KR20220056103A - Nucleic acids-dediated pattern replication and methods for manufacturing 2d material using the same - Google Patents

Abstract

Provided are: a nucleic acid-mediated pattern replication; and a method for manufacturing a two-dimensional (2D) material using the same. The method for manufacturing a 2D material according to one embodiment of the present invention comprises the steps of: preparing a first material having a first nucleic acid patterned on a surface thereof; combining the first nucleic acid with a linker-nucleic acid; replicating a pattern of the first material to the surface of a second material by combining the first nucleic acid and a second nucleic acid, which is modified on the surface of the second material, through the linker-nucleic acid; separating the first material; and applying a third material on a pattern replicated to the surface of the second material.

Description

Nucleic acid-mediated pattern duplication and two-dimensional material manufacturing method using the same

The following description relates to nucleic acid-mediated pattern replication and a two-dimensional material manufacturing method using the same.

Contact-based patterning techniques used to fabricate micrometer-to-nanometer-scale structures include nanoimprint lithography, dip pen nanolithography, and microcontact printing. In these existing technologies, since only one type of material can be patterned in one process, it is inevitable to repeat the process in order to fabricate a substrate on which multiple types of materials are complexly patterned. In addition, there is a difficulty in that the manufacturing time and the complexity of the process increase in proportion to the number of patterning materials, such as the need to accurately align the substrate every time the process is repeated.

In addition, in the case of contact-based patterning technologies such as microcontact printing and nanoimprint lithography, only one pattern per one mold (stamp) can be repeatedly formed, and in order to form several different patterns, a mold for each pattern have to create

In addition, conventional processes such as photolithography cannot be applied to curved or flexible substrates, and soft lithography techniques including nanoimprint lithography can form resist materials (thermoplastic and thermosetting polymers, ultraviolet photocurable materials, etc.) ) and the types of substrates are limited.

In addition, most micro-nano patterning techniques can only transfer a pattern to a substrate made of a hard material. In addition, the resolution of the pattern made in this way is limited by the resolution of the original pattern and the resolution of the process.

[Prior art literature]

Japanese Laid-Open Patent No. 2015-008308

A method for fabricating a two-dimensional material capable of multi-patterning in a single process is provided.

To provide a two-dimensional material fabrication method capable of selectively and/or continuously replicating an original pattern.

A method for fabricating two-dimensional materials capable of high-resolution patterning based on single-molecule contact is provided.

A two-dimensional material fabrication method that can be applied to various patterning materials and substrates is provided.

A two-dimensional material fabrication method capable of achieving higher resolution than the original pattern is provided.

A method for manufacturing a two-dimensional material, the method comprising: preparing a first material in which a first nucleic acid is patterned on a surface; binding a linker-nucleic acid to the first nucleic acid; copying the pattern of the first material to the surface of the second material by binding the first nucleic acid and the modified second nucleic acid to the surface of the second material through the linker-nucleic acid; separating the first material; and applying a third material over the pattern copied to the surface of the second material.

According to one aspect, the preparing step may be characterized in that the target material particles or molecules on the surface of the first material are labeled with the first nucleic acid including the first sequence.

According to another aspect, the linker-nucleic acid comprises a third nucleic acid comprising a second sequence complementary to the first sequence of the first nucleic acid and a fourth sequence complementary to the third sequence of the second nucleic acid. It may be characterized as having a structure in which 4 nucleic acids are linked by a linker.

According to another aspect, in the binding step, the linker-nucleic acid to the first nucleic acid by binding the third nucleic acid of the second sequence included in the linker-nucleic acid with the first nucleic acid of the first sequence. It may be characterized by complementary binding.

According to another aspect, in the copying step, by physical contact between the surface of the first material and the surface of the second material, the linker-nucleic acid includes the fourth nucleic acid of the fourth sequence and the second material. By binding the second nucleic acid of three sequences, the pattern of the first material (position information of individual target material particles or target molecules) may be copied to the surface of the second material.

According to another aspect, the separating step recognizes and cuts out a specific sequence of the linker-nucleic acid linker portion, the bond between the first nucleic acid and the third nucleic acid, or the bond between the second nucleic acid and the fourth nucleic acid may be characterized in that the bond between the first material and the second material is cut using an enzyme or a chemical reaction.

According to another aspect, in the separating step, the bond between the first nucleic acid and the third nucleic acid is cut off while maintaining the bond between the second nucleic acid and the fourth nucleic acid using a difference in binding force between the nucleic acids. can be characterized as

According to another aspect, the third material is a material comprising at least one of a metal, a polymer, a ceramic, a biomolecule, and a cell whose surface is modified with the first sequence of the first nucleic acid or a new nucleic acid containing the first sequence It may be characterized in that it comprises at least one of a single chemical molecule to which a particle, or a first sequence of the first nucleic acid or a new nucleic acid comprising the first sequence, is chemically bound.

According to another aspect, the third material may be the same material as the target material particle or single molecule on the surface of the first material.

According to another aspect, the third material may be a material different from the target material particles or single molecules on the surface of the first material.

According to another aspect, in the applying step, the third material is the first sequence of the first nucleic acid or the remaining on the surface of the second material as the surface is modified with a new nucleic acid comprising the first sequence. It may be characterized in that it binds to the first nucleic acid.

According to another aspect, the preparing comprises preparing the first material including a plurality of patterns, wherein two or more patterns among the plurality of patterns are composed of nucleic acids having a sequence orthogonal to each other, and the first The nucleic acid is one of the nucleic acids of the sequence orthogonal to each other, and the binding step comprises combining a plurality of linker-nucleic acids including a nucleic acid different from a nucleic acid included in the plurality of patterns, and the different linker-nucleic acids They may be characterized in that they include a nucleic acid of a sequence complementary to each of the nucleic acids of the sequence orthogonal to each other.

According to another aspect, the copying includes physically contacting the first material with a surface of the second material to copy the plurality of patterns of the first material to the surface of the second material, The surfaces of the two materials may be characterized in that they are modified with nucleic acids corresponding to each of the nucleic acids of the sequences orthogonal to each other.

According to another aspect, in the applying step, a plurality of different third materials having surfaces modified with each of the nucleic acids of the sequence orthogonal to each other are applied on the pattern copied to the surface of the second material, characterized in that can do.

According to another aspect, in the preparing step, a first material including N (wherein N is a natural number) patterns is prepared, the nucleic acids constituting the different patterns are orthogonal to each other, and the separated first material By recycling , it may be characterized in that it is possible to form patterns of 2 or more M (the M is 2 ^N -1) or less.

According to another aspect, when recycling the first material, the plurality of linker-nucleic acids combined with the nucleic acids including the N patterns include (N-1) or less types of nucleic acids having different sequences. can be characterized. According to another aspect, the second material comprises a flexible material, the first step of shrinking the flexible material to which the pattern is replicated; a second step of duplicating the pattern on another second material by using the contracted flexible material again as a first material; and a third step of repeating the first step and the second step A (where A is a natural number) times.

a pattern mediated by a first nucleic acid replicated from the surface of the original material to the surface of the two-dimensional material; a second nucleic acid modified on the surface of the two-dimensional material; a linker connecting the first nucleic acid and the second nucleic acid-nucleic acid; and an additional material applied to the surface of the two-dimensional material and bound to the first nucleic acid of the pattern.

It is possible to provide a method for fabricating a two-dimensional material capable of multi-patterning in a single process.

In addition, it is possible to provide a two-dimensional material fabrication method capable of selectively and/or continuously replicating the original pattern.

In addition, it is possible to provide a method for fabricating a two-dimensional material capable of high-resolution patterning based on single-molecule contact.

In addition, it is possible to provide a method for manufacturing a two-dimensional material that can be applied to various patterning materials and substrates.

In addition, it is possible to provide a two-dimensional material manufacturing method capable of achieving a higher resolution than the original pattern.

1 to 6 are views showing an example of a two-dimensional material manufacturing method according to an embodiment of the present invention.
7 is a diagram illustrating an example of a conventional contact-based patterning technique.
8 is a diagram illustrating an example of duplicating a complex pattern composed of a plurality of materials or a plurality of single molecules at once using a pair of nucleic acids orthogonal to each other according to an embodiment of the present invention.
9 is a diagram illustrating an example of duplicating patterns of different combinations from one original pattern according to an embodiment of the present invention.
FIG. 10 is a diagram illustrating an example of improving patterning resolution through repeated replication to a flexible material and shrinkage of the flexible material according to an embodiment of the present invention.
11 is a diagram illustrating an example of replicating a pattern of an original hydrogel to another hydrogel surface through an interaction between complementary nucleic acids according to an embodiment of the present invention.
12 to 15 are diagrams illustrating examples of experimentally implemented pattern replication using nucleic acids according to an embodiment of the present invention.
16 to 18 are diagrams illustrating an example in which the distribution of biomolecules on the surface of a tissue is copied to a new material surface according to an embodiment of the present invention.
19 is a diagram illustrating an example in which a pattern of a neuron nuclear protein expressed in a mouse brain is copied on a hydrogel surface according to an embodiment of the present invention.
20 and 21 are diagrams illustrating an example in which the resolution of a duplicated pattern is improved by using a contractible hydrogel according to an embodiment of the present invention.
22 is a flowchart illustrating an example of a method for manufacturing a two-dimensional material according to an embodiment of the present invention.

Specific structural or functional descriptions of the embodiments are disclosed for purposes of illustration only, and may be changed and implemented in various forms. Accordingly, the actual implementation form is not limited to the specific embodiments disclosed, and the scope of the present specification includes changes, equivalents, or substitutes included in the technical spirit described in the embodiments.

Although terms such as first or second may be used to describe various elements, these terms should be interpreted only for the purpose of distinguishing one element from another. For example, a first component may be termed a second component, and similarly, a second component may also be termed a first component.

When a component is referred to as being “connected to” another component, it may be directly connected or connected to the other component, but it should be understood that another component may exist in between.

The singular expression includes the plural expression unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to designate that the described feature, number, step, operation, component, part, or combination thereof exists, and includes one or more other features or numbers, It should be understood that the possibility of the presence or addition of steps, operations, components, parts or combinations thereof is not precluded in advance.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present specification. does not

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings.

Embodiments of the present invention are two-dimensional pattern replication and material manufacturing technology, which reproduces the chemical pattern of the original material surface to the surface of another material through a specific interaction between two nucleic acid strands having complementary sequences. provide technology. Here, a molecular sequence can be made as a nucleic acid, and all substances that strongly interact only between specific molecular sequences can be used. The most representative example is DNA, and there are locked nucleic acid (LNA) and peptide nucleic acid (PNA), which are one of RNA and modified nucleic acid. As such, as nucleic acids, all substances that strongly interact only between specific molecular sequences in addition to 'DNA, RNA, LNA, and PNA' can be used. In addition, by precisely positioning various materials (eg, nanoparticles, metals, polymers, ceramics, biomolecules, cell material particles, or single chemical molecules) on the duplicated pattern, complex two-dimensional materials can be easily produced in a single process. provide technology that can In addition, metals, polymers, ceramics, biomolecules, etc. can be attached to or grown from these material particles or chemical molecules. In the method for manufacturing a two-dimensional material according to an embodiment, the two-dimensional material may be manufactured using the interaction between nucleic acid strands. The main idea is to develop a contact-based two-dimensional patterning technique that exploits the specific interaction between two nucleic acid strands with complementary sequences.

1 to 6 are views showing an example of a two-dimensional material manufacturing method according to an embodiment of the present invention. The two-dimensional material manufacturing method according to the embodiment of FIGS. 1 to 6 shows an example of duplicating the pattern of the material surface using nucleic acids and manufacturing the two-dimensional material using the same. 1 to 6 schematically illustrate an example of replicating the simplest single type of pattern that can be implemented, and the size of the nucleic acid is exaggerated for better understanding. Actual nucleic acids are within a few nanometers in length, and are very small compared to new materials (substrates) or patterning materials from which patterns are replicated. Pattern duplication using nucleic acids and a method of manufacturing a two-dimensional material using duplicated patterns can basically go through four steps.

First, FIG. 1 shows an enlarged view of the original material in which the target material particles or target molecules to be replicated are patterned on the surface, and FIG. 2 is the target pattern labeling step, which is a nucleic acid containing a specific sequence 'A' on the surface of the original material. It shows the process of specifically labeling target material particles or target molecules in Alternatively, all kinds of materials that have already been patterned on the surface with nucleic acids (eg, commercially available DNA chips, tissue sections labeled with specific proteins with nucleic acids, etc.) may be used as the original.

3 is a linker-nucleic acid binding step, showing a process of binding a linker-nucleic acid having a sequence 'A*' complementary to 'A'. This linker-nucleic acid has the sequence 'B*' at the opposite end.

FIG. 4 shows a process of physically contacting the surface of another material modified with a nucleic acid having a sequence 'B' complementary to the terminal sequence 'B*' of the linker-nucleic acid as a pattern transcription step. As a result, positional information (pattern) on the target molecule on the surface of the original material is mediated by the specific interaction between A-A* and B-B* as 'nucleic acid labeled target molecule - linker - nucleic acid - modified nucleic acid on the surface of a new material' It may be delivered in the form of a complex. Then the original material surface is separated.

In this case, at least one of the surface of the original material and the surface of another material to be patterned may be a hydrogel (or a soft surface material). If the original material and the other material to be copied are both materials with a hard surface, the duplication may not work. In this case, the method of duplicating the pattern of the material of the hard surface with the hydrogel (or the material of the soft surface) with the hydrogel (or the material of the soft surface), and then duplicating the pattern of the material of the hard surface again with another material of the hard surface. can

5 and 6 are surface functionalization steps, and material particles (eg, metals, polymers, ceramics, biomolecules, cells, nanoparticles, etc.) whose surface is modified with a nucleic acid containing an 'A' sequence or an 'A' sequence An example of the process of applying a chemical molecule to which a containing nucleic acid is linked is shown on a copied pattern. At this time, if the target material particles or target molecules are repositioned, the original material can be copied as it is, and if a different type of material particles or molecules is applied, a two-dimensional material with the same distribution (pattern) but patterned with new material particles or molecules is obtained. can be (Fig. 5). At this time, the 2 materials may be the same material as the original 1 material (FIG. 5), or may be different materials (FIG. 6).

In addition, according to embodiments of the present invention, a complex two-dimensional material composed of one or more materials, which was not possible with the existing two-dimensional material manufacturing technology, can be easily manufactured using several orthogonal nucleic acid pairs. In particular, it is possible to simultaneously reproduce a complex pattern on the surface of a new material without limiting the type and number of branches, and furthermore, it becomes possible to selectively reproduce only a desired pattern even if the original material has one or more complex types of patterns.

In the two-dimensional material manufacturing method according to an embodiment, a complex pattern may be simultaneously replicated using orthogonal nucleic acid pairs.

7 is a diagram illustrating an example of a conventional contact-based patterning technique, and FIG. 8 is a diagram illustrating an example of duplicating a complex pattern at once using a nucleic acid pair orthogonal to each other according to an embodiment of the present invention. .

In the method for manufacturing a two-dimensional material according to an embodiment of the present invention, a pattern can be replicated using interactions between complementary nucleic acid strands, and a pair of nucleic acids can replicate one type of pattern. Since only complementary nucleic acid strands can specifically interact with a nucleic acid strand, multiple orthogonal (=non-complementary) pairs of nucleic acids can be processed simultaneously and used to replicate and pattern different patterns, respectively.

For example, when using a nucleic acid consisting of 20 bases, 4 ²⁰ = 1,099,511,627,776 (about 1 trillion) nucleic acid strands are possible, which corresponds to 500 billion orthogonal nucleic acid pairs. In other words, 5000 different It is possible to duplicate and pattern billions of patterns simultaneously.

Therefore, using the embodiments of the present invention, multi-patterning can be easily performed without the complicated process of repeating the same process and aligning the substrate, and there is an advantage that there is no practical limit to the number of patterns that can be transferred.

In addition, in the two-dimensional material manufacturing method according to an embodiment, different patterns may be selectively duplicated from a single original pattern.

9 is a diagram illustrating an example of duplicating patterns of different combinations from one original pattern according to an embodiment of the present invention. The two-dimensional material manufacturing method according to the present embodiment may replicate only some of the different patterns on the original surface using different nucleic acid pairs or by selectively combining them. For example, if you have 4 different types of patterns in the original, copy each pattern singly (number of cases = 4), combine two (number of cases = 6), or combine three Alternatively (the number of cases = 4), all four patterns can be duplicated (the number of cases = 1), so a total of 15 different patterns can be duplicated from one original pattern.

In addition, according to embodiments of the present invention, it is possible to easily achieve pattern replication on the surface of a soft material such as hydrogel, which has been limited by existing two-dimensional material manufacturing methods, and repeats pattern replication and hydrogel contraction by applying this. By doing so, the resolution of the pattern can be dramatically improved. In other words, in the method for manufacturing a two-dimensional material according to an embodiment, the resolution may be improved through repetitive pattern duplication and contraction of the hydrogel.

10 is a diagram illustrating an example of improving patterning resolution through repeated replication and hydrogel shrinkage according to an embodiment of the present invention. One of the advantages of the two-dimensional material manufacturing method according to the present embodiment is that there is no limitation on the new material from which the pattern can be copied. In particular, unlike existing technologies, the two-dimensional material manufacturing method according to this embodiment can be applied to soft materials such as hydrogels. If a contractible hydrogel is used, the pattern is contracted in the isotropic direction after pattern transfer. It is possible to achieve high resolution.

To this end, in the two-dimensional material manufacturing method according to this embodiment, the original pattern is reproduced on the surface of a hydrogel (eg sodium polyacrylate hydrogel) in a 4-fold expanded state, the hydrogel is contracted to its original size, and the The pattern can be transferred to another expanded hydrogel surface, and the process can be re-contracted. As such, each time the pattern replication-hydrogel contraction is repeated, the size of the pattern is reduced by 4n times, which has the effect of improving the patterning resolution by up to 4n times, making it possible to pattern at a much higher resolution than the original pattern. For example, if you make an original pattern with a level of 100 micrometers or nanometers, and shrink the pattern three times, you can obtain a pattern with a level of about 1.5 micrometers or nanometers.

In the patterning technology, as the size of the pattern decreases, the complexity of the process increases, and accordingly, the patterning time and cost exponentially increase. On the other hand, by using the two-dimensional material manufacturing method according to the present embodiment, an original pattern is made with a size that can be patterned relatively easily, and by replicating and shrinking it, a pattern of several nanometers that is difficult to achieve with existing patterning technology can be formed. This can significantly save time and money required to achieve a similar level of patterning resolution.

As such, the embodiments of the present invention have a differentiated advantage that can complexly overcome the limitations of the existing two-dimensional material manufacturing technology, such as patterning efficiency and resolution problems, limited pattern complexity, and limited applicability to rigid materials.

More specifically, in the two-dimensional material manufacturing method according to embodiments of the present invention, multi-patterning is possible in a single process. Contact-based patterning techniques used to fabricate micro-nano structures include nanoimprint lithography, dip pen nanolithography, and microcontact printing. In these existing technologies, since only one type of material can be patterned in one process, it is inevitable to repeat the process in order to fabricate a substrate on which multiple types of materials are complexly patterned. In addition, there is a difficulty in that the manufacturing time and the complexity of the process increase in proportion to the number of patterning materials, such as the need to accurately align the substrate every time the process is repeated. In contrast, in the two-dimensional material manufacturing method according to the embodiments of the present invention, the replication of the pattern is made through a nucleic acid characterized by interaction with sophisticated programmability and high specificity. If an orthogonal nucleic acid pair that does not affect is used, it is possible to simultaneously replicate various types of patterns in a single process, and there is an advantage that there is no limitation in the type and number of molecules that can be replicated.

In addition, the method for manufacturing a two-dimensional material according to embodiments of the present invention enables selective and/or continuous duplication of an original pattern. In the case of contact-based patterning technologies such as microcontact printing and nanoimprint lithography, only one pattern per one mold (stamp) can be repeatedly formed, and in order to form several different patterns, a mold for each pattern is manufactured must do it. On the other hand, the two-dimensional material manufacturing method according to the embodiments of the present invention can be duplicated by selectively combining each pattern from one complex original pattern, and it is possible to reuse the separated original by re-modifying it with nucleic acid after pattern duplication. . Accordingly, it is possible to significantly reduce the time and cost spent in the existing technology to form a plurality of multi-patterns.

In addition, the two-dimensional material fabrication method according to embodiments of the present invention is capable of high-resolution patterning based on single-molecule contact. The resolution of the two-dimensional material manufacturing method according to the embodiments of the present invention is determined by physical factors such as the linker-nucleic acid length and the distribution density of the modified nucleic acid on the replication surface, and the pattern of the original surface is a single molecule unit of contact. can be transferred through Transcription mediated by physical interactions at the molecular level enables more sophisticated replication than photolithography, which is limited by optical diffraction in structures of 100 nm or less.

In addition, in the two-dimensional material manufacturing method according to embodiments of the present invention, it is possible to apply various patterning materials and substrates. Conventional processes such as photolithography cannot be applied to curved or flexible substrates, and soft lithography technology including nanoimprint lithography uses resist materials (thermoplastic and thermosetting polymers, ultraviolet photocurable materials, etc.) that can form patterns and The types of substrates are limited. In contrast, in the two-dimensional material manufacturing method according to embodiments of the present invention, any material whose surface is modified with nucleic acid can be used as a substrate, and any material capable of linking nucleic acids can be applied as a patterning material. there is. The fabrication of biochips and sensors using nucleic acids has already been widely used, and by using the well-known surface modification and conjugation technology using nucleic acids, a wide range of materials such as metals, polymers, glass, ceramics, and biological samples are used. Or molecules can be applied to the present technology.

In addition, the two-dimensional material manufacturing method according to embodiments of the present invention can achieve a higher resolution than the original pattern. Most of the micro-nano patterning techniques can transfer the pattern only to a substrate made of a hard material. In addition, the resolution of the pattern made in this way is limited by the resolution of the original pattern and the resolution of the process. On the other hand, in the two-dimensional material manufacturing method according to embodiments of the present invention, patterning is possible even on a substrate made of a flexible material such as hydrogel. By applying this, it is possible to create a smaller and more sophisticated pattern than the original pattern by duplicating the master pattern on a physically contractible hydrogel, and then proceeding with the hydrogel contraction.

The idea of the embodiments of the present invention was successfully implemented experimentally, and the possibility of various material manufacturing techniques using the same was further demonstrated. In addition, by verifying that the most useful and differentiated application method of the embodiments of the present invention, the protein pattern on the surface of biological tissue can be used to manufacture artificial tissue mimetics, it is commercialized as a commercialization model with differentiated competitiveness in the future. It has been confirmed that it can be

Main Result 1: Implementation of pattern replication by applying embodiments of the present invention

Using the embodiments of the present invention, the shape of the original polygonal hydrogel was directly transferred to the surface of a new material, and it was confirmed that the nucleic acid-mediated pattern transfer is actually well implemented and works effectively.

11 is a diagram illustrating an example of replicating a pattern of an original hydrogel to another hydrogel surface through an interaction between complementary nucleic acids according to an embodiment of the present invention. According to FIG. 11 , there is a nucleic acid 'A' of a specific sequence on the surface of the triangular original hydrogel, and another nucleic acid 'B' on the surface of the new hydrogel whose pattern is to be replicated. When a linker-nucleic acid having an A* sequence and a B* sequence complementary to A of the original hydrogel is linked, and a new hydrogel is brought into contact with it, B* at the opposite end of the linker-nucleic acid and B on the surface of the new hydrogel are complementary interact negatively. After physically separating the two hydrogels, the triangular-shaped original hydrogel pattern is duplicated on the surface of the copy hydrogel.

In addition, in an embodiment of the present invention, a control group using a hydrogel modified with a nucleic acid that is not complementary to the linker-nucleic acid as a copy was placed, and it was confirmed that the pattern was not transcribed in the corresponding experimental group. Through this, it was confirmed that the replication of the pattern occurred only by the specific interaction between the two complementary nucleic acid strands.

12 to 15 are diagrams illustrating examples of experimentally implemented pattern replication using nucleic acids according to an embodiment of the present invention. In the control group, when a non-complementary nucleic acid was used, it was confirmed that the pattern was not transcribed (see FIG. 15 ), and it was confirmed that the pattern transcription occurred only by a specific interaction between complementary nucleic acid strands. Here, the scale bar is 1 mm.

Key Result 2: Replication of protein expression patterns on the surface of brain tissue to a novel material surface

By using the embodiments of the present invention, a biological tissue can be used as an original (mold, master), and a pattern of a specific biomolecule on the surface of a tissue can be copied as it is to the surface of another material.

16 to 18 are diagrams illustrating an example in which the distribution of biomolecules on the surface of a tissue is copied to a new material surface according to an embodiment of the present invention. After selectively labeling the target protein with the nucleic acid-linked antibody, the pattern of the target protein can be copied as it is on the surface of the hydrogel, which is a new material, by applying the two-dimensional material manufacturing method according to the embodiments of the present invention. Here, scale bar = 1 mm.

In this example, the neuronal nuclear protein (NeuN) strongly expressed in the hippocampal region of a rat brain section was selectively immunolabeled and then transcribed onto the surface of a new hydrogel using a nucleic acid-mediated pattern transcription method. The characteristic patterns (CA1, CA2, CA3, DG regions) of the hippocampus, which are densely populated with neurons, were clearly transcribed.

19 is a diagram illustrating an example in which a pattern of a neuron nuclear protein expressed in a mouse brain is copied on a hydrogel surface according to an embodiment of the present invention. In FIG. 19, the left side shows the expression pattern of the target protein transcribed on the copy nucleic acid-hydrogel (purple) (white), and the right side shows the target protein specifically stained on the surface of the original brain slice (black) (white) are respectively shown. Here, scale bar = 1 mm.

Key Result 3: Improved resolution of replicated patterns through hydrogel shrinkage

After the pattern is transcribed into a contractible hydrogel using nucleic acid, the hydrogel can be contracted to improve the resolution of the pattern.

20 to 21 are diagrams illustrating an example in which the resolution of a duplicated pattern is improved by using a contractible hydrogel according to an embodiment of the present invention. After copying the triangular-shaped pattern onto the surface of the new hydrogel, the hydrogel was contracted in the isotropic direction to realize a pattern smaller than the original pattern. After duplicating the pattern on the surface of a flexible material, it was shown that the resolution can be further increased by using the properties of the material. Here, scale bar = 1 mm.

In this example, sodium polyacrylate hydrogel capable of repeating expansion and contraction depending on the salt concentration was used. It was confirmed that the actual resolution of the pattern could be improved by replicating the pattern with the 4-fold expanded hydrogel surface, shrinking it, and then repeating the process of replicating the pattern with another expanded hydrogel again.

20 and 21 are diagrams illustrating another example in which the resolution of a duplicated pattern is improved by using a contractible hydrogel according to an embodiment of the present invention. In this embodiment, after the triangular pattern was copied to the new hydrogel surface, the hydrogel was contracted in the isotropic direction to realize a smaller pattern than the original pattern. After duplicating the pattern on the surface of a flexible material, it was shown that the resolution can be further increased by using the properties of the material.

22 is a flowchart illustrating an example of a method for manufacturing a two-dimensional material according to an embodiment of the present invention. The two-dimensional material manufacturing method according to this embodiment includes the steps of preparing a first material in which a first nucleic acid is patterned on the surface (2210), a linker-binding nucleic acid to the first nucleic acid (2220), a linker- through the nucleic acid Copying the pattern of the first material to the surface of the second material by binding the first nucleic acid and the modified second nucleic acid to the surface of the second material (2230), separating the first material (2240), and the third material may include applying 2250 to the pattern copied on the surface of the second material.

Step 2210 may be, for example, an example of the process of labeling the target pattern described above with reference to FIG. 2 . For example, by labeling a target molecule on the surface of the first material with a first nucleic acid comprising a first sequence, a first material having a patterned surface of the first nucleic acid may be generated. Alternatively, a first material in which the first nucleic acid is patterned on the surface may be prepared by a separate method. The surface of the first material may be flat, rough, and may have physical irregularities or patterns.

Step 2220 may be, for example, an example of the linker-DNA binding process described above with reference to FIG. 3 . In this case, the linker-nucleic acid is a third nucleic acid comprising a second sequence complementary to the first sequence of the first nucleic acid and a fourth nucleic acid comprising a fourth sequence complementary to the third sequence of the second nucleic acid by a linker. It can have one structure. In this case, the first nucleic acid of the first sequence and the linker-nucleic acid may be bound to the first nucleic acid by binding the third nucleic acid of the second sequence included in the linker-nucleic acid.

Step 2230 may be an example of the process of pattern transfer (contact) described above with reference to FIG. 4 . In one example, by physical contact between the surface of the first material and the surface of the second material, the linker-nucleic acid includes a fourth nucleic acid of a fourth sequence and a second nucleic acid of a third sequence, thereby forming a pattern of the first material can be copied to the surface of the second material.

Step 2240 may be an example of a process of separating the first material as the original material. or a linker portion connecting the third nucleic acid and the fourth nucleic acid in the first nucleic acid or the second nucleic acid or the third nucleic acid or the fourth nucleic acid or the linker-nucleic acid, or a combination of nucleic acids above (eg, the first nucleic acid and the third nucleic acid; The nucleic acid link between the two materials may be chemically cut using an enzyme or a chemical reaction that recognizes and cuts a specific sequence of the second nucleic acid and the fourth nucleic acid). Alternatively, after using such an enzyme, the two materials can be physically separated. Alternatively, a physical or chemical method capable of severing only the bond between the first nucleic acid and the third nucleic acid while maintaining the bond between the second nucleic acid and the fourth nucleic acid by using the difference in the binding force between the nucleic acids may be used. The separated first material may be recycled to generate another pattern after reattaching the first nucleic acid or as it is, according to an embodiment.

The third material applied in step 2250 may include at least one of metals, polymers, ceramics, biomolecules, and cells whose surface is modified with the first sequence of the first nucleic acid. The third material whose surface is modified with the first sequence of the first nucleic acid may be associated with the first nucleic acid comprising the pattern copied to the surface of the second material. In this case, the third material may be the same material as the target molecule on the surface of the first material, or a material different from the target molecule on the surface of the first material.

In some embodiments, step 2210 may prepare a first material including a plurality of patterns. In this case, two or more patterns among the plurality of patterns may be composed of nucleic acids having sequences orthogonal to each other. As previously described, sequences orthogonal to each other may refer to sequences in which specific interactions do not occur because they are not complementary to each other. In this case, the first nucleic acid may be one of the nucleic acids having a sequence orthogonal to each other. In this case, in step 2220, a plurality of linker-nucleic acids including nucleic acids different from the nucleic acids included in the plurality of patterns may be combined. In this case, the different linker-nucleic acids may include a nucleic acid having a sequence complementary to each of the nucleic acids having a sequence orthogonal to each other. As an example, in FIG. 8, multi-panning is possible in a single process by simultaneously processing pairs of nucleic acids orthogonal to each other. Shows an example in which a plurality of linkers are combined with nucleic acids. Similarly, in step 2230, the plurality of patterns of the first material may be copied to the surface of the second material by physically contacting the first material with the surface of the second material. In this case, the surfaces of the two materials may be modified with a nucleic acid corresponding to each of the nucleic acids having a sequence orthogonal to each other. Meanwhile, in step 2250 , a plurality of different third materials, each of which has a surface modified with each of the nucleic acids having an orthogonal sequence, may be applied on the pattern copied to the surface of the second material. In this case, a plurality of different third materials may be combined with nucleic acids having an orthogonal sequence to each other.

As another embodiment, in operation 2210, a first material including N (N is a natural number) patterns may be prepared. In this case, the nucleic acids constituting the different patterns may be orthogonal to each other. In this case, by recycling the separated first material, it is possible to form patterns of 2 or more M (M is 2 to the N power) or less. For example, in FIG. 9 , it has been described that a total of 15 (2 ⁴ -1) types of patterns can be formed with one original composed of four patterns. In this case, the fact that a maximum of 15 types of patterns can be formed may mean that 15 or less types of patterns can be formed. Meanwhile, when the first material is recycled, the plurality of linker-nucleic acids combined with the nucleic acids included in the N patterns may include (N-1) or less types of nucleic acids having different sequences. As an example, the example of FIG. 9 shows an example of forming a new type of pattern while decreasing the number of types of nucleic acids linked through the linker-nucleic acid by one. In this way, different third materials can be attached to the different nucleic acids replicated on the second surface.

In yet another embodiment, the second material may include a soft material. In this case, the first step of shrinking the flexible material from which the pattern is replicated, the second step of using the contracted flexible material again as the first material, and the second step of replicating the pattern on another second material, and the first and second steps are A The patterning resolution may be improved through the third step of repeating (A is a natural number) times. As an example, FIG. 10 shows an example in which patterning resolution can be dramatically improved by repeating pattern duplication and shrinkage of hydrogel as a flexible material.

In yet another embodiment, the second material may include a soft material. In this case, the first step of expanding, bending, or deforming the flexible material from which the pattern is replicated, the second step of duplicating the pattern to another second material by using the expanded, bent, or deformed flexible material again as the first material to greatly enlarge the original pattern, or to duplicate the original pattern with a curved or serpentine surface.

In another embodiment, in step 2250, metal, polymer, ceramic, biomolecules, etc. may be attached to the third material or grown therefrom.

On the other hand, the two-dimensional material manufactured according to the embodiments of the present invention includes a pattern mediated by a first nucleic acid replicated from the surface of the original material to the surface of the two-dimensional material, a second nucleic acid modified on the surface of the two-dimensional material, and a first nucleic acid It may include a linker-nucleic acid connecting the second nucleic acid and an additional material applied to the surface of the two-dimensional material and bound to the first nucleic acid of the pattern. Here, the additional material may correspond to the third material described with reference to FIG. 22 . When the original material includes a plurality of patterns, it will be readily understood that the plurality of patterns may be duplicated in the two-dimensional material.

In addition, according to the embodiments of the present invention, it is possible to transfer the pattern by using the nucleic acid pattern replicated on the surface as an etch mask. Recently, it has been known that nucleic acid can be used as an etching mask. Accordingly, it is possible to transfer the physical pattern to the surface of the second material by using the nucleic acid transferred to the pattern as an etching mask. Through this, it is possible to convert the chemical pattern formed on the surface of the first material into a physical pattern on the surface of the second material. In this process, it is possible to form a physical pattern that is smaller, larger, curved, or modified than the chemical pattern on the surface of the first material by using the hydrogel. As such an etching mask, a nucleic acid may be directly used, or a third material copied to the surface of the second material through this embodiment may be used. In addition, it is possible to post-process the material grown from the replicated surface, such as by sintering it to connect them to each other to make them conductive. This post-processed third material may be utilized as an etching mask.

As such, according to the embodiments of the present invention, it is possible to precisely reproduce and process patterns of biomolecules using biological tissue as an original, which was impossible with conventional techniques. Using this, it will be possible to create a mimic of living tissue and combine biotechnology such as cell-sheet technology that can stack tissues in order, making it possible to create implantable-level artificial organs. . As such, it is expected that the embodiments of the present invention will be utilized as a base technology to construct an artificial tissue and organ supply system that will open a new paradigm in the future healthcare industry.

The two-dimensional material manufacturing method according to embodiments of the present invention is expected to provide a large-capacity artificial living tissue manufacturing method essential for artificial tissue and organ transplantation systems that will open a new paradigm in the future healthcare industry.

Artificial organ biotechnology is a potent application field of the two-dimensional material manufacturing method according to embodiments of the present invention, and since the distribution of various biomolecules on the tissue surface can be copied as it is, it greatly contributes to making artificial tissues and organs at a transplantable level. can do. According to the two-dimensional material manufacturing method according to the embodiments of the present invention, it is possible to make a single-molecule level nanobiochip by precisely fixing various biomolecules on various substrates, which is a precise biosensor for disease diagnosis or customized treatment. It is expected to be useful in the field of bio-health, such as as a In addition, the use of polymer materials including high biocompatibility hydrogels as substrates can contribute to the production of various biomaterials for medical use, which were difficult to make with existing micro-nano structure fabrication techniques.

The global tissue engineering product market, which constitutes artificial organ biologics, formed a size of $89 billion in 2016 and occupies the largest portion of the tissue engineering and regeneration-related markets. As the development initiative for bio artificial organs is rapidly transferring from early public institutions to biotechnology companies, the artificial organ market using regenerative medicine such as tissue engineering, stem cells, and therapeutic cloning is expected to form a large market in the medical industry in the future. is judged to be The two-dimensional material manufacturing method according to the embodiments of the present invention is combined with existing technologies for manufacturing artificial tissues and organs, such as 3D printing technology and decellularization technology, to produce various types of artificial organs at a transplantable level. It will play a role in overcoming limitations.

The biohealth industry is an industry that produces products or provides services for the human body based on biotechnology and medical/pharmaceutical knowledge. The global bio-health market is expected to expand rapidly due to an aging population and an increase in health demand around the world. Nanobiochips such as single-molecule resolution protein chips manufactured according to the two-dimensional material manufacturing method according to embodiments of the present invention are widely used as biosensors for disease diagnosis and customized treatment, test platforms for clinical research of biopharmaceuticals, etc. It is expected to build the technological infrastructure necessary for the bio-health industry in the future.

By applying the two-dimensional material manufacturing method according to the embodiments of the present invention, a tissue section is duplicated on a hydrogel having excellent biocompatibility and fused with a cell sheet technology to utilize it as an artificial tissue. This method does not require a separate three-dimensional support for culturing organs, and has the advantage of achieving a much higher level of resolution than technology based on 3D printing. In addition, it is expected to be applied as a new concept regenerative medical platform that can be fused with a patient-specific medical system by engineering the microenvironment of organs and tissues in the human body.

By using the two-dimensional material manufacturing method according to the embodiments of the present invention, various metals and ceramics, which are used as an aid in diagnosing or treating diseases medically, can be arranged together with biomolecules, and a new concept of composite biomaterials can be produced. It can be easily manufactured. Most of the materials used as substitutes for the human body in the medical composite biomaterial market are related to hard tissue, and although these materials are stable and strong, they remain as heterogeneous materials, so they are easy to cause pain to patients, and there is a problem that reoperation is required over time. The two-dimensional material manufacturing method according to the embodiments of the present invention fixes particles such as various metals and ceramics as needed to medical biomaterials, which were mainly made of a single material due to problems such as manufacturing cost, to produce a biocomposite material at a precise level. It can be used as a base technology to provide at a lower price.

Recently, the development of artificial tissues that can replace small and medium-sized animal experiments performed for product approval in the bio-health field is attracting attention. In this field, it is very difficult to secure each cell line and establish conditions for co-culture of various cells, which acts as a major obstacle to the technology. On the other hand, the two-dimensional material manufacturing method according to the embodiments of the present invention can easily solve the problem by duplicating the surface to be tested as it is. It is expected to provide an experimental platform for research.

Nucleic acids used to transcribe patterns in embodiments of the present invention are stable biomolecules, capable of sophisticated design and mass synthesis, and are already widely used in fields such as biology, medicine, and materials engineering. Therefore, the embodiments of the present invention can be flexibly combined with existing technologies using nucleic acids, such as biochip manufacturing technology, drug screening technology, and diagnostic technology, and can be applied to commercialized technologies for various purposes and can be used immediately.

The artificial organ bio market is pursuing various strategies to mass-produce these artificial organs, going beyond the development of implantable-level artificial organs. The two-dimensional material production method according to the embodiments of the present invention can contribute to mass production by duplicating the transformed xenogeneic organ or the intermediate stage tissue of the cell-based artificial organ as it is, domestic and foreign artificial organ bio companies or hospitals. It can be effectively supplied to demanding institutions such as In addition, it can be effectively used to develop biomaterials tailored to patients, which required relatively sophisticated manufacturing technology, going beyond the limited product lines such as intraocular lenses, dental implants, artificial joints and cartilage, which have been used for a long time. It will expand product lines and increase market competitiveness.

According to an embodiment of the present invention, it is possible to form various physical patterns from one original pattern to another pattern. In the conventional semiconductor process, only one physical pattern can be formed from one mask pattern. In an embodiment of the present invention, since original patterns can be formed with nucleic acids of different sequences, it is possible to form thousands or tens of thousands of patterns depending on which nucleic acid is a complementary linker-nucleic acid. In addition, by using the contraction, expansion, and deformation of the hydrogel, it is possible to create duplicate patterns of various sizes from one original pattern, which is expected to overcome the limitations of the existing semiconductor process. Since the present invention can be all carried out in water, it can be used for patterning molecules such as proteins or nucleic acids on the surface, and thus can be used to manufacture an antibody-based sensor or nucleic acid chip.

As such, according to embodiments of the present invention, it is possible to provide a method of manufacturing a two-dimensional material capable of multi-patterning in a single process. In addition, it is possible to provide a method for producing a two-dimensional material capable of selectively and/or continuously replicating the original pattern. In addition, it is possible to provide a method for fabricating a two-dimensional material capable of high-resolution patterning based on single-molecule contact. In addition, it is possible to provide a method for manufacturing a two-dimensional material that can be applied to various patterning materials and substrates. In addition, it is possible to provide a two-dimensional material manufacturing method capable of achieving a higher resolution than the original pattern. As described above, although the embodiments have been described with reference to the limited embodiments and drawings, various modifications and variations are possible from the above description by those skilled in the art. For example, the described techniques are performed in an order different from the described method, and/or the described components of the system, structure, apparatus, circuit, etc. are combined or combined in a different form than the described method, or other components Or substituted or substituted by equivalents may achieve an appropriate result.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (22)

In the two-dimensional material manufacturing method,
preparing a first material having a first nucleic acid patterned thereon;
binding a linker-nucleic acid to the first nucleic acid;
copying the pattern of the first material to the surface of the second material by binding the first nucleic acid and the modified second nucleic acid to the surface of the second material through the linker-nucleic acid;
separating the first material; and
applying a third material over the pattern copied to the surface of the second material;
A two-dimensional material manufacturing method comprising a. According to claim 1,
The preparing step is
A method for producing a two-dimensional material, characterized in that the target material particles or molecules on the surface of the first material are labeled with the first nucleic acid comprising a first sequence. According to claim 1,
The linker-nucleic acid comprises a third nucleic acid comprising a second sequence complementary to the first sequence of the first nucleic acid and a fourth nucleic acid comprising a fourth sequence complementary to the third sequence of the second nucleic acid as a linker. A two-dimensional material manufacturing method characterized in that it has a connected structure. 4. The method of claim 3,
The combining step is
The first nucleic acid of the first sequence and the linker-nucleic acid include the third nucleic acid of the second sequence, and the linker-nucleic acid is coupled to the first nucleic acid. 4. The method of claim 3,
The copying step is
By physical contact between the surface of the first material and the surface of the second material, the fourth nucleic acid of the fourth sequence and the second nucleic acid of the third sequence included in the linker-nucleic acid are bound to the second material. A method for fabricating a two-dimensional material, characterized in that the pattern of one material is copied to the surface of the second material. 4. The method of claim 3,
The separating step is
The first material using an enzyme or chemical reaction that recognizes and excises a specific sequence of the linker-nucleic acid linker portion, the bond between the first nucleic acid and the third nucleic acid, or the bond between the second nucleic acid and the fourth nucleic acid and cutting the bond between the second materials. 4. The method of claim 3,
The separating step is
A method for producing a two-dimensional material, characterized in that the bond between the first nucleic acid and the third nucleic acid is cut off while maintaining the bond between the second nucleic acid and the fourth nucleic acid by using a difference in binding force between the nucleic acids. According to claim 1,
The third material is a first sequence of the first nucleic acid or a material particle including at least one of a metal, a polymer, a ceramic, a biomolecule, and a cell whose surface is modified with a new nucleic acid comprising the first sequence, or the first A method for manufacturing a two-dimensional material, comprising a single chemical molecule to which a first sequence of nucleic acids or a new nucleic acid comprising the first sequence is chemically bound. According to claim 1,
and the third material is the same material as the target material particles or molecules on the surface of the first material. According to claim 1,
and the third material is a material different from the target material particles or molecules on the surface of the first material. According to claim 1,
In the applying step,
wherein the third material binds to the first nucleic acid remaining on the surface of the second material as the surface is modified with the first sequence of the first nucleic acid or a new nucleic acid comprising the first sequence. production method. According to claim 1,
The preparing step is
Preparing the first material comprising a plurality of patterns,
Two or more patterns among the plurality of patterns are composed of nucleic acids of sequences orthogonal to each other,
The first nucleic acid is one of the nucleic acids of the sequence orthogonal to each other,
The combining step is
A plurality of linker-nucleic acids comprising a nucleic acid different from the nucleic acid included in the plurality of patterns is combined;
The different linker-nucleic acids include a nucleic acid of a sequence complementary to each of the nucleic acids of the mutually orthogonal sequence
A method for producing a two-dimensional material, characterized in that 13. The method of claim 12,
The copying step is
copying the plurality of patterns of the first material to the surface of the second material by physically contacting the first material to the surface of the second material;
A method for producing a two-dimensional material, characterized in that the surface of the second material is modified with nucleic acids corresponding to each of the nucleic acids of the mutually orthogonal sequence. 13. The method of claim 12,
The applying step is
A method for producing a two-dimensional material, characterized in that by applying a plurality of different third materials, each of which has a surface modified with each of the nucleic acids having an orthogonal sequence, on the pattern copied to the surface of the second material. According to claim 1,
The preparing step is
Prepare a first material including N (where N is a natural number) patterns,
Nucleic acids constituting different patterns are orthogonal to each other,
By recycling the separated first material, two or more M (the M is 2 ^N -1) or less types of patterns can be formed. 16. The method of claim 15,
When the first material is recycled, the plurality of linker-nucleic acids combined with the nucleic acids included in the N patterns include (N-1) or less types of nucleic acids having different sequences. Way. According to claim 1,
the second material comprises a soft material;
a first step of shrinking the flexible material from which the pattern is replicated;
a second step of duplicating the pattern on another second material by using the contracted flexible material again as a first material; and
A third step of repeating the first step and the second step A times (where A is a natural number)
A two-dimensional material manufacturing method, characterized in that it further comprises. a pattern mediated by a first nucleic acid replicated from the surface of the original material to the surface of the two-dimensional material;
a second nucleic acid modified on the surface of the two-dimensional material;
a linker connecting the first nucleic acid and the second nucleic acid-nucleic acid; and
an additional material applied to the surface of the two-dimensional material and bound to the first nucleic acid of the pattern
A two-dimensional material comprising 19. The method of claim 18,
The linker-nucleic acid comprises a third nucleic acid comprising a second sequence complementary to the first sequence of the first nucleic acid and a fourth nucleic acid comprising a fourth sequence complementary to the third sequence of the second nucleic acid as a linker. having a connected structure
A two-dimensional material characterized by 20. The method of claim 19,
Binding of the linker-nucleic acid to the first nucleic acid as the first nucleic acid of the first sequence and the third nucleic acid of the second sequence included in the linker-nucleic acid are combined
A two-dimensional material characterized by 20. The method of claim 19,
As the fourth nucleic acid of the fourth sequence and the second nucleic acid of the third sequence included in the linker-nucleic acid are bound by physical contact between the surface of the original material and the surface of the two-dimensional material, the original material that the pattern of is copied to the surface of the two-dimensional material
A two-dimensional material characterized by 19. The method of claim 18,
The original material includes a plurality of patterns,
Two or more patterns among the plurality of patterns are composed of nucleic acids of sequences orthogonal to each other,
The first nucleic acid is one of the nucleic acids of the sequence orthogonal to each other,
The nucleic acid included in the plurality of patterns and the plurality of linker-nucleic acids comprising different nucleic acids are combined, whereby the first nucleic acid and the linker-nucleic acid are combined,
The different linker-nucleic acids include a nucleic acid of a sequence complementary to each of the nucleic acids of the mutually orthogonal sequence
A two-dimensional material characterized by

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