KR102177124B1 - A dopaminergic patterning method by subtractive transfer method - Google Patents

Abstract

The present invention relates to a method of manufacturing a dopamine pattern, characterized by a method of manufacturing a dopamine pattern through a subtractive transcription method, and a clean and perfect dopamine pattern without collapse of the pattern can be produced in large quantities through simple manipulation, and a hydrophobic substrate Since it is formed on the top, it has the advantage that it can be used in many ways.

Description

Method for preparing dopamine pattern by subtractive transfer method {A dopaminergic patterning method by subtractive transfer method}

The present invention relates to a method for manufacturing a dopamine pattern using a subtractive transfer method, and more particularly, to a method of forming a dopamine pattern on a non-elastic substrate using a subtractive transfer method.

The excellent surface adhesion ability of mussels living in the sea has been widely studied as a subject that can be used in various areas. Catechol, a functional group of mussel adhesive substances, plays an important role in this surface adhesion ability. Therefore, dopamine composed of catechol also exhibits excellent adhesion properties, and can be applied to various fields such as metal plating, cell culture, etc., and has strengths in cost and process.

In the metal plating or cell culture, it is important to accurately and uniformly form a certain pattern. To this end, elastomeric membranes have been used as cell seeing molds, and polydimethylsiloxane (PDMS) stamps are used to stamp polylysine or to coat PEG (polyethylene glycol) on a substrate. Techniques such as lithography methods, inkjet printing, magnetically labeled cells and dielectrophoresis have been developed. Among them, the soft lithography method is most widely used for patterning dopamine.

When a dopamine pattern is prepared by the above-described methods and a pattern of cells or nanomaterials is formed by using it, a problem of pairing between the patterns occurs, as well as sagging of the pattern, or the PDMS substrate during the curing process. Various problems such as deformation/damage of the pattern occurred due to the contraction. That is, there are many difficulties in forming a dopamine pattern layer only with conventional patterning methods, and there are many problems in using this for patterning cells. Furthermore, even in the case of the soft lithography method, the biggest limitation exists in that it has very low transfer efficiency for a substrate having a low surface energy.

Therefore, there is an urgent need to develop a new method capable of accurately and consistently forming a dopamine pattern layer even on a substrate having a low surface energy (high water contact angle).

Patent Document 1. Korean Patent Registration No. 10-1721071

The present invention has been devised in view of the above problems, and an object of the present invention is to provide a method for effectively manufacturing a dopamine pattern on a substrate having a low surface energy.

Another object of the present invention is to provide a method of manufacturing a pattern of cells or nanomaterials using a physically coated dopamine pattern.

The present invention provides a method for producing a dopamine pattern comprising the following steps to achieve the above object.

1) engraving a certain pattern on the hard substrate by engraving;

2) treating an oxygen plasma to modify the surface of the hard substrate to be superhydrophilic;

3) coating an amine-based polymer layer on the surface of the hard substrate on which the surface-modified pattern is formed;

4) disposing a hard substrate coated with an amine-based polymer layer on a dopamine-coated thermoplastic substrate, wherein the amine-based polymer layer faces the dopamine layer; And

5) After pressing and heating the thermoplastic substrate in the direction of the hard substrate, when the two substrates are separated, the dopamine layer is transferred and removed from the thermoplastic substrate except for a certain pattern of the hard substrate, and the dopamine pattern is removed from the thermoplastic substrate. The step in which the layer is formed.

After the step 1), a method of manufacturing a dopamine pattern by a subtractive transfer method, further comprising: washing the hard substrate.

The hard substrate may be poly methyl methacrylate (PMMA).

The thermoplastic substrate may be any one or more selected from the group consisting of a polystyrene (PS) substrate, a polycarbonate (PC) substrate, and a polyethylene terephthalate (PET) substrate.

The amine-based polymer is polyethyleneimine (PEI), polyamines, polyamideamine, polyvinylamine, polyamidoimine, polyallylamine, and polylysine. -L-lysine) and chitosan (Chitosan) may be one or more polymers selected from the group or a copolymer thereof.

The pressurization conditions in step 5) may be 0.05 to 5.0 MPa.

The heating condition in step 5) may be 60 to 90°C.

Pressurization and heating in step 5) may be performed for 1 to 10 minutes.

In order to achieve the above object, the present invention provides a step of coating a substrate on which a dopamine pattern layer prepared from the method for producing a dopamine pattern is formed with a dispersion in which micromaterials, nanomaterials, or cells are dispersed; It provides a method of manufacturing a material pattern.

The nanomaterial may be any one or more selected from the group consisting of luminescent nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, and metal oxide nanoparticles.

In the case of the metal nanoparticles, it may be coated by an electroless plating method.

The present invention relates to a novel strategy for producing a hydrophilic dopamine pattern on a substrate having a non-elastic and hydrophobic surface. By using the interaction of dopamine with the amine functional group of PEI through a subtractive transcription method, the pattern is clean and perfect without collapse. Dopamine patterns can be produced in large quantities through simple manipulation.

Since the dopamine pattern manufactured through the above-described process is formed on a hydrophobic substrate, it can be used in many ways. For example, it can be applied to selectively patterning micromaterials, nanomaterials or cells.

1 is a process diagram schematically showing a process of manufacturing a dopamine pattern according to the present invention.
2A is a photograph of a dopamine pattern layer prepared using various thermoplastic substrates (PC, PMMA, PS, PET) and various transfer temperatures (room temperature, 40°C, 50°C, 60°C, 70°C).
2B is a photograph of each surface when dopamine is transferred under a transfer temperature of 60° C. using a PEI-coated PMMA substrate having an intaglio pattern from a dopamine-coated PC substrate.
3A is a schematic diagram of an experimental process for confirming surface properties.
3B is a fluorescence microscope image of the surface of the PMMA substrate to which nothing was treated.
3C is a fluorescence microscope image of the surface of a PMMA substrate treated with O2-plasma.
3D is a fluorescence microscope image of the surface of a PEI coated PMMA substrate.
4A is a diagram showing a dopamine patterning method according to the subtractive transcription method according to the present invention.
4B is a graph showing the measurement of water contact angles with respect to the dopamine-coated thermoplastic substrate prepared from Experimental Example 3 and the dopamine-removed substrate.
4C is a photograph of a surface after performing a dopamine patterning process using a PMMA substrate that is not treated with anything and a PMMA substrate coated with PEI.
FIG. 4D is a measurement of water contact angles on PS substrates from which dopamine is removed, prepared by performing a dopamine patterning process using a PMMA substrate that is not treated with anything and a PEI-coated PMMA substrate.
FIG. 5 shows the results of XPS analysis on a PEI-coated PMMA substrate, a PEI-coated PMMA substrate, and a dopamine-coated PS substrate in Example 1; 5A is a broad XPS scan spectrum, FIG. 5B is a table showing the atomic ratio (%) of elements for each substrate, and FIG. 5C is a high-resolution spectrum for O1s and N1s core levels.
6A schematically illustrates a process of culturing HUVECs cells on a PS substrate on which a dopamine pattern layer ('GU') prepared in Example 1 is formed.
6B is a photograph taken with an optical microscope after culturing HUVECs cells on the PS substrate on which the dopamine pattern layer ('dot pattern') prepared in Example 1 is formed for 2 days.
6C is a photograph taken with an optical microscope after culturing HUVECs cells on the PS substrate on which the dopamine pattern layer ('dot pattern') prepared in Example 1 is formed for 4 days.
6D is a photograph of HUVECs cells cultured for 4 days on the PS substrate on which the dopamine pattern layer ('GU') prepared in Example 1 is formed, followed by an optical microscope.
FIG. 6E shows HUVECs cells were cultured for 4 days on the PS substrate on which the dopamine pattern layer ('GU') prepared in Example 1 was formed, and then the living cells were stained with green-fluorescent calcein-AM (green fluorescence) for fluorescence. It was observed under a microscope.
7A is a diagram illustrating a manufacturing process of a microwell structured PS substrate according to Example 4. FIG.
7B is an optical microscope (OM) image of a microwell structured PS substrate (d = 300 μm) according to Example 4.
7C is a fluorescence image for confirming the glass beads enclosed in the PS substrate of the microwell structure according to Example 4.
7D is an enlarged optical microscope image to show that glass beads (d = 40 μm) are enclosed in the wells of the PS substrate having a microwell structure according to Example 4.
8A is a photograph of the surface of the PS substrate on which the dopamine pattern layer prepared in Example 1 is formed before and after being coated with silver with an optical microscope, and FIG. 8A(a) is a PS substrate on which the dopamine pattern layer is formed, and FIG. 8A ( b) is a PEI-coated PMMA substrate to which dopamine is transferred, FIG. 8A(c) is a PS substrate on which a dopamine pattern layer is formed after being coated with silver by an electroless plating method, and FIG. 8A(d) is a dopamine coated with silver. It is a transferred PEI coated PMMA substrate.
8B is a SEM image showing the surface of the PS substrate on which the dopamine pattern layer prepared from Example 1 is formed before and after coating with silver. FIG. 8B(a) is a surface on which the dopamine pattern layer is formed among the PS substrate on which the dopamine pattern layer is formed. The boundary between the surface from which the dopamine was removed was photographed, and FIG. 8B(b) is an enlarged SEM image of the surface of the dopamine pattern layer on the PS substrate on which the dopamine pattern layer was formed, and FIG. 8B(c) is a dopamine pattern layer formed thereon. It is an SEM image of the silver layer coated on the PS substrate, and Fig. 8B(d) is an enlarged image of the silver layer.

Hereinafter, various aspects and various embodiments of the present invention will be described in more detail.

Throughout this specification, when a layer or substrate is said to be “on” another layer or substrate, this means that it is not only when a layer or substrate is in contact with the other layer or substrate, but also is another layer between two layers or two substrates. Or it includes the case where another description exists. In addition, when a part "includes" a certain component, it means that other components may be further included rather than excluding other components unless otherwise specified.

One aspect of the present invention relates to a method for producing a dopamine pattern comprising the following steps.

1) engraving a certain pattern on the hard substrate by engraving;

2) treating an oxygen plasma to modify the surface of the hard substrate to be superhydrophilic;

3) coating an amine-based polymer layer on the surface of the hard substrate on which the surface-modified pattern is formed;

4) disposing a hard substrate coated with an amine-based polymer layer on a dopamine-coated thermoplastic substrate, wherein the amine-based polymer layer faces the dopamine layer; And

5) After pressing and heating the thermoplastic substrate in the direction of the hard substrate, when the two substrates are separated, the dopamine layer is transferred and removed from the thermoplastic substrate except for a certain pattern of the hard substrate, and the dopamine pattern is removed from the thermoplastic substrate. The step in which the layer is formed.

First, 1) a certain pattern 10 is engraved on the hard substrate 110 in an intaglio.

The step of forming the pattern 10 is not particularly limited as long as it is a method generally used in the art, but may be preferably formed using computer numerical control milling. CNC milling is one of the milling cutting methods using computer numerical control, and the CNC milling machine can precisely perform plane cutting, curved surface cutting, groove cutting, and cutting processing of a workpiece. Since CNC milling is commonly known in the cutting field, a detailed description will be omitted.

The hard substrate 120 may be poly methyl methacrylate (PMMA).

The shape of the pattern 10 is not particularly limited as long as it can be engraved on the hard substrate, and may be designed in various shapes without being specified in width, length, depth, or shape. As in the experimental examples described later, it may be formed in the form of'GU' or a symbol representing a person.

After step 1), the step of washing the hard substrate 120 may be further included. The washing step is a step for removing impurities remaining on the hard substrate, and may be performed through a process of sufficiently drying in a vacuum dryer after washing with distilled water, methanol, ethanol, isopropanol, etc. for 5 to 20 minutes. .

Next, 2) oxygen plasma is treated to modify the surface of the hard substrate 120 to be superhydrophilic. Surface treatment may be performed using the oxygen plasma device. In the plasma process, a treatment time of 30 to 300 seconds is appropriate, and the power may be 60 W. If the plasma treatment is performed in less than 30 seconds, partial hydrophilization proceeds, resulting in a problem of lowering efficiency when transferring the pattern. If it exceeds 300 seconds, the physical properties of the substrate may be deteriorated and process costs may increase. In addition, if the power is increased, the plasma treatment time can be shortened, so the treatment time can be appropriately adjusted according to the power.

Thereafter, 3) an amine-based polymer layer is coated on the surface of the hard substrate 120 on which the surface-modified pattern is formed. In forming the dopamine pattern layer, since the hard substrate is hardly provided with a functional group capable of mediating the transfer of dopamine, the amine-based polymer is provided on the surface of the hard substrate 120 to coat the dopamine layer. When the transfer step with the thermoplastic substrate 140 is performed, only the desired pattern 10 area is thermoplastically formed by easily interacting with the remaining dopamine except for the pattern 10 area on the hard substrate 120 There is an advantage that it can be removed and left on the substrate 140.

The amine-based polymer is polyethyleneimine (PEI), polyamines, polyamideamine, polyvinylamine, polyamidoimine, polyallylamine, and polylysine. -L-lysine) and chitosan (Chitosan) may be one or more polymers selected from the group or a copolymer thereof, preferably polyethyleneimine (PEI).

Next, 3) a hard substrate 120 coated with an amine-based polymer layer is disposed on the thermoplastic substrate 140 coated with dopamine, and the amine-based polymer layer is positioned to face the dopamine layer.

The thermoplastic substrate may be any one or more selected from the group consisting of a polystyrene (PS) substrate, a polycarbonate (PC) substrate, and a polyethylene terephthalate (PET) substrate.

The step of coating dopamine on the thermoplastic substrate may be provided by preparing a coating agent in which dopamine is dissolved, and forming a dopamine layer by applying or immersing the coating agent on the thermoplastic substrate.

Due to the Schiff base reaction between the amine functional group of the amine polymer and the catechol group of dopamine, the pattern 10 does not collapse, and the dopamine layer other than the pattern 10 is accurately, completely and rapidly transferred. Can be removed.

Finally, 4) after pressing and heating the thermoplastic substrate 140 and the hard substrate 120, when the two substrates are separated, a dopamine layer from the thermoplastic substrate 140 is formed in a constant pattern of the hard substrate 140 ( Except for 10), it is transferred and removed, and a dopamine pattern layer is formed on the thermoplastic substrate 150.

In more detail, when the hard substrate 120 is removed and separated from the thermoplastic substrate 140, the dopamine layer of the thermoplastic substrate 140 excluding the constant pattern 10 of the hard substrate 120 is formed on the hard substrate ( It is transferred by bonding with the PEI layer of 160) and removed from the thermoplastic substrate 140. As a result, a dopamine layer is left on the thermoplastic substrate 150 along the pattern 10 of the hard substrate 120, and a dopamine layer patterned in a desired pattern 10 is formed on the thermoplastic substrate 150. .

In step 5), the pressurization conditions are preferably 0.05 to 5.0 MPa.

The heating condition in step 5) is preferably 60 to 90°C. If it is less than 60°C, the dopamine layer is not properly transferred from the dopamine-coated thermoplastic substrate 140 to the hard substrate 120, and the residue It was confirmed that there is a problem that remains as it is. If it exceeds 90°C, deformation of the substrate or deformation of the pattern may be caused, and a problem of increasing cost may occur.

In step 5), the pressurization and heating are preferably performed for 1 to 10 minutes, but if it is performed in less than 1 minute, sufficient contact is not made, so that the dopamine layer from the dopamine-coated thermoplastic substrate 140 is transferred to the hard substrate 120 ), and if it exceeds 10 minutes, primary amine and secondary amine are formed as intermediate products of dopamine polymerization, so that the desired physical properties of the dopamine pattern layer cannot be obtained.

Through the step 5), by selectively and efficiently removing portions other than the pattern 10 from the dopamine layer physically coated on the thermoplastic substrate 140, only the pattern 10 can be left on the surface. Accordingly, the present invention enables patterning of high-contrast dopamine through a subtractive transcription method.

That is, the amine-based polymer coated on the hard substrate 120 forms a covalent bond through a strong Schiff-base reaction with dopamine, thereby subtraction of dopamine in a region other than the pattern 10. will be.

When the dopamine layer is removed by the hard substrate 120 through this, the original hydrophobicity and transparency of the thermoplastic substrate 140 are restored. It was confirmed that dopamine was completely removed through an experiment to be described later. In contrast, dopamine remains in the pattern 10, and the dopamine pattern layer 150 maintains hydrophilicity as it is.

As described above, since the dopamine pattern layer 150 manufactured according to the present invention is a hydrophobic region except for the dopamine pattern layer 150, and the dopamine pattern layer 150 is hydrophilic, substantially between the pattern and the external region. Even if the separator is not formed on the surface, a physical barrier is formed due to the difference in wettability. That is, when treating an aqueous solution in which cells or nanomaterials are dispersed, it has a great advantage that cells or nanomaterials can be patterned along the dopamine pattern layer 150 without a separate process step.

Unlike soft lithographs using conventional elastomer stamps, the above-described manufacturing method transfers dopamine with high accuracy and homogeneity without deformation of the pattern using an inelastic mold, thereby accurately patterning dopamine on a hydrophobic substrate with only a simple operation. You can see that you can be angry. The thickness of the dopamine pattern layer prepared through the above process can be easily adjusted by controlling the thickness of the dopamine layer coated on the thermoplastic substrate.

The dopamine pattern layer prepared by the above-described method can be used to develop a microwell array or to pattern cells for a cell culture platform.

Another aspect of the present invention is the step of immersing or applying a micromaterial or nanomaterial or a dispersion in which cells are dispersed on the substrate 160 on which the dopamine pattern layer is formed prepared through the above-described process; It relates to a method of manufacturing a material pattern.

In the present invention, by effectively patterning the dopamine pattern on a substrate having a low surface energy (high water contact angle) through the method for producing a dopamine pattern previously, each pattern acts as a physical barrier due to the difference between the hydrophilic and hydrophobic surfaces. These may be spatially separated and formed. Therefore, since cells can be simply patterned with such characteristics, it is possible to provide a cell culture platform in large quantities.

The dispersion is preferably an aqueous solution, and may be distilled water or a buffer.

The nanomaterial may be any one or more selected from the group consisting of luminescent nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, and metal oxide nanoparticles.

The dispersion in which the cells are dispersed may further include a cell culture medium. The culture medium may be appropriately selected according to the cell, and the cell is not particularly limited as long as the cell is also a cell capable of two-dimensional culture.

The micromaterial may be a spherical bead having a diameter of 1 to 100 μm.

In the case of the metal nanoparticles, it may be coated by an electroless plating method. This can be patterned by simply selectively depositing metal on the basis of the dopamine pattern layer 160 without using a complex electric device. In an embodiment of the present invention, silver was used as the metal.

As confirmed in the experimental examples to be described later, it can be seen that the pattern made of micromaterials, nanomaterials, or cells prepared through the above process is not deposited at all outside the dopamine pattern layer.

In addition, since the dopamine pattern layer according to the present invention exhibits no toxicity in cell culture, it was confirmed that it can be used as a method for producing a new cell pattern and a cell culture platform.

Hereinafter, the present invention will be described in more detail through examples, etc., but the scope and contents of the present invention are reduced or limited by the examples below and cannot be interpreted. In addition, if based on the disclosure content of the present invention including the following examples, it is clear that the present invention for which no specific experimental results are presented can be easily carried out by a person skilled in the art, and such modifications and modifications are attached to the patent. It is natural to fall within the scope of the claims.

<Experimental material>

Dopamine hydrochloride, PEI (branch, Mw ~ 25,000), silver nitrate (AgNO ₃ ), D-(+)-glucose and ammonium hydroxide solution (NH ₄ OH, 28.0-39.0%) were purchased from Sigma-Aldrich. . Tris-HCl (1 M, pH 8.0) was purchased from Biosesang (Korea). PMMA, PC, PS and PET substrates were purchased and used from Goodfellow (Huntingdon, UK, UK).

Human umbilical vein endothelial cells (HUVECs), endothelial cell medium (ECM), penicillin/streptomycin (P/S) solution, endothelial cell growth supplement (ECGS), trypsin/EDTA solution 0.25% (T/E) , Trypsin neutralization solution (TNS) Trypan blue solution 0.4% (TB, trypan blue solution), Dulbecco's phosphate buffered saline (DPBS, Dulbecco's phosphate buffered saline) and fetal bovine serum (FBS) ) Was purchased from ScienCell Research Laboratories (California, USA).

The LIVE/DEADㄾ Viability/Cytotoxicity Kit for analyzing cell viability and the Green Fluorescent Carboxylate-Modified Microspheres (also known as'Fluorescent Beads') are from Thermo Fisher Scientific. (Massachusetts, USA) was purchased and used. In addition, 30-50 μm glass beads were purchased from Polysciences (Pennsylvania, USA).

<Example 1> Preparation of dopamine pattern layer

Referring to FIG. 1, first, a certain pattern was engraved on the surface of the PMMA substrate by using a computer numerical control (CNC) milling machine (TINYROBO, Korea) (step 1). Thereafter, the PMMA substrate on which the intaglio pattern was engraved was thoroughly cleaned to remove plastic dust. The PMMA substrate with the intaglio pattern was treated with oxygen plasma at 60 W for 1 minute (Femto Science, Korea) (step 2), and 1% at room temperature for 30 minutes to impart amine-rich functionalities. It was immersed in an aqueous PEI solution (step 3).

Next, a 10 mM Tris-HCl (pH 8) buffer solution was prepared and mixed with dopamine hydrochloride to prepare a dopamine solution having a concentration of 2 mg mL ^-1 . The PS substrate (thermoplastic substrate) was immersed in the dopamine solution, left to stand at room temperature for 20 hours, washed with distilled water, and dried with air to prepare a dopamine-coated substrate.

The dopamine-coated PS substrate and the PEI-coated PMMA substrate prepared in step 3 were in close contact with each other (step 4), and heated to 60° C. or higher using a custom pneumatic press for 10 minutes at a pressure of 0.15 MPa. , By separating the two substrates, a PS substrate on which a dopamine pattern layer was formed and a PEI-coated PMMA substrate on which dopamine was transferred were obtained (step 5).

As a result of visually checking the surfaces of the two substrates, it was confirmed that the dopamine layer (brownish) excluding a certain pattern was transferred from the dopamine-coated PS substrate to the PEI-coated PMMA substrate. That is, since the dopamine layer remains along the intaglio pattern existing on the PEI-coated PMMA substrate, it was confirmed that the dopamine pattern layer was formed on the following PS substrate.

<Example 2> Preparation of a PC substrate having a dopamine pattern layer

A PC substrate having a dopamine pattern layer was prepared in the same manner as in Example 1, except that PC was used as the thermoplastic substrate.

<Example 3> Preparation of a PET substrate having a dopamine pattern layer

A PET substrate having a dopamine pattern layer was prepared in the same manner as in Example 1, except that PET was used as the thermoplastic substrate.

<Example 4> Microwell Array Preparation

A PS substrate having a microwell structure was manufactured in the same manner as in Example 1, except that a PS substrate having a microwell structure and a PMMA substrate having no intaglio pattern were used as the thermoplastic substrate.

The PS substrate having the microwell structure was manufactured by engraving a recess having a diameter of 300 µm on the PS substrate using a CNC milling machine.

In order to inject dopamine into the microwell of the PS substrate having the microwell structure, an additional process is required. First, in order to coat dopamine in a hydrophobic micro-well without air bubbles, the cavities were filled with ethanol. Thereafter, it was put in a batch filled with water, ethanol was removed in the concave portion, and water was filled in the vacancy. Next, the substrate was immersed in the dopamine solution for 20 hours, so that dopamine was coated on both the recess and the substrate surface.

After the process is the same as in Example 1, but briefly described, the dopamine-coated PS substrate (microwell structure) was brought into close contact with the PEI-coated PMMA substrate, followed by heating under pressure at 0.15 MPa and 60° C. for 10 minutes, and then When separated, the dopamine layer in addition to the concave portion is removed by the PEI-coated PMMA substrate, thereby obtaining a microwell array in which only the concave portion is coated with the dopamine layer.

Thereafter, the aqueous solution in which the fluorescent beads were dispersed or the aqueous solution in which the glass beads were dispersed was repeatedly applied and aspirated on the microwell array with a micropipette, and confirmed with a fluorescence microscope and an optical microscope. I confirmed that the glass beads were filled. This will be described in more detail through experimental examples to be described later.

<Experimental Example 1> Optimization of transfer conditions

In order to optimize the conditions for preparing the dopamine pattern layer, the experiment was performed as follows.

The PMMA substrate was thoroughly cleaned to remove plastic dust, treated with oxygen plasma at 60 W for 1 minute (Femto Science, Korea), and then 1 for 30 minutes at room temperature to impart amine-rich functionalities. By immersion in% PEI aqueous solution, a PEI coated PMMA substrate was prepared.

Next, a 10 mM Tris-HCl (pH 8) buffer solution was prepared and mixed with dopamine hydrochloride to prepare a dopamine solution having a concentration of 2 mg mL ^-1 , and various thermoplastic substrates (PC, PMMA, PS, etc.) were prepared in the dopamine solution. PET) was each soaked and left at room temperature for 20 hours, washed with distilled water, and dried with air to prepare a dopamine-coated substrate.

The dopamine-coated substrate and the PEI-coated PMMA substrate are in close contact with each other for 10 minutes at a pressure of 0.15 MPa, under various transfer temperatures (room temperature, 40° C., 50° C., 60° C., 70° C.), customized pneumatic press machine Was used. After separating the two substrates, each surface was photographed and shown in FIG. 2.

2A is a photograph of a dopamine pattern layer prepared using various thermoplastic substrates (PC, PMMA, PS, PET) and various transfer temperatures (room temperature, 40°C, 50°C, 60°C, 70°C).

2B is a photograph of each surface when dopamine is transferred under a transfer temperature of 60° C. using a PEI-coated PMMA substrate having an intaglio pattern from a dopamine-coated PC substrate.

In FIGS. 2A and 2B,'a' is a thermoplastic substrate on which dopamine was originally coated, and a dopamine layer is removed from the surface through a transfer process. 'b' is a PMMA substrate coated with PEI, and transfer is made from'a' to'b', which is indicated by an arrow. Scale bar = 50 μm.

As shown in Figure 2A, polydopamine (Polydopamine) has a unique dark brown. In this experiment, the appearance and relocation of the brown color present on each substrate surface were observed to confirm whether dopamine was coated and transferred. In the case of using PEI-coated PMMA, it was confirmed that the dopamine transfer was well performed due to the abundance of primary and secondary amines, and the dopamine layer transferred from the thermoplastic substrate to the PEI-coated PMMA layer was uniformly performed overall. It can be seen that the higher the transfer temperature, regardless of the thermoplastic material, the more dominant. In particular, it was confirmed that the dopamine layer on the thermoplastic surface was completely transferred to the PEI-coated PMMA substrate at a temperature of 50°C or higher.

However, when the transfer temperature is lowered to 40° C. or lower or at room temperature, it can be visually confirmed that the dopamine layer cannot be transferred from the thermoplastic substrate to the PEI-coated PMMA substrate. This is expected to be due to the increased reactivity between the amine group and the dopamine layer at high temperatures.

2B is a photograph of a dopamine layer coated on a flat polycarbonate (PC) surface as described above, and then transferred to a PEI-coated PMMA substrate having an intaglio pattern. Referring to FIG. 2B, it can be seen that the PEI-coated PMMA substrate was transferred from the surface of the polycarbonate (PC) substrate, but selectively transferred only to a flat area excluding the engraved pattern. After transfer printing, the dimensions of the dopamine pattern remaining on the PC substrate were well matched with the dimensions of the engraved portion of the PEI-coated PMMA, and it was confirmed that no distortion of the pattern occurred during the transfer process. In addition, it can be seen that the present invention not only obtains a pattern of high quality and high resolution, but also overcomes problems caused by conformal contact even when a hard substrate is used.

<Experimental Example 2> Surface analysis of PEI-coated PMMA substrate

The PMMA substrate was thoroughly cleaned to remove plastic dust, thereby preparing an untreated PMMA substrate (Pristine PMMA). Next, the PMMA substrate was treated with oxygen plasma (Femto Science, Korea) at 60 W for 1 minute to prepare an O2-plasma-treated PMMA substrate. Finally, in order to impart amine-rich functionalities, the O2-plasma-treated PMMA substrate was immersed in 1% PEI aqueous solution at room temperature for 30 minutes to prepare a PEI-coated PMMA substrate.

It was attempted to evaluate whether or not the PEI layer was successfully formed on the PMMA surface through the above process through microspheres modified with carboxylate (hereinafter referred to as'fluorescent beads').

3A is a schematic diagram of an experiment process for confirming surface characteristics, and an experiment method will be described with reference to this as follows. A PMMA substrate without any treatment (Pristine PMMA), an O2-plasma-treated PMMA substrate, and a PEI-coated PMMA substrate prepared through the above process were prepared, and microspheres modified with carboxylate on the entire surface of each (hereinafter ,'Fluorescent beads') were evenly treated. After 5 minutes, it was washed with distilled water, and the fluorescence was measured through a blue fluorescent filter, and then analyzed with ProgRes® CapturePro software.

Fluorescent beads modified with carboxylate are negatively charged by carboxylate and are bound to the amine group of PEI. Therefore, when the fluorescent beads are treated on the PMMA surface, it is possible to evaluate whether PEI is uniformly or successfully formed on the PMMA surface by confirming with fluorescence (green fluorescence) whether the fluorescent beads are immobilized. At this time, the microspheres (fluorescent beads) modified with carboxylate were diluted 100 times and used.

3B is a fluorescence microscope image of the surface of the PMMA substrate that is not treated with anything, FIG. 3C is a fluorescence microscope image of the surface of the PMMA substrate treated with O2-plasma, and FIG. 3D is a fluorescence microscope image of the surface of the PMMA substrate coated with PEI. . Scale bar is 500 μm.

As shown in FIGS. 3B, C, and D, when fluorescent beads were treated on an untreated PMMA substrate (Pristine PMMA), O2-plasma-treated PMMA substrate, and PEI-coated PMMA substrate, only PMMA coated with PEI ( 3D) only showed a strong fluorescence signal. In the untreated PMMA substrate and the O2-plasma treated PMMA substrate, only negligible weak signals were observed.

Through the difference between the above results, since the carboxylate group of the fluorescent bead forms a bond by electrostatic interaction with the amine group of PEI, the fluorescent beads having a carboxylate group through the expressed green fluorescence are PEI-coated PMMA substrate. It can be seen that the phase is uniform and has been successfully fixed. That is, it can be seen that the PEI layer is uniformly formed on the surface of the PMMA substrate.

<Experimental Example 3> Contact angle analysis-1

In order to optimize the conditions for preparing the dopamine pattern layer, the experiment was performed as follows.

The PMMA substrate was thoroughly cleaned to remove plastic dust, treated with oxygen plasma at 60 W for 1 minute (Femto Science, Korea), and then 1 for 30 minutes at room temperature to impart amine-rich functionalities. By immersion in% PEI aqueous solution, a PEI coated PMMA substrate was prepared.

Next, a thermoplastic substrate (PC, PMMA, PS, PET) without any treatment is prepared (Step I, Pristine). A 10 mM Tris-HCl (pH 8) buffer solution was prepared and mixed with dopamine hydrochloride to prepare a dopamine solution having a concentration of 2 mg mL ^-1 , and various thermoplastic substrates (PC, PMMA, PS, PET) were added to the dopamine solution. Each was immersed and left at room temperature for 20 hours, washed with distilled water, and dried with air, to prepare a dopamine-coated substrate (step II, dopamine coated).

The dopamine-coated substrate and the PEI-coated PMMA substrate were brought into conformal contact with each other, and a custom pneumatic press was used at a pressure of 0.15 MPa for 10 minutes and under a temperature condition of 60° C. The two substrates were separated to prepare a PEI-coated PMMA substrate to which dopamine was transferred (Dopamine-transferred to PEI-coadted PMMA) and a substrate from which dopamine was removed (step III, Dopamin-subtracted). A water contact angle was measured on each of the substrates using a sessile drop technique, which was used with a Phoenix 10 system (Surface Electro Optics, Korea). Water contact angles were measured at two or more locations per each substrate, and the average was calculated and shown in FIG. 4.

4A is a diagram showing a dopamine patterning method according to the subtractive transcription method according to the present invention. Here, "I" means a thermoplastic substrate (Pristine) to which no treatment has been applied. "II" means a thermoplastic substrate coated with dopamine for 20 hours. "III" means a thermoplastic substrate (Dopamin-subtracted) from which the dopamine layer has been removed through a transfer process.

4B is a graph showing the measurement of water contact angles with respect to the dopamine-coated thermoplastic substrate prepared from Experimental Example 3 and the dopamine-removed substrate.

When dopamine is patterned by the subtractive transfer method according to the present invention, it was investigated whether all of the dopamine from which the pattern was removed from the dopamine-coated substrate is transferred to the PEI-coated PMMA substrate. To this end, it was confirmed through the change in the contact angle of the surface of the substrate obtained from steps Ⅰ, Ⅱ, and Ⅲ of Experimental Example 3.

As shown in FIG. 4B, the water contact angles of the PC, PMMA, PS and PET substrates (Step I) that were not treated with anything were 79.1 ± 0.7°, 74.3 ± 1.1°, 85.1 ± 1.4°, and 83.6 ± 1.1°, respectively.

After dopamine was coated, the contact angle was all reduced to about 40° due to the hydrophilicity of dopamine.

Next, after the dopamine was removed through step Ⅲ, the water contact angles of the PC, PMMA, PS and PET substrates recovered to the initial level at 78.6 ± 0.5°, 69.4 ± 0.8°, 85.4 ± 1.4°, and 81.2 ± 0.7°, respectively. You can see that it is. That is, when dopamine is patterned by the subtractive transfer method according to the present invention, that is, all dopamine from the dopamine-coated substrate excluding the pattern is transferred to the PEI-coated PMMA substrate.

<Experimental Example 4> Analysis of the contact angle according to the PEI coating layer-2

When dopamine is patterned by the subtractive transfer method according to the present invention, the effect of the surface of the PMMA substrate was examined. To this end, dopamine patterning was performed as in Experimental Example 3 using a PMMA substrate that was not treated with anything and a PEI-coated PMMA substrate, and each dopamine prepared therefrom was removed on a PS substrate ('b','d'). The water contact angle was analyzed. In addition, the water contact angle was analyzed for each of the PMMA substrates ('a' and'c') to which the dopamine obtained through the above process was transferred.

FIG. 4C is a photograph of a surface after performing a dopamine patterning process using a PMMA substrate that is not treated with anything and a PEI-coated PMMA substrate. FIG. 4D is a measurement of water contact angles on PS substrates from which dopamine is removed, prepared by performing a dopamine patterning process using a PMMA substrate that is not treated with anything and a PEI-coated PMMA substrate.

As shown in Fig. 4C, when a PMMA substrate that has not been treated with anything was used, dopamine transfer occurred very partially. On the other hand, in the case of using a PEI-coated PMMA substrate, it was confirmed that dopamine transfer was uniformly performed throughout.

4D, it was confirmed that the PEI-coated PMMA substrate ('c') to which dopamine was transferred had a remarkably increased water contact angle to 72.84 ± 0.6 °. Considering that the water contact angle of the dopamine-coated substrate is 40°, this is an unexpected result.

As shown in the results of Experimental Example 3, it can be said that it is contradictory that the dopamine surface will naturally be hydrophilic. This is believed to be due to the fact that dopamine exhibits different properties depending on its structural form. That is, it can be considered that the delocalization of the lone electron pair occurs along the conjugated form of the amine group and the catechol group present in the PEI layer, affecting the degree of hydrogen bond formation with water.

Therefore, it can be seen that in order to pattern dopamine by the subtractive transfer method according to the present invention, it is necessary to coat the PMMA substrate with PEI.

<Experimental Example 5> XPS analysis

XPS analysis (X-ray photoelectron spectroscopy, Axis-His, Kratos Analytical) was performed. Measurements were made using AxisHsi (Kratos Analytical, UK) equipped with an aluminum X-ray source (mono gun, 1486.6 eV) with 40 eV of pass energy. Before data acquisition, the pressure in the chamber was less than 5 × 10 ^-9 Torr, and the voltage and current of the anode were 13 kV and 18 mA, respectively. The take-off angle was 45°. The binding energy of C1s (284.5 eV) was used as a reference, and the energy resolution for measuring the binding energy was approximately 0.1 eV.

FIG. 5 shows the results of XPS analysis on a PEI-coated PMMA substrate, a PEI-coated PMMA substrate, and a dopamine-coated PS substrate in Example 1; 5A is a broad XPS scan spectrum, FIG. 5B is a table showing the atomic ratio (%) of elements for each substrate, and FIG. 5C is a high-resolution spectrum for O1s and N1s core levels.

As shown in Fig. 5A, since the PS substrate is composed of only carbon and hydrogen, the O1s and N1s peaks observed in the XPS spectrum can be said to represent dopamine coated on the PS substrate, through which dopamine on the PS substrate. You can see that it is coated properly.

In the PEI-coated PMMA substrate, a distinct and strong N1 peak can be observed, indicating that PEI has been successfully bonded to the PMMA substrate surface. In addition, it was confirmed that in the PEI-coated PMMA substrate to which dopamine was transferred, the C1s peak intensity was higher than that of others.

As shown in Fig. 5B, the atomic ratio was calculated from the XPS spectrum for each, the dopamine-coated PS substrate had an N/C ratio of 0.13, but the dopamine-transferred PEI-coated PMMA substrate had an N/C of 0.084. It was found to have a C ratio. That is, even though both substrates have the same dopamine layer on their surface, it was analyzed that the N/C ratio of the PEI-coated PMMA substrate to which dopamine was transferred was lower. In this way, when dopamine was transferred, only the C peak intensity increased, not the N peak or O peak, is considered to be an unexpected phenomenon caused by the organic coupling relationship between the PEI layer and the dopamine layer transfer process.

As shown in FIG. 5C, the peaks of O1s and N1s for each substrate were plotted by high-resolution XPS scans, indicating that the spectrum of PMMA coated with PEI and the spectrum of PS coated with dopamine showed distinct differences in peak shift and intensity. Confirmed.

In the PEI-coated PMMA substrate, it was confirmed that the primary amine of PEI interacted with oxygen bound to the PMMA surface through O2-plasma treatment, and the primary amine was reduced. On the other hand, it is believed that the dopamine-transferred PEI-coated PMMA substrate completely covers the PEI spectrum. Although the dopamine-transferred PEI-coated PMMA substrate had similar spectral characteristics at the O1s and N1s peaks than the dopamine-coated PS substrate, it showed a remarkable difference in the ratio of the peaks.

Specifically, the dopamine-coated PS substrate had a higher ratio of primary amine to tertiary amine, but the PEI-coated PMMA substrate to which dopamine was transferred had a higher ratio of tertiary amine than the primary amine. It is believed that when dopamine is transferred to the surface of the PEI-coated PMMA substrate, more tertiary amines are formed than primary amines (the secondary amine has no significant change). If the transfer process was performed for more than 10 minutes, it was possible to obtain a result that the primary and secondary amines were increased.

It is believed that the dopamine patterning method according to the subtractive transcription method of the present invention results in a high concentration of a tautomer-related tertiary amine as a dopamine polymerization intermediate through a dopamine transfer process of less than 10 minutes. Through this, it can be understood that the water contact angle is increased in the dopamine layer coated on the PEI-coated PMMA substrate through the transfer process. In other words, since the possibility of forming hydrogen bonds is reduced, a high water contact angle is obtained.

<Experimental Example 6> Preparation of cell pattern

Through the dopamine patterning method according to the subtractive transfer method of the present invention, a PS substrate (Example 1) on which a dopamine pattern layer is formed was prepared, and it was examined whether a cell pattern can be produced through it. After culturing HUVECs on the surface of the PS substrate on which the dopamine pattern layer prepared in Example 1 was formed, it was confirmed whether or not survival.

1) cell culture

First, T/E, TNS and DPBS and medium (1% P/S, 1% ECGS, and 5% FBS were diluted in ECM) were prepared at room temperature. Thereafter, HUVECs cells were cultured in a cell culture flask to which DPBS was added, and rinsed using a mixed solution of DPBS (10 mL) and T/E (1 mL). In order to separate the cells, the mixture was stirred and cultured at 37° C. for 2 minutes to obtain a cell mixture. The cell mixture was transferred to a 50 ml tube containing 5 ml of FBS and 5 ml of TNS, and centrifuged at 1000 rpm for 5 minutes. The solution was removed there, and the cells were resuspended in the medium. We stained dead cells with trypan blue and counted the number of living cells using a hemocytometer to determine viable cell concentration.

After preparing the HUVEC (5000 cells cm ^-2 ) cells obtained through the above process, put them in a culture dish containing each substrate, and cultured in a humidified condition at 37°C and 5% CO ₂ to culture cells patterned cells The platform was prepared. The culture medium was changed every 3 days. After 2 or 4 days of culture, the cell culture platform was taken out and carefully washed with DPBS to check the cell pattern, and the cell viability was confirmed by calcein AM staining through the LIVE/DEADㄾ kit. Specifically, a 4 μM calcein AM solution (ex/em ~495 nm/~515 nm) diluted with DBPS solution was prepared, added to the cell culture platform, reacted for 20 minutes, observed with a fluorescence microscope, and then ProgResㄾ Analyzed with CapturePro software.

2) result

6A schematically illustrates a process of culturing HUVECs cells on a PS substrate on which a dopamine pattern layer ('GU') prepared in Example 1 is formed. 6B is a photograph taken with an optical microscope after culturing HUVECs cells on a PS substrate on which a dopamine pattern layer ('dot pattern') prepared in Example 1 is formed, and FIG. 6C is prepared from Example 1 This is a photograph taken with an optical microscope after culturing HUVECs cells for 4 days on a PS substrate on which the dopamine pattern layer ('dot pattern') is formed.

6D is a photograph of HUVECs cells cultured for 4 days on the PS substrate on which the dopamine pattern layer ('GU') prepared in Example 1 is formed, followed by an optical microscope. FIG. 6E shows HUVECs cells were cultured for 4 days on the PS substrate on which the dopamine pattern layer ('GU') prepared in Example 1 was formed, and then the living cells were stained with green-fluorescent calcein-AM (green fluorescence) for fluorescence. It was observed under a microscope. Scale bars are Fig. 6B, C = 100 μm, Fig. 6D, E = 300 μm.

The PS substrate with the dopamine pattern layer prepared from Example 1 prepared through the present invention may be used as a platform for cell culture. Since the surface of the thermoplastic substrate (PS substrate) that is not treated with anything has a cell-antifouling function, it is difficult to attach cells immediately, and there are many difficulties in controlling the cells to be cultured within a certain pattern.

However, the PS substrate on which the dopamine pattern layer is formed prepared in Example 1 has the advantage of being able to selectively culture cells within a certain pattern because the cells have high affinity for the dopamine surface.

6A to 6C, it was confirmed that HUVECs cells can be selectively attached, cultured, and grown in the dopamine pattern layer on the PS surface on which the dopamine pattern layer is formed, and even though the number of cells increased on the 4th day, the dopamine pattern layer was still formed. It can be seen that it is selectively cultured without deviating. Even if the cells leave the dopamine pattern layer, the cells cannot adhere/proliferate on the PS surface, so it was confirmed that each pattern acts as a separate culture space.

6D, it was confirmed that, in addition to the dot pattern of a certain arrangement, various pattern layers can be formed, and cells can be cultured in the various pattern layers.

In addition, as a result of confirming the live cells with calcein AM among HUVECs cells cultured on the PS substrate on which the dopamine pattern layer ('GU') prepared in Example 1 is formed through FIG. 6E, the'GU' pattern and the living HUVECs cells are accurately It was confirmed that they were consistent. That is, the PS substrate on which the dopamine pattern layer ('GU') prepared in Example 1 is formed can culture cells according to the pattern and has little cytotoxicity, so it can be sufficiently utilized as a new platform for cell culture.

<Experimental Example 7> Microwell Array

Through the dopamine patterning method according to the subtractive transfer method of the present invention, a PS substrate (Example 4) having a microwell structure was manufactured, and its application possibility as a microwell array was examined. After immobilization of fluorescent beads or glass beads in the microwell structure PS substrate prepared by the manufacturing method of the present invention, it was confirmed with an optical microscope and a fluorescence microscope.

7A is a diagram illustrating a manufacturing process of a microwell structure PS substrate according to Example 4, and FIG. 7B is an optical microscope (OM) image of a microwell structure PS substrate (d = 300 μm) according to Example 4. .

7C is a fluorescence image for confirming the glass beads enclosed in the PS substrate of the microwell structure according to Example 4, and FIG. 7D is the glass beads in the well of the PS substrate of the microwell structure according to Example 4 (d = 40 μm) is an enlarged optical microscope image to show that it is enclosed. The scale bar is 400 μm.

As shown in FIG. 7A, in the PS substrate having a microwell structure according to Example 4, each well is hydrophilic, but the surface from which the dopamine layer is removed through transfer is hydrophobic. That is, it is confirmed that the surface can be selectively prepared to be hydrophilic or hydrophobic through the patterning process according to the present invention, and through this property, the fluorescent beads or glass beads can be sealed or fixed only in the wells using an aqueous solution. I did.

In fact, as a result of analyzing the microwell structure PS substrate (Example 4) in which the fluorescent beads were immobilized on the well by the above-described method, the optical image (Fig. 7B) and the fluorescent image (Fig. 7C) were analyzed, the fluorescent beads were effectively fixed only inside the well. It was confirmed that it is.

According to FIG. 7D, it can be seen that glass beads having a diameter of 30-40 μm can be fixed only to the wells of the PS substrate having the microwell structure in the same manner. The microwell structure PS substrate prepared in Example 4 can be used as a cell culture platform that can be used for multi-molecule testing or single cell capture.

<Experimental Example 8> Silver coating through electroless plating

Through the dopamine patterning method according to the subtractive transfer method of the present invention, a PS substrate (Example 1) having a dopamine pattern layer formed thereon was prepared, and it was examined whether it can be applied to selective electroless plating of silver. Silver (Ag) was coated on the surface of the PS substrate on which the dopamine pattern layer prepared in Example 1 was formed by an electroless plating method using silver nitrate (AgNO ₃ ). At this time, the pattern of the dopamine pattern layer was used as a'human shape symbol'.

Specifically, a plating solution of AgNO3 aqueous solution (50 mM, 4 mL) was prepared, and 2 M NH ₄ OH was added dropwise to the plating solution, followed by mixing well until brown aggregates were formed and disappeared. Thereafter, glucose (400 μL of 16.7 MM) was added as a reducing agent, and the PS substrate (Example 1) on which the dopamine pattern layer was formed was immersed, and then removed after 15 minutes and washed with distilled water (termination of the reaction).

In order to analyze the PS substrate coated with silver by the electroless plating using SEM and EDX, first, the substrate surface was coated with platinum using a Cressington sputter coater 108auto (Cressington Scientific Instruments, UK), and this was JSM-7500F SEM ( JEOL Ltd., Japan) and an X-act EDX device (Oxford Instruments, Mumbai) to obtain SEM and EDX data.

8A is a photograph of the surface of the PS substrate on which the dopamine pattern layer prepared in Example 1 is formed before and after being coated with silver with an optical microscope, and FIG. 8A(a) is a PS substrate on which the dopamine pattern layer is formed, and FIG. 8A ( b) is a PEI-coated PMMA substrate to which dopamine is transferred, FIG. 8A(c) is a PS substrate on which a dopamine pattern layer is formed after being coated with silver by an electroless plating method, and FIG. 8A(d) is a dopamine coated with silver. It is a transferred PEI coated PMMA substrate.

8B is a SEM image showing the surface of the PS substrate on which the dopamine pattern layer prepared from Example 1 is formed before and after coating with silver. FIG. 8B(a) is a surface on which the dopamine pattern layer is formed among the PS substrate on which the dopamine pattern layer is formed. The boundary between the surface from which the dopamine was removed was photographed, and FIG. 8B(b) is an enlarged SEM image of the surface of the dopamine pattern layer on the PS substrate on which the dopamine pattern layer was formed, and FIG. 8B(c) is a dopamine pattern layer formed thereon. It is an SEM image of the silver layer coated on the PS substrate, FIG. 8B(d) is an enlarged image of the silver layer, and FIG. 8B(e) is EDX data for FIG. 8B(d). The scale bar in Figure 8A is 1 mm.

8A(a) and (b), it is possible to compare before and after silver is coated by electroless plating on the PS substrate on which the dopamine pattern layer prepared in Example 1 is formed. After the silver was coated, it was confirmed that the dopamine pattern layer lost transparency and became opaque by the silver coating over time.

On the contrary, in the PEI-coated PMMA substrate to which dopamine was transferred, silver was plated on the remaining surfaces except for the pattern (FIGS. 8A(c), 8A(d)).

In addition, looking at FIG. 8B(a), the surface from which dopamine is removed from the PS substrate on which the dopamine pattern layer is formed is clean and flat, which can be seen that the remaining dopamine layer, not the pattern, was completely and cleanly removed from the substrate through the transfer process. .

It can be seen more clearly from FIG. 8B that the silver coating was well made through electroless plating on the dopamine pattern layer prepared in Example 1. In particular, it was confirmed that the silver nanoparticles were densely and densely coated along the dopamine pattern layer through electroless plating through FIG. 8B(d). This can also be confirmed from the result of confirming the main component of the surface as EDX (energy dispersive X-ray spectrometry) data (FIG. 8B(e)).

Claims (11)

1) engraving a certain pattern on the hard substrate by engraving;
2) treating an oxygen plasma to modify the surface of the hard substrate to be superhydrophilic;
3) coating an amine-based polymer layer on the entire surface of the hard substrate on which the surface-modified pattern is formed;
4) disposing a hard substrate coated with an amine-based polymer layer on a dopamine-coated thermoplastic substrate, wherein the amine-based polymer layer faces the dopamine layer; And
5) After pressing and heating the thermoplastic substrate in the direction of the hard substrate, when the two substrates are separated, the dopamine layer is transferred and removed from the thermoplastic substrate except for a certain pattern of the hard substrate, and the dopamine pattern is removed from the thermoplastic substrate. Method for producing a dopamine pattern by a subtractive transcription method comprising the step of forming a layer. The method of claim 1,
After the step 1), the method of manufacturing a dopamine pattern using a subtractive transfer method, further comprising: washing the hard substrate. The method of claim 1,
The hard substrate is a method of manufacturing a dopamine pattern by a subtractive transfer method, characterized in that poly methyl methacrylate (PMMA). The method of claim 1,
The thermoplastic substrate is a polystyrene (PS) substrate, a polycarbonate (PC) substrate, and a dopamine pattern by a subtractive transfer method, characterized in that at least one selected from the group consisting of a polyethylene terephthalate (PET) substrate Method of manufacturing. The method of claim 1,
The amine-based polymer is polyethyleneimine (PEI), polyamines, polyamideamine, polyvinylamine, polyamidoimine, polyallylamine, and polylysine. -L-lysine) and chitosan (Chitosan) at least one polymer selected from the group or a copolymer thereof, a method for producing a dopamine pattern by a subtractive transcription method, characterized in that. The method of claim 1,
The pressurization condition in step 5) is a method for producing a dopamine pattern by a subtractive transcription method, characterized in that 0.05 to 5.0 MPa. The method of claim 1,
The heating condition in step 5) is a method of manufacturing a dopamine pattern by subtractive transfer method, characterized in that 60 to 90 ℃. The method of claim 1,
In the step 5), the pressurization and heating are performed for 1 to 10 minutes. delete delete delete

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