KR101079718B1 - Method of immobilization and patterning of biomolecules onto biodegradable polymeric materials using radiation - Google Patents

Abstract

The present invention relates to a method of forming a biomolecule pattern on a biodegradable polymer material using radiation, and more particularly, by irradiating a biodegradable polymer to radiation and reacting the functional groups on the surface of the polymer generated with the biomolecule, The present invention relates to a method of immobilizing molecules and a method of forming a biomolecule pattern using the same.

According to the present invention, the biomolecules can be immobilized on the surface of the biodegradable polymer material without affecting the bulk physical properties of the biodegradable polymer material, and the process of introducing the biomolecules is very simple and is applied to various kinds of biomolecules. Since the present invention is applicable, it can be usefully used in the manufacturing field of biocompatible materials for tissue engineering, bio devices, food packaging materials and the like.

Biodegradable polymer materials, biomolecules, radiation, ion beams, functional groups, covalent bonds, immobilization, pattern formation

Description

Method of immobilization and patterning of biomolecules onto biodegradable polymeric materials using radiation}

The present invention relates to a method of immobilizing a biomolecule on a biodegradable polymer material using radiation and more specifically to a pattern formation method. A method of immobilizing molecules to form biomolecular patterns.

Polymer materials are widely used in many fields, including the high-tech information electronics industry, biomaterials industry, precision machinery industries such as automobiles and aviation, and living industries, such as building materials and packaging materials. In addition, since polymer materials can be processed in large quantities at low cost, there is an increasing interest in applications to medical engineering materials, biosensors, or tissue engineering. In order to apply to these fields, it is necessary to introduce biomolecules such as cells, proteins, enzymes, DNA, and the like onto polymer materials. However, general purpose polymer materials are basically non-hydrophilic and have low chemical reactivity, so that the introduction of biomolecules is very difficult.

Many surface modification methods have been developed to date, and representative surface modification methods for the polymer materials currently used include wet chemical treatment with base or acid, surface coating with silane coupling agent, corona discharge, plasma treatment and graft. There are many physical and chemical methods, including polymerization, metal deposition, ion implantation, surface grafting (SM Desai et al, Advances in Polymer Science, 169, 231 (2004)).

Specifically, Korean Patent Publication No. 10-2003-26076 discloses a method of forming a functional group on the surface of a polyurethane and immobilizing antithrombogenic protein by gas plasma treatment, and Korean Patent Publication No. 10-2003-76109 discloses a plasma generation method. A method of immobilizing anti-thrombotic proteins by covalent bonds through carboxyl groups to polytetrafluoroethylene by generating plasma by injecting a dissolved gas is disclosed. US Patent Publication No. 2003-0129740 discloses a method for forming a pattern of a functional group for fixing a biomaterial. The present invention discloses a method for immobilizing a biomolecule on a sample surface by generating and maintaining a low temperature atmospheric plasma. Meanwhile, Korean Patent Publication No. 10-2007-7577 discloses an apparatus and method for patterning animal cell fixation collagen using an electro-hydraulic printing method, and a cell chip manufactured thereby, and a Korean Patent Registration 10-0722321 No. describes a method of forming a protein pattern which induces a protein to be immobilized at a selective position by capillary lithography and the like and a protein chip produced thereby.

Representative methods for introducing biomolecules onto materials include: 1) electrostatic interaction, 2) physical entrapment with hydrogels, and 3) ligand / receptor parining. 4) covalent immobilization. Among these, covalent bonds can provide the most stable bond between material and biomolecule, which is a very desirable method. (J. M. Goddard et al. Prog. Polym. Sci., 32, 698 (2007)).

Recently, in the production of bio devices such as biosensors, biochips, and microfluidic chips, a technology capable of forming circuits using biomolecules such as cells and proteins is used. These methods include microarrays, electronic arrays, photolithography, and ink jet printing, and different methods are used by companies that manufacture biodevices. Among these, the production of bio devices using the photolithography method has the advantage of being able to manufacture devices with high integration by applying the microfabrication technology used in semiconductor fabrication, but the yield of photoreaction is not high, and it meets the needs of users. There is a problem that the manufacturing of the device is difficult. Methods such as microarray and inkjet printing allow the gene to be injected without touching the surface of the chip electrically, which can produce a large number of chips with quantitative genes. There are technical problems such as exchanging genetic material in cartridges that are required to produce DNA chips. In addition, microcontact printing, dip pen nanolithography, nanoshaving, electron beam lithography, focused ion beam, Research using nanoimprinting and the like is underway (Senaratne et al., Biomacromolecules, 6, pp 2427 (2005)). However, the above methods are still in the research stage, are very slow, can not pattern various types of biomolecules, and also have to go through several steps to pattern the biomolecules, There is a problem that requires much more research to be practical because there is a difficulty in fixing.

Therefore, while the present inventors are studying a method for improving biocompatibility on a biodegradable polymer material, the present invention can generate functional groups such as carboxylic acid on the biodegradable polymer material through irradiation, and directly generate biomolecules into the generated functional group. It was found that can be introduced by a covalent bond, to complete the present invention.

An object of the present invention is to provide a method for immobilizing a biomolecule on a biodegradable polymer material using radiation and a method for forming a biomolecule pattern using the same.

In order to achieve the above object, the present invention comprises the steps of irradiating the surface of the biodegradable polymer material to generate a functional group (step 1); And reacting the polymer material of the step 1 with a biomolecule (biomolecule) to fix the biomolecule by generating a covalent bond between the functional group and the biomolecule generated on the surface of the polymer (step 2). Provided are a method for immobilizing biomolecules on a biodegradable polymer material and forming a pattern using radiation.

According to the present invention, it does not affect the bulk physical properties of the polymer material and can easily introduce biomolecules or form a biomolecule pattern on the polymer material at low cost, and has excellent biodegradability and biocompatibility, and is applicable to various kinds of biomolecules. Since this is possible, it can be very usefully used in the manufacturing field of biodevices and biocompatible materials for tissue engineering.

Hereinafter, the present invention will be described.

The present invention comprises the steps of irradiating the surface of the biodegradable polymer material to generate a functional group (step 1); And

By reacting the polymer material of the step 1 and the biomolecule (biomolecule), and covalently bonded to the functional group and the biomolecules generated on the surface of the polymer to fix the biomolecule (step 2) A method of immobilizing biomolecules and forming patterns on biodegradable polymeric materials is provided.

Hereinafter, the present invention will be described in detail step by step.

First, the step 1 according to the present invention is a step of generating functional groups on the surface of the biodegradable polymer material by irradiating radiation, specifically, by irradiating the surface of the biodegradable polymer material to activate the surface of the biodegradable polymer material carboxyl It is a step of generating functional groups, such as an acidic radical, a hydroxyl group, and a ketone group.

The polymer material in step 1 is biodegradable polymers, such as polyester-based, polycarbonate, polyether carbonate polymer. Representative biodegradable polymer materials include natural polymers such as cellulose, chitin and starch, poly (3-hydroxyalkanoate) made by microorganisms, and poly (3-hydroxyvalerate-co-3-hydroxybutyrate ) polymer, polyglycolic acid (poly (glycolic acid)), polylactic acid (poly (lactic acid)), poly-caprolactone (polycaprolactone), poly hydroxy butyric acid, polyglycolic Lai de, polyethylene adipate (poly, such as ( ethylene adipate)), poly (trimethylene adipate), polybutylene adipate, poly (butylene adipate), poly (ethylene terephthalate), polybutylene terephthalate (poly ( synthetic polymers such as butylene terephthalate), polytrimethylcarbonate, and poly (1,4-dioxane-2-one) (poly (1,4-dioxane-2-one)).

In step 1, the biodegradable polymer material may induce the above-mentioned functional group by irradiation of relatively low energy (1 keV to 1 MeV) than the non-biodegradable polymer material. From this point of view, it is preferable to irradiate radiation in the range of 1 keV to 1 MeV. In addition, in order to prevent thermal deformation or decomposition, it is preferable to irradiate the radiation while maintaining the temperature of the polymer material at room temperature.

When the ion beam is irradiated in step 1, it is preferable to adjust the ion beam current density to 1 μA / cm ² or less, and the injection element may use gases such as carbon, oxygen, hydrogen, argon, helium, neon or xenon, and the like. It is preferable that ion beam energy is 1-300 keV. The total ion dosage is preferably 1 × 10 ⁹ to 1 × 10 ¹⁶ ions / cm ² . When the total ion irradiation amount is less than 1 × 10 ⁹ ions / cm ² , there is a problem in that the surface of the polymer material cannot be effectively activated, and when the total ion irradiation amount exceeds 1 × 10 ¹⁶ ions / cm ² , thermal deformation or decomposition of the polymer material is caused. There is a problem that occurs.

When irradiating the electron beam in the step 1, the energy of the electron beam is preferably 1 keV ~ 1 MeV and the total irradiation amount is 5 ~ 1000 kGy. If the total irradiation amount is less than 1 kGy, there is a problem that can not effectively activate the surface of the polymer material, if more than 1000 kGy there is a problem that thermal deformation or decomposition of the polymer material occurs.

Step 2 according to the present invention is a step of immobilizing the biomolecule on the polymer material obtained in step 1. Specifically, first, the polymer material derived from the functional group obtained in step 1 is N, N'-dicyclohexylcarbodiimide (N, N'-dicyclohexylcarbodiimide; DCC), 1-ethyl-3 '-(3-dimethylamino Propyl) immersed in buffer solution dissolved in 1-ethyl-3 '-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for 0.5 to 1 hour Pretreatment. Thereafter, the biomolecules, such as proteins and DNA, are immersed in a solution dissolved in a buffer solution for 6 to 24 hours to react to obtain a biodegradable polymer material having a biomolecule pattern formed by covalent immobilization of the biomolecules on the biodegradable polymer material. Can be.

The pattern formation may confirm pattern formation by immersing and reacting a polymer material in which the biomolecule is immobilized in a buffer solution in which a fluorescent material is tagged protein or c-DNA.

In addition, the biomolecule of step 2 may be carbohydrates, proteins, nucleic acids or lipids.

Furthermore, the present invention can selectively generate functional groups on the surface of the polymer material by irradiating radiation on the surface of the polymer material using a mask during the irradiation of step 1 above. That is, by using the mask, pattern formation can be performed so that the biomolecule can be selectively fixed to only portions other than the mask.

Hereinafter, an Example demonstrates this invention in detail. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example  One: Polycaprolactone  Formation of protein patterns on the surface (1)

<Step 1> Selective activation of the surface of the biodegradable polymer material

Polycaprolactone (PCL, Aldrich) was dissolved in chloroform solvent to prepare a 10% by weight solution, and then cast on a glass plate to prepare a polycaprolactone film. Neon ions were injected into the prepared film through a mask (SUS, 400 mesh). The ion implantation apparatus used in the implantation step is a 300-keV ion implanter, and irradiated with ions of 1 x 10 ⁹ to 1 x 10 ¹⁶ ions / cm ² with ion beam energy of 50 to 150 keV. After ion beam irradiation, the film was left in air for 24 hours.

Step 2 Formation of Protein Pattern

The polymer film ion-implanted in step 2 was immersed in a solution of 15 mM NHS (N-hydroxysuccinimide) and 45 mM EDC (1-ethyl-3- (3- (dimethylaminopropyl) carboimide) dissolved in PBS (phosphate-buffered saline). The reaction was carried out for 30 minutes.

Subsequently, the film was immersed in a solution in which biotin amine ((+)-Biotinyl-3,6,9-trioxaundecane diamine, biotin-amine, Pierce) was dissolved in PBS, reacted at room temperature for 6 hours, and then several times with distilled water. Washed.

The film was again immersed in a solution in which fluorescent-tagged streptavidin was dissolved in PBS containing 0.1% (w / v) BSA (bovine serum albumin) and 0.02% (v / v) Tween 20. After reacting at room temperature for an hour, the polymer material was formed by washing with PBS solution and distilled water several times to form a protein pattern.

Example  2: Polycaprolactone  Superficial DNA  Formation of the pattern (2)

<Step 1> Selective activation of the surface of the polymer material

Step 1 was carried out in the same manner as step 1 of Example 1.

<Step 2> DNA  Formation of patterns

In step 2, the ion beam-treated polymer film was immersed in a solution in which 15 mM NHS and 45 mM EDC were dissolved in PBS and reacted for 30 minutes. The film was again immersed in a solution dissolved in PBS in a probe p-DNA formed of 5'-CGA CCA CTT TGT CAA GCT CA-Amino-3 'nucleotides (nucleotides) for 6 hours and then washed several times with distilled water. The film was again immersed in a solution of 5'-Cy5-TGA GCT TGA CAA AGT GGT CG-3 'nucleotides tagged with fluorescent material in a solution dissolved in PBS for 6 hours at 30 ° C. After washing, the polymer material was washed with PBS solution and distilled water several times to form a DNA pattern.

Example  3: Polylactic acid  Formation of protein patterns on the surface (3)

<Step 1> Selective activation of the surface of the polymer material

It was carried out in the same manner as in Step 1 of Example 1 except that polylactic acid was used as the polymer material.

Step 2 Formation of Protein Pattern

Step 2 was carried out in the same manner as step 2 of Example 1.

Example  4: Polylactic acid  Superficial DNA  Formation of patterns (4)

<Step 1> Selective activation of the surface of the polymer material

Step 1 was carried out in the same manner as step 1 of Example 3.

Step 2 Formation of Protein Pattern

Step 3 was carried out in the same manner as step 2 of Example 2.

Example  5: Poly ( Hydroxybutyl acid ) Formation of protein patterns on the surface ((5)

<Step 1> Selective activation of the surface of the polymer material

It was carried out in the same manner as in Step 1 of Example 1, except that poly (hydroxybutyl acid) was used as the polymer material.

Step 2 Formation of Protein Pattern

Step 2 was carried out in the same manner as step 2 of Example 1.

Example  6: Poly ( Hydroxybutyl acid Superficial DNA  Formation of patterns (6)

<Step 1> Selective activation of the surface of the polymer material

Step 1 was carried out in the same manner as step 1 of Example 5.

<Step 2> DNA  Formation of patterns

Step 2 was carried out in the same manner as step 2 of Example 2.

Example  7: Polyglycolide  Formation of protein patterns on the surface (7)

<Step 1> Selective activation of the surface of the polymer material

It was carried out in the same manner as in Step 1 of Example 1 except that polyglycolide was used as the polymer material.

Step 2 Formation of Protein Pattern

Step 2 was carried out in the same manner as step 2 of Example 1.

Example  8: Polyglycolide  Superficial DNA  Formation of patterns (8)

<Step 1> Selective activation of the surface of the polymer material

Step 1 was carried out in the same manner as step 1 of Example 7.

<Step 2> DNA  Formation of patterns

Step 2 was carried out in the same manner as step 2 of Example 2.

The polymer material and biomolecule used in each of the above examples are shown in Table 1 below.

Polymeric Materials and Biomolecules Used in Examples of the Present Invention Polymer material Biomolecule Example 1 Polycaprolactone protein Example 2 Polycaprolactone DNA Example 3 Polylactic acid protein Example 4 Polylactic acid DNA Example 5 Polyhydroxybutyl acid protein Example 6 Polyhydroxybutyl acid DNA Example 7 Polyglycolide protein Example 8 Polyglycolide DNA

Experimental Example  1: On the surface of the polymer material by the ion beam irradiation of step 1 Functional group  Confirmation of creation

In order to determine whether a functional group such as carboxylic acid was formed on the surface of the activated polymer film through Step 1 of Example 1, it was analyzed using an infrared spectrometer (FT-IR). The control was compared with a polymer film that did not irradiate neon ions. The analysis results are shown in FIG. 2. FIG. 2 Polycapro without irradiating ion beams, as can be seen from the change of functional groups of pure polycaprolactone and selectively activated polycaprolactone at 1 × 10 ¹⁵ ions / cm ² using infrared spectroscopy (FT-IR) In the case of polycaprolactone (Example) irradiated with ion beams compared to lactone (control), it was confirmed that peaks for carboxyl groups (-COOH) that did not appear in pure polycaprolactone were generated at 3,500 cm ^-1 and 1,650 cm ^-1 . Through this, it was confirmed that the surface activation was successful through the ion beam.

Experimental Example  2: optional biomolecule of step 2 Attachment  Confirm

In order to confirm the adhesion of biomolecules to the polymer film, a protein-attached film according to Example 1 and a DNA-attached film according to Example 2 were analyzed using a fluorescence microscope and a confocal microscope. . The analysis results are shown in FIGS. 3 and 4. As shown in Figure 3 and 4, it was confirmed that the protein or DNA attached selectively to the unmasked portion.

As a result, when the method of the present invention is applied, biomolecules can be easily introduced by direct covalent bonds with functional groups such as carboxylic acids of a polymer material. It can be usefully used in the manufacturing field of biocompatible materials.

1 is a schematic diagram showing a process of forming a biomolecular pattern on the surface of a selectively activated biodegradable polymer material according to the present invention.

FIG. 2 is a graph showing the change of functional groups of pure polycaprolactone and selectively activated polycaprolactone at 1 × 10 ¹⁵ ions / cm ² using infrared spectroscopy (FT-IR).

Figure 3 is a fluorescence micrograph showing the protein pattern on the polycaprolactone surface prepared by selectively activated at 1 X 10 ¹⁵ ions / cm ² .

Figure 4 is a fluorescence micrograph showing the DNA pattern on the surface of polycaprolactone prepared by selectively activated by selectively activated at 1 X 10 ¹⁵ ions / cm ² .

Claims (8)

1 × 10 ⁹ to 1 × 10 ¹⁶ ions / cm ² ion beam or 5 kGy to 1000 kGy on the surface of any one biodegradable polymeric material selected from the group consisting of biodegradable polyesters, polycarbonates and polyethercarbonates Irradiating an electron beam to generate a functional group (step 1); And The biodegradable polymer using radiation, comprising the step of reacting the polymer material of the step 1 and the biomolecule, and covalently bonding the functional group and the biomolecule produced on the surface of the polymer (step 2) Methods of immobilizing biomolecules on a material and forming patterns. delete The method of claim 1, wherein the biodegradable polymeric material of step 1 is selected from the group consisting of cellulose, chitin, starch, poly (3-hydroxyalkanoate), poly (3-hydroxyvalerate-co-3-hydroxybutyrate), Polyglycolic acid (poly (glycolic acid)), polylactic acid (poly (lactic acid), polycaprolactone, polyhydroxybutyl acid, polyglycolide, polyethylene adipate, poly Trimethylene adipate (poly (trimethylene adipate)), polybutylene adipate (poly (butylene adipate)), poly (ethylene terephthalate), polybutylene terephthalate Biodegradability using radiation, characterized in that any one selected from the group consisting of polytrimethylcarbonate and poly (1,4-dioxane-2-one) (poly (1,4-dioxane-2-one)) Biomolecules on Polymer Materials Immobilization and pattern formation method. delete delete delete The method of claim 1, wherein the biomolecule of step 2 is at least one selected from the group consisting of carbohydrates, proteins, nucleic acids, and lipids. 8. The biomolecule according to any one of claims 1, 3 and 7, wherein the functional group and the biomolecule selectively generated on the surface of the polymer material are reacted by irradiating radiation on the surface of the polymer material using a mask during the irradiation of step 1. Biomolecules are selectively immobilized to form a biomolecule.

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