KR101571829B1 - Manufacturing method of microarray chip using radioactive ray - Google Patents

Abstract

The method of manufacturing a microarray chip using radiation according to the present invention is excellent in preventing nonspecific adsorption and is effective in inducing specific adsorption of cells to a desired site. In addition, it can be applied to glass, silicon and various polymer substrates, but the manufacturing process is simple using radiation. It is also suitable for cell application because it does not use other toxic substances such as crosslinking agent, catalyst, activator through radiation crosslinking. Therefore, it can be applied to industrial fields such as cell-based new drug development, pathogen detection, It is also applicable to academic fields such as intercellular, interactions between cells and matrix.

Description

Technical Field [0001] The present invention relates to a method of manufacturing a microarray chip using radiation,

The present invention relates to a method of manufacturing a microarray chip using radiation, and more particularly, to a method of manufacturing a microarray chip in which a polymer resin is applied on various substrates, followed by irradiation with radiation in two steps, will be.

In order to identify various complex biological interactions as well as cell growth, genetic functions, and biological signaling processes at the molecular level, there is a need for a rapid and cost-effective way to analyze complex, diverse and widely distributed data . In order to analyze these data, researches on the development of biochips are being actively continued.

A biochip is a dense accumulation of biomaterials such as DNA, protein, and other cells on a solid surface such as silicon, glass, or polymer. By using this information to obtain biological information, it is possible to obtain information on gene expression patterns, genetic defects, DNA- - It is used as a useful tool to carry out the purpose of protein interaction, disease diagnosis and so on.

The biochip can be divided into microarrays and microfluidics chips depending on the type. The microarray chip arranges thousands or tens of thousands of DNAs or proteins at regular intervals and attaches them to the solid substrate. Which is a biochip capable of analyzing the binding pattern. Recently, it has been recognized that there are limitations in identifying complex and diverse life phenomena with a single molecule level approach such as DNA or protein, so that cells are directly applied to a microarray to observe more diverse and complex physiological activities Is actively proceeding.

Two methods have typically been used to form a cell microarray. First, there is a method of directly spotting cells on a substrate using a robot or the like, and secondly, a substrate in which an area is defined in array form using physicochemical surface treatment so that cells can grow only in a limited region To cultivate the cells. However, since the first method is able to temporarily form a cell array, but the physicochemical compartment is not defined, the orientation of the cell array is liable to be lost. Therefore, the developers mainly use the second method, that is, Culture.

The most important prerequisite for manufacturing a substrate for cell microarray is that the surface on which the cell attaching portion and the non-attaching portion are clearly distinguished is formed on the same substrate. In order to realize such a surface, there has been conventionally used a photolithography such as Korean Patent Registration No. 10-0561874, a layer-by-layer deposition method in which a cell attachment material is laminated, a microfluidic channel method, a microcontact printing method microcontact printing). However, these methods have problems in reproducibility due to complicated substrate manufacturing processes, take a long time to manufacture, and are not suitable for environment formation for application to cells using toxic organic solvents such as crosslinking agents. In addition, when a cell-attaching peptide or an extracellular matrix protein is additionally coated or immobilized on a biologically inert substrate surface to facilitate cell attachment, it is difficult to specifically apply a cell-adhesive substance to only a desired segment, There is a disadvantage that the possibility of adsorption increases.

Korean Registered Patent No. 10-0561874 (March 10, 2006)

Development of Monolayer and Microarray of Protein Using Hyperbranched Dendritic Polymer, Korean Institute of Chemical Engineers Volume 7 Bulletin 1, p.128

In order to solve the above problems, the present invention provides a method of manufacturing a microarray chip using radiation, which induces cell adsorption at a desired position by selectively forming a biomolecule adsorptive pattern on an anti-adsorption thin film by using a simple and clean process .

The present invention relates to a method of manufacturing a microarray chip using radiation.

One aspect of the present invention is

a) coating at least one polymer solution on a substrate to form a polymer thin film;

b) curing the polymer thin film by first irradiating the substrate of step a) with radiation; And

c) placing a mask on the cured polymer thin film surface and irradiating it with radiation to form a biomolecule adsorption pattern;

Wherein the total irradiation amount in the second irradiation is higher than the total irradiation amount in the first irradiation, and more particularly, to a method of manufacturing a microarray chip using radiation.

The method of manufacturing a microarray chip using radiation according to the present invention is excellent in preventing nonspecific adsorption and is effective in inducing specific adsorption of cells to a desired site. In addition, it can be applied to glass, silicon and various polymer substrates, but the manufacturing process is simple using radiation. It is also suitable for cell application because it does not use other toxic substances such as crosslinking agent, catalyst, activator through radiation crosslinking. Therefore, it can be applied to industrial fields such as cell-based new drug development, pathogen detection, It is also applicable to academic fields such as intercellular, interactions between cells and matrix.

1 is a schematic view showing a process of forming a cell microarray according to an embodiment of the present invention.
FIG. 2 is a SEM showing a pattern of a microarray chip according to an embodiment of the present invention.
3 shows FT-IR spectrum of a microarray chip according to an embodiment of the present invention.
FIG. 4 shows a cell growth curve on the surface of a cell microarray chip according to an embodiment of the present invention.
5 is a photograph of a cell micrograph of the surface of a cell microarray chip according to an embodiment of the present invention.
6 shows the result of culturing H1299 cells on a cell microarray chip according to the production method of the present invention and staining with methylene blue staining reagent.

Hereinafter, a method for manufacturing a microarray chip using radiation according to the present invention will be described in detail with reference to specific examples or embodiments. It should be understood, however, that the invention is not limited thereto and that various changes and modifications may be made without departing from the spirit and scope of the invention.

Unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Also, the singular forms as used in the specification and the appended claims are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The present inventors have conducted studies on the cell adsorption behavior in the polymer thin film preventing the adsorption of biomolecules, but the polymer thin film formed at the low irradiation condition effectively inhibits cell adsorption and, in a high irradiation condition, And then the polymer film is formed on the surface of the polymer thin film by irradiating the polymer film with a low radiation dose and then irradiated with a high radiation dose to form a biomolecule adsorption pattern at a desired position, It was confirmed that the microarray can be manufactured, and the present invention was completed.

Hereinafter, the present invention will be described in more detail in a step-by-step manner.

A method of manufacturing a microarray chip using radiation according to the present invention can be started by coating at least one polymer solution on a substrate to form a polymer thin film.

The substrate is not limited to the type as long as it is commonly used in a microarray chip, and includes, for example, a polymer composed of an ester polymer, a silicone polymer, an acrylic polymer, an olefin polymer and copolymers thereof or an inorganic material such as glass And preferably at least one material selected from the group consisting of polyethylene terephthalate, polybutylene terephthalate, polysilane, polydimethylsiloxane, polysilazane, polyacrylate, polymethyl methacrylate, polyethyl acrylate , Polyethyl methacrylate, polyethylene naphthalate, cyclic olefin polymer, polyethylene, polypropylene, polyimide, polystyrene, polyacetal, polyetheretherketone, polyester sulfone, polytetrafluoroethylene, polyvinyl chloride, polyvinyl Liden Floride , A perfluoroalkyl polymer, and a combination thereof.

The polymer solution refers to a form in which a polymer is dissolved in a solvent to facilitate processing. The polymer solution is not limited to a polymer capable of crosslinking by radiation and capable of inhibiting the adsorption of biomolecules. In general, biomolecules such as cell lines, antigens, antibodies, etc., and DNAs and RNAs maintain their system through specific binding through the three-dimensional structure and chemical interaction with the physical form of the molecules, but besides these specific interactions And tend to physically adsorb to other surfaces.

The polymer used in the present invention is a polymer having a tendency to suppress nonspecific adsorption of biomolecules, and generally has a large volume in a solution, high degree of freedom, and high mobility of a polymer main chain, thereby inhibiting protein attachment. Or a brush having a high hydrophobicity or a modified end, a block copolymer capable of forming a self-assembled monolayer, and the like.

Examples of the polymer include polyethylene oxide, polyethyleneglycol, poly (ethyleneoxide-co-propyleneoxide-co-ethyleneoxide) But are not limited to, polydimethylsiloxane, polyalkylsiloxane, poly (meth) acrylate, polyalkyl (meth) acrylates, polycarbonates, One or two or more selected from the group consisting of polycyclic olefins, polyimides, polyurethanes and dextrans can be used.

As the polymer according to the present invention, poly (ethylene oxide-co-propylene oxide-co-ethylene oxide) is preferably used. The poly (ethylene oxide-co-propylene oxide-co-ethylene oxide) may have a structure represented by the following formula (1).

[Chemical Formula 1]

(Wherein n and m are each an integer of 10 to 130)

Commercially sold examples of the poly (ethylene oxide-co-propylene oxide-co-ethylene oxide) include F127 (n = , m = 39), f68 (n = 75, m = 30), P123 (n = 20, m = 70), P105 .

As the solvent constituting the polymer solution, a polar solvent or a nonpolar solvent commonly used in the art can be used, but it is preferable to use a solvent having low cytotoxicity due to the characteristics of the cell microarray chip. As the solvent, for example, water; Alcohols such as methanol, ethanol and ethylene glycol; And the like.

The polymer solution according to the present invention may comprise 1 to 70% by weight of the polymer and 30 to 99% by weight of a solvent. When the content of the polymer is less than 1% by weight, the viscosity of the solution is too low to form a thin film. When the content of the polymer is more than 70% by weight, the application is possible but the viscosity of the solution is too large, Do not.

In the polymer solution according to the present invention, it is not necessary to add other additives in addition to the polymer and solvent. Generally, a bio-chip such as a microarray or a microfluidic chip is produced by casting a polymer solution and crosslinking it. If necessary, additives such as a crosslinking agent, a crosslinking aid, a catalyst, an adhesion promoter, a surfactant, and a viscosity controlling agent are added. These additives remain in the biochip after production, and can act as a cytotoxic substance. However, since the microarray manufactured according to the present invention is manufactured by crosslinking by radiation without the addition of such an additive, biomolecules can be effectively adsorbed compared to the conventional technology.

When the polymer solution is prepared as described above, it is coated on the substrate surface to form a polymer thin film. The method of applying the polymer solution can be applied to any method as long as it can coat the substrate surface with a uniform thickness. For example, a suitable method such as a spraying method, a roll coating method, a spin coating method, a slit die coating method, a bar coating method, and an ink jet method can be used. Double spin coating method or slit die coating method, Can be obtained.

Next, the polymer thin film coated on the surface of the substrate can be cured by irradiating the first irradiation with radiation as in step b).

In the present invention, the radiation may be any one or two or more selected from ion beams, electron beams, gamma rays, alpha rays and beta rays, and it is preferable to irradiate ion beams.

When the ion beam is irradiated in the radiation, the energy of the ion beam is preferably 1 to 300 keV, and the current density of the ion beam is preferably adjusted to 1 μA or less. In addition, the injected element may be any one or two or more selected from carbon, oxygen, hydrogen, argon, helium, neon, and xenon.

It is preferable that the total ion irradiation amount during the ion beam irradiation is 1 x 10 ⁹ to 5 x 10 ¹⁴ ions / cm 2. The chemical change in the thin film surface by the ion beam if not occur a sufficient cross-linking to the polymer material, and a problem that is not the thin film is fixed, more than 5 × 10 ¹⁴ ions / ㎠ occurs when the total ion dose is less than 1 × 10 ⁹ ions / ㎠ The effect of preventing biomolecule adsorption is reduced, and nonspecific cell adsorption occurs during cell culture.

When the polymer thin film is cured as described above, a biomolecule adsorption pattern can be formed by placing a mask on the surface of the cured polymer thin film and irradiating it with a second radiation as in step c).

The above step will be described in more detail. The array type mask is fixed on the polymer thin film, and the surface of the polymer thin film irradiated with the radiation is chemically changed by selectively irradiating the radiation to form functional groups capable of fixing biomolecules, A step of transferring a microarray of various shapes and sizes having good orientation properties to a polymer thin film by forming a pattern that is easy to adhere.

The mask is for forming a biomolecule adsorptive pattern on the polymer thin film and may be any material that does not affect the polymer thin film during the removal process, such as a metal hard mask. In particular, the method of manufacturing a microarray according to the present invention is advantageous in that a photoresist such as a photosensitive polymer is applied, and a process of removing the photoresist after light irradiation is unnecessary.

As in the first irradiation, the secondary irradiation may be any one or two or more selected from among ion beams, electron beams, gamma rays, alpha rays and beta rays, and preferably, the ion beams are irradiated. In addition, the injected element may be any one or two or more selected from carbon, oxygen, hydrogen, argon, helium, neon and Xeon during the irradiation of radiation.

In the second irradiation, the energy of the ion beam is preferably 1 to 300 keV, and the current density of the ion beam is preferably controlled to 1 μA or less.

In the secondary irradiation, the total ion irradiation amount during ion beam irradiation is preferably 1 x 10 < ¹⁴ > to 1 x 10 < ¹⁶ > ions / When the total ion irradiation dose is less than 1 x 10 ¹⁴ ions / cm 2, sufficient chemical change does not occur in the polymer thin film even in the additional irradiation, and biomolecular adsorption pattern is not formed properly. In the case of exceeding 1 × 10 ¹⁶ ions / cm 2 There is a problem in that browning is induced in the secondarily irradiated pattern and the observation of the cells after the cell culture is not easy.

In addition, the method for manufacturing a microarray chip using radiation according to the present invention may further include a step (d) of forming a cell microarray by culturing the cells on the microarray chip manufactured through step c).

The microarray chip thus prepared is placed in a cell culture container, and the culture medium in which the cells are previously suspended is filled in the culture container so that the substrate is sufficiently immersed in the culture medium. Then, the microarray chip is cultured in an animal cell incubator for an appropriate time to form a cell microarray .

The biomolecule used in step d) is not limited in its kind, and examples thereof include proteins (mainly enzymes or antibodies), nucleic acids, hormones, enzyme substrates, antigens, polysaccharides, lipids, bacteria, yeast, Preferably human fibroblast, L929 (mouse fibroblast), HEK293 (human embryonic kidney fibroblast), HaCaT (human cervical cancer), MCF-7 (breast cancer), HUVECs (human umbilical vein endothelial cells) It is advisable to use a variety of adherent cells.

Referring to FIG. 6, a cell microarray chip manufactured according to the present invention can be used to manufacture a multi-scale microarray chip having various shapes and sizes. Particularly, the shape of the pattern can be manufactured not only in a general shape such as a circle, a rectangle and a line, but also in a letter form. In addition, it was confirmed that the deposition of cells with dye such as methylene blue was adsorbed only on the pattern formed by irradiation, and it was confirmed that the radiation adhered to the specific attachment and growth direction of the cells.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples. However, the following examples and comparative examples are merely illustrative of the present invention, and the present invention is not limited by the following examples and comparative examples.

(Whether a biomolecule adsorptive pattern is formed)

The surface of the prepared specimen was observed through a scanning electron microscope (SEM, JSM-7500F, JEOL).

(Infrared spectroscopic analysis (FT-IR))

The prepared specimens were observed through FT-IR (640-IR, Varian).

(Biomolecule adhesion and growth)

H1299 cells were cultured in the prepared specimens, and the number of cells was measured according to the incubation time, and the growth curves thereof were shown.

(Example 1)

A cell microarray was prepared by the following steps a) to d). The physical properties of the prepared microarray were measured and shown in FIGS. 2 to 5. FIG.

a) Polymer thin film  formation

Poly (ethylene oxide-co-propylene oxide-co-ethylene oxide) (HO (CH ₂ CH ₂ O) ₉₈ - (CH (CH ₃ ) CH ₂ O) ₆₇ - (CH ₂ CH ₂ O) ₉₈ H, (Ethylene oxide-co-propylene oxide-co-ethylene oxide) solution of 7 wt% (wt%) was dissolved in 9.3 g of an aqueous solvent to prepare a solution of the poly (ethylene oxide- To form a polymer thin film.

b) Polymer thin film  Hardening

Hydrogen (H ⁺ ) ions were irradiated onto the entire surface of the poly (ethylene oxide-co-propylene oxide-co-ethylene oxide) polymer thin film coated as described above. The irradiation apparatus used was irradiated with an ion beam having an energy of 200 keV with an ion beam irradiation apparatus at a total dose of 2 × 10 ¹⁴ ions / cm 2.

c) Biomolecule adsorption pattern formation

Hydrogen ions were secondarily irradiated by placing a pattern mask (SUS (steel use stainless steel, stainless steel mask, cell size: 50 占 50 占 퐉, cell spacing: 50 占 퐉) on the cured polymer thin film. , And irradiated with a total dose of 5 x 10 < ¹⁴ > ions / cm < 2 > at an energy of 200 keV to form a biomolecule adsorptive pattern.

d) Cells Microarray  formation

The cell microarray prepared in the step c) was allowed to not float in the cell culture container. Then, the cells in the subculture were suspended in a single cell, and the cell suspension at a concentration of 1 × 10 ⁵ cells / ml was put in a culture container so that the substrate was sufficiently immersed, and then cultured for 72 hours to form a cell microarray. In this step, RPMI640 medium containing 10% fetal bovine serum and 1% antibiotic (penicillin streptomycin) was used as the cell suspension medium. Cell culture was performed in an animal cell incubator maintained at 5% CO ² and 37 ° C. .

(Example 2)

The procedure of Example 1 was repeated except that poly (ethylene oxide-co-propylene oxide-co-ethylene oxide) was replaced with polyethylene oxide (Sigma-aldrich, To produce a specimen.

(Example 3)

(Ethylene oxide-co-propylene oxide-co-ethylene oxide) in Example 1 was replaced with polyethylene glycol (Sigma-aldrich, Poly (ethylene glycol), Mn 1,000) The test specimens were prepared through the steps.

(Example 4)

The procedure of Example 1 was repeated except that poly (ethylene oxide-co-propylene oxide-co-ethylene oxide) was replaced with dextran (Sigma-aldrich, Dextran, Mw 25,000) .

(Example 5)

The same procedure as in Example 1 was carried out except that poly (ethylene oxide-co-propylene oxide-co-ethylene oxide) was replaced with polydimethylsiloxane (Dow corning, Sylgard 184A) Respectively.

(Example 6)

The specimen was prepared in the same manner as in Example 1 except that the total irradiation amount of the ion beam was 6 × 10 ¹⁴ ions / cm 2 in the second irradiation.

(Example 7)

The specimen was prepared in the same manner as in Example 1 except that the total irradiation amount of the ion beam was 7 × 10 ¹⁴ ions / cm 2 at the second irradiation in Example 1.

(Comparative Example 1)

In Example 1, a specimen was prepared using the poly (ethylene oxide-co-propylene oxide-co-ethylene oxide) in Example 1, and the same procedure as in Example 1 was performed except that the primary and secondary curing were not performed Specimens were prepared.

(Comparative Example 2)

In Example 1, a specimen was prepared by using poly (ethylene oxide-co-propylene oxide-co-ethylene oxide) in Example 1, except that the specimen was not subjected to secondary curing, .

As shown in FIG. 2, when the biomolecule adsorptive pattern was formed on the sample prepared in Example 1, it was confirmed that the portion irradiated with the ion beam remained unremoved by the development as shown in FIG. Therefore, it can be seen that the thin film was successfully cured by the ion beam, and it was confirmed that the biomolecule adsorption pattern was successfully formed on the surface of the polymer thin film cured through (b) to (d).

3, the FT-IR spectroscopy (FT-IR) of the specimen was confirmed. The CH (2869 cm ^-1 ) and CO (1106 cm ^-1 ) I can confirm that it appears. The specimens ((c) to (e)) prepared in Examples 1, 6, and 7 were prepared in the same manner as in Example 1, except that -OH (3470 cm ^-1 ) And C = O (1728 cm ^-1 ) peaks were newly formed, and the peak size was small in the first irradiated thin film but relatively large in the second irradiated thin film, and the chemical characteristics of the polymer thin film I could confirm the change.

In addition, when the cell adhesion and growth of the test piece were confirmed through FIG. 4, the cells were hardly grown in the test piece prepared in Comparative Example 2 ((b)), and the test pieces prepared in Examples 1, 6 and 7 (c) to (e)) show that the growth curve of the cells over time shows a very similar tendency to the polystyrene container for cell culture ((a)).

Fig. 5 is a micrograph of the cultured cells after 3 days of cultivation. In the specimen prepared through Comparative Example 2 (b), almost no cells were grown, and the specimen prepared through Example 7 (c) , It was confirmed that adhesion and growth were almost similar to the polystyrene container for cell culture ((a)).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Those skilled in the art will recognize that many modifications and variations are possible in light of the above teachings.

Accordingly, the spirit of the present invention should not be construed as being limited to the embodiments described, and all of the equivalents or equivalents of the claims, as well as the following claims, belong to the scope of the present invention .

Claims (6)

(meth) acrylate, a polyalkyl (meth) acrylate, a polycyclic olefin, a polyimide, a polyurethane, and / or a copolymer of ethylene and at least one selected from the group consisting of polyethylene oxide, polyethylene glycol, poly (ethylene oxide-co-propylene oxide-co-ethylene oxide) Dextran, a polymeric adsorption-preventive polymer solution containing one or two or more polymers selected from dextran,
b) curing the biomolecule adsorption-preventive polymer thin film by first irradiating the substrate of step a) with a total dose of 1 × 10 ⁹ to 2 × 10 ¹⁴ ions / cm 2; And
c) placing a mask on the surface of the cured biomolecule adsorption-preventive polymer thin film and secondly irradiating a total dose of 5 × 10 ¹⁴ to 7 × 10 ¹⁴ ions / cm 2 to form a biomolecule adsorption pattern;
Wherein the method comprises the steps of: delete The method according to claim 1,
Wherein the polymer is poly (ethylene oxide-co-propylene oxide-co-ethylene oxide) represented by the following formula (1).
[Chemical Formula 1]

(Wherein n and m are each an integer of 10 to 130) The method according to claim 1,
Wherein the radiation is one or more selected from ion beams, electron beams, gamma rays, alpha rays and beta rays. delete delete

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