal pressure.

  Several examples relating to the present invention will be described below, but the present invention is not intended to be limited to those shown in the examples. Further, matters other than matters specifically mentioned in the present specification and necessary for the implementation of the present invention can be grasped as design matters for those skilled in the art based on the prior art in this field. The present invention can be carried out based on the contents disclosed in this specification and common technical knowledge in the field.

In addition, the organic molecular material (copolymer) of the aspect by which PEG modification was given to the peptide used in the following examples uses PEG modification type resin (for example, TentaGelPAP-resin made by RAPPpolymere). This was prepared by a general Fmoc solid phase synthesis method. Further, ¹ H-NMR was used for primary structure analysis of the obtained organic molecular material, and circular dichroism (CD) spectrum was used for secondary structure analysis in the solution.

<Example 1>
Peptide chain shown in SEQ ID NO: 1, ie, leucine (L) and glutamic acid (E) are alternately linked to a total of 16 residues, and a peptide (LELE) ₄ -PEG having a molecular weight of 7000 attached to the C-terminus is adjusted to pH 11. An aqueous peptide solution was prepared by dissolving in the prepared sodium hydroxide solution so that the peptide concentration was 6.0 × 10 ⁻⁵ . A mica substrate (1 cm × 1 cm) was immersed in 2 mL of this peptide aqueous solution and left at 25 ° C. for 16 hours. The substrate pulled up from the solution was immersed in pure water to clean the surface and dried under normal pressure.

When the secondary structure of the peptide chain portion in the peptide aqueous solution was evaluated by CD spectrum, it was found to be a random coil structure.
Further, when the surface of the obtained substrate was observed with an atomic force microscope (AFM), as shown in FIG. 4, peptide fibers having a constant width of nano size were dispersed (uniformly) at approximately equal intervals by a PEG spacer. The formation of the monomolecular film arranged in this manner was confirmed. The width of this peptide fiber was about 5 nm. This substantially coincided with the width of the β-sheet that can be formed by the peptide chain represented by (LELE) ₄ above.

<Example 2>
A silicon carbide substrate (0.7 cm × 0.7 cm) was immersed in 2 mL of (LELE) ₄ -PEG / sodium hydroxide aqueous solution prepared in Example 1, and left at 25 ° C. for 3 days. The substrate pulled up from the solution was immersed in pure water to clean the surface and dried under normal pressure.
When the surface of the obtained substrate was observed by AFM, a monomolecular film in which peptide material fibers having a width of about 5 nm were mainly oriented in the vertical direction was confirmed as shown in FIG.
In addition, since the surface of the silicon carbide base material has a stepped shape at the level of the atomic level, as shown in FIG. 3, it is considered that the fibers grew in a substantially constant direction with this step line as the main starting point. .

<Example 3>
1. Peptide chain shown in SEQ ID NO: 2, that is, peptide (RADA) ₄ consisting of arginine (R), aspartic acid (D) and alanine (A) is dissolved in 2,2,2-trifluoroethanol (TFE). A peptide solution having a concentration of 2 × 10 ⁻⁵ mol / L was prepared. 0.1 mL of this solution is supplied along the bottom of one wall surface of a Teflon (registered trademark) container, and a mica substrate (2 cm × 2 cm) is held along the wall surface by a method as shown in FIG. ). After the substrate was immersed in the solution for 10 minutes at 25 ° C., the pulled substrate was washed by immersing it in TFE and dried under normal pressure.
When the surface of the obtained substrate was observed by AFM, as shown in FIG. 6, formation of a monomolecular film composed mainly of a plurality of peptide fibers having a width of about 5 nm grown mainly in the longitudinal direction was confirmed.

<Example 4>
Peptide chain shown in SEQ ID NO: 3, ie, leucine (L) and glutamic acid (E) in a total of 16 residues linked at a ratio of 3: 1 (PELL having a molecular weight of 7000 at the C-terminus (LELL) ₄ -PEG Was dissolved in an aqueous sodium hydroxide solution adjusted to pH 11 to prepare an aqueous peptide solution so that the peptide concentration was 6.0 × 10 ⁻⁵ . A mica substrate (1 cm × 1 cm) was immersed in 2 mL of this peptide aqueous solution and left at 25 ° C. for 16 hours. The substrate pulled up from the solution was immersed in pure water to clean the surface and dried under normal pressure.

When the secondary structure of the peptide chain portion in the peptide aqueous solution was evaluated by CD spectrum, it was found to be a random coil structure.
In addition, when the surface of the obtained substrate was observed with an atomic force microscope (AFM), a single molecule in which peptide fibers having a constant width of nano size were dispersed (uniformly) at regular intervals by a PEG spacer. Formation of the film was confirmed. The width of this peptide fiber was about 5 nm. This almost coincided with the width of the β-sheet that can be formed by the peptide chain represented by (LELL) ₄ above.

Specific examples of the present invention have been described in detail above, but these are merely examples and do not limit the scope of the claims. The technology described in the claims includes various modifications and changes of the specific examples illustrated above.

<Sequence Listing Free Text>
SEQ ID NO: 1 Synthetic peptide SEQ ID NO: 2 Synthetic peptide SEQ ID NO: 3 Synthetic peptide

It is a schematic diagram which shows one aspect | mode of the method of supplying a peptide material solution to the base-material surface using a capillary phenomenon. It is a schematic diagram which shows one aspect | mode of the other method of supplying a peptide material solution to the base-material surface using a capillary phenomenon. It is a schematic diagram which shows the mode of the thin film formation in the base-material surface which provided the growth starting point of (beta) -sheet. 2 is an atomic force microscope (AFM) image of a monomolecular film of the peptide material obtained in Example 1. FIG. 2 is an atomic force microscope (AFM) image of a monolayer of peptide material obtained by Example 2. FIG. 4 is an atomic force microscope (AFM) image of a monolayer of peptide material obtained by Example 3. FIG.

Explanation of symbols

10, 12 Containers for thin film production 20, 22, 24, 26 Base material 30 Gap 40 Peptide material solution

Claims (9)

A method for producing a two-dimensional fine thin film on a substrate,
As an organic molecular material for forming the thin film, it has at least a peptide chain,
The peptide chain is, have at least a hydrophilic amino acid residues and hydrophobic amino acid residues, can be adsorbed on the substrate, when it is intake wearing an amino acid sequence capable of forming a β- sheet structure ,
Formula _{(X 1 -X 2 -X 3 -X} 4) n ( where, n is an integer of 2 or _more, X _1, X 2, at least one of _{X 3} and _{X 4} are the same to each other or A step of preparing an organic molecular material characterized by having an amino acid sequence structure represented by different hydrophilic amino acid residues and the rest being the same or different hydrophobic amino acid residues. When,
Preparing a solution containing the organic molecular material at a concentration at which the peptide chain can maintain an α-helix or a random coil structure and in which the organic molecular material is individually released and dispersed;
The solution is supplied to the surface of the base material, the organic molecular material in the solution is adsorbed on the base material, and the peptide chains of the organic molecular material when adsorbed on the base material form β-sheets, respectively. Providing a monomolecular film formed on the substrate;
A method for producing a two-dimensional fine thin film including _2. The two-dimensional thin film according to claim 1 , wherein X ₁ and X ₃ are hydrophilic amino acid residues that are the same or different from each other, and X ₂ and X ₄ are hydrophobic amino acid residues that are the same or different from each other. Manufacturing method. _2. The two-dimensional thin film according to claim 1 , wherein X ₁ and X ₃ are the same or different hydrophobic amino acid residues, and X ₂ and X ₄ are the same or different hydrophilic amino acid residues. Manufacturing method. Each of the hydrophilic amino acid residues is independently an amino acid residue selected from the group consisting of aspartic acid, glutamic acid, arginine, lysine, histidine, serine, threonine, asparagine, glutamine and tyrosine, and the hydrophobic amino acid residue There are each independently valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, an amino acid residue selected from the group consisting of glycine and alanine, making the two-dimensional micro-thin film according to any one of claims 1 to 3 Method. At least water or a lower alcohol solvent is used as a solvent for preparing the solution, and the concentration of the organic molecular material contained in the solution is 1 × 10 ⁻⁶ to 1 × 10 ⁻⁴ mol / L. The method for producing a two-dimensional fine thin film according to any one of claims 1 to 4 , wherein the solution is prepared. The method for producing a two-dimensional fine thin film according to any one of claims 1 to 5 , wherein at least one surface of the substrate is made of a material selected from the group consisting of silicon carbide, silica glass, alumina, and mica. Supplying the solution to the substrate surface by immersing said substrate in said solution, a method for manufacturing a two-dimensional micro-thin film according to any one of claims 1-6. The method for producing a two-dimensional fine thin film according to any one of claims 1 to 6 , wherein the solution is supplied to the surface of the substrate by capillary action. A substrate with living tissue adaptability, comprising a peptide thin film produced by the method for producing a two-dimensional fine thin film according to any one of claims 1 to 8 on the surface.