KR101248120B1 - Polyelectrolyte multilayer and biosensor - Google Patents

Abstract

The present invention relates to a polymer electrolyte multilayer membrane comprising a gamma-cyclodextrin or a derivative thereof in an alternating stacked structure of an anionic polymer electrolyte layer and a cationic polymer electrolyte layer, a method of manufacturing the same, and a biosensing device or a chemical sensitive device using the same.
The multilayer membrane of the present invention has excellent wettability, excellent cell adsorption, and, when gamma-cyclodextrin or a derivative thereof is included in the multilayer membrane, the characteristics of the included gamma-cyclodextrin or its derivatives are determined through contact angle. It can be usefully applied to a biosensing device or a chemical sensitive device that can measure the presence and specific content of a specific material by capturing a specific material or inducing optical activity of the specific material. The effect will be very big.

Description

POLYELECTROLYTE MULTILAYER AND BIOSENSOR}

The present invention relates to a polymer electrolyte multilayer membrane and a biosensing device.

Recently, bioengineering has emerged. Bioengineering refers to the characteristics of cells through interaction between the support and the cells where cells are attached and grow, unlike conventional methods of controlling hormones, growth factors, and serum contained in cell culture in studying or inducing cell functions. It refers to the regulation of phosphorus attachment, proliferation, differentiation and extracellular matrix secretion. This requires the development of materials with biocompatibility and the development of chemical surface modification.

Therefore, a variety of multilayer membranes including a multilayer membrane having not only biocompatibility but also improving cell adhesion, and a multilayer membrane having both capturing ability and cell adhesion for a specific material, and technologies for biosensors including the same Development is required.

In order to meet the above demands, an object of the present invention is to provide a polymer electrolyte multilayer film having excellent biocompatibility and having a selective capturing ability to a material, and a biosensing device including the multilayer film.

In order to achieve the above object, the present invention provides a multilayer membrane including cyclodextrin or a derivative thereof in an alternating stacked structure of an anionic polymer electrolyte layer and a cationic polymer electrolyte layer.

Moreover, this invention provides the manufacturing method of the said multilayer film in order to achieve the said objective.

In addition, the present invention provides a biosensing device comprising the multi-layer film in order to achieve the above object.

In addition, the present invention provides a chemical sensitive device including the multilayer film in order to achieve the above object.

The inventors of the present invention prepare a multilayer film using a layer-by-layer (LBL) deposition technique using a polymer electrolyte that is safe in terms of immunity and toxicity, and can secure antimicrobial and antibacterial properties, and the cyclodextrin or its When the derivative is included or adsorbed, the present invention was completed by confirming that it is not only suitable for cell growth and hygroscopicity, but also capable of capturing a specific material or inducing optical activity of a specific material depending on application conditions.

Hereinafter, the present invention will be described in more detail.

In the present specification, cyclodextrin refers to a cyclic oligosaccharide in which glucopyranose units have α- (1,4) bonds. The cyclodextrin is obtained by degrading starch by an enzyme called cyclodextrin glucanotransferase (CGTase), followed by an intermolecular transglycosylation.

In addition, in the present specification, gamma-cyclodextrin (γ-cyclodextrin) means a cyclic oligosaccharide in which eight glucopyranose units are bonded.

As shown in FIG. 1, a hydroxyl group bonded to carbon 2 (C 2) and carbon 3 (C 3) of the cyclodextrin is expanded to the outside of the ring, and is bonded to carbon 6 (C 6). The bound hydroxyl groups also extend outwards in the opposite direction, making the outer ring hydrophilic. In addition, the oxygen of hydrogen and ether bonded to carbon 3 (C3) and carbon 5 (C5) are located in the inner direction of the ring, and the cavity of the ring is hydrophobic. . Cyclodextrins have hydrophilic properties with high solubility in polar solvents such as water, and the hydrophobic properties of the ring form molecular size pores in the molecule, and thus have different properties in one molecule. You have a micro heterogeneous environment.

In the present specification, a polymer electrolyte (Polyelectrolyte) refers to a polymer that can be dissolved in a solvent and dissociate, specifically, a polymer that can be dissolved in water and can be positively charged or negatively charged. The polymer electrolyte includes a cationic polymer electrolyte (polyanion). And anionic polymer electrolytes (polycation) may be included.

The cationic polymer electrolyte may mean a polymer electrolyte in which the chemical functional group of the polymer chain loses an anion or obtains hydrogen ions in an aqueous solution, and thus may be positively charged. The degree of ionization may be affected by the acidity or basicity of the surrounding environment. In addition to various weakly electrolytic cationic polymers, neutrally water-soluble polymers include neutral water-soluble polymers such as polyacrylamide, which are not ionized well in the pH range of general water and have a positive charge when the acidity of the surroundings is very high.

The anionic polymer electrolyte may mean a polymer electrolyte in which a polymer functional group of the polymer chain loses a cation or loses hydrogen ions, and thus may be negatively charged. The degree of ionization may vary depending on the acidity or basicity of the surrounding environment. Weak electrolyte anionic polymers are included.

In the present specification, 1 bilayer means a multilayer film in which one layer of each of two polymer electrolyte layers having different kinds is stacked. For example, the three bilayers are repeatedly immersed three times in a polymer electrolyte solution containing an anionic polymer electrolyte and a polymer electrolyte solution containing a cationic polymer electrolyte, thereby anionic polymer electrolyte layer and cationic polymer electrolyte layer. This means a multilayer film in which three double films stacked one by one are laminated.

In one aspect of the invention, the invention relates to a multilayer film.

The multilayer membrane may be a polymer electrolyte multilayer membrane, and specifically, the multilayer membrane may be a multilayer membrane including an alternating stack structure of a cationic polymer electrolyte layer and an anionic polymer electrolyte layer, and preferably, the cationic polymer electrolyte layer and anionic It may be a multi-layered film including an alternating layered structure of the polymer electrolytically etched layer and including the cyclodextrin or a derivative thereof, preferably gamma-cyclodextrin or a derivative thereof.

The anionic polymer electrolyte layer is preferably poly methacrylic acid (PMA), poly acrylacid (PAA), polystyrene sulfonate (PSS) and hyaluronic acid (HA) It may be made of any one selected from the group consisting of, more preferably may be made of polymethacrylic acid or polyacrylic acid, the cationic polymer electrolyte layer is preferably poly allylamine hydrochloride (poly (allylamine hydrochloride) , PAH), poly acrylamide (PAAm) and poly 4-vinylbenzlytrimethylammonium chloride (poly (4-vinylbenzlytri-methyl ammonium chloride, PVTAC) may be made of any one selected from the group consisting of, more preferably Polyallylaminehydrochloride or poly 4-vinylbenzyltrimethylammonium chloride It may be made of a rod.

In the group consisting of an anionic polyelectrolyte and poly allylamine hydrochloride, poly acrylamide and poly 4-vinylbenzyltrimethylammonium chloride, which is any one selected from the group consisting of polymethacrylic acid, polyacrylic acid, polystyrene sulfonate and hyaluronic acid Any one of the selected cationic polymer electrolytes is a weak polyelectrolyte whose charge density is sensitively changed according to pH, and the pH change of the polymer electrolyte directly affects the chain structure of the polymer electrolyte. The change in the chain structure affects various properties such as the structure or morphology of the polymer membrane or the wettability. Therefore, the anionic polymer electrolyte layer and the cationic polymer electrolyte layer corresponding to the polymer weak electrolyte can easily modify the structure of the electrolyte layer by adjusting the pH to form a multilayer film having a desired structure, or a desired material on the multilayer film. Has the advantage of being easy to adsorb.

The polymethacrylic acid may include not only polymethacrylic acid itself but also salts thereof. Specifically, at least one selected from the group consisting of the polymethacrylic acid and polymethacrylic acid salts. The molecular weight of the polymethacrylic acid is not particularly limited, but may be, for example, 10,000 to 200,000 or 50,000 to 150,000.

The polyacrylic acid may include not only polyacrylic acid itself but also a salt thereof. Specifically, it may be at least one selected from the group consisting of the polyacrylic acid and polyacrylic acid salt. The molecular weight of the polyacrylic acid is not particularly limited, but may be, for example, 10,000 to 100,000 or 30,000 to 90,000.

The hyaluronic acid is a kind of glucosamide glycan consisting of a disaccharide unit in which D-glucuronic acid and N-acetylglucosamine are connected by β (1 → 3) glycosidic bonds, and the hyaluronic acid has a chemical and physical structure. There are no species differences, humans have a metabolic system and are the safest biomaterials in terms of immunity and toxicity.

The hyaluronic acid may include not only hyaluronic acid itself but also all salts thereof. Specifically, the hyaluronic acid may be one or more selected from the group consisting of hyaluronic acid and hyaluronic acid salts. The hyaluronic acid salt includes both inorganic salts such as sodium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronic acid and the like and organic salts such as tetrabutylammonium hyaluronic acid. The molecular weight of the hyaluronic acid is not particularly limited, but may be, for example, 100,000 to 10,000,000.

The polyallylamine hydrochloride may be chemically bonded with hyaluronic acid or polyacrylic acid, and the molecular weight of the polyallylamine hydrochloride is not particularly limited, but may be, for example, 10,000 to 100,000 or 30,000 to 80,000.

The poly acrylamide may be chemically bonded with hyaluronic acid or polyacrylic acid, and the molecular weight of the poly acrylamide is not particularly limited, but may be, for example, 100,000 to 10,000,000.

When the cationic polyelectrolyte is polyacrylamide, the multi-layered film may have cross-linking formed by performing a crosslinking reaction between the polymer electrolytes in order to impart stability to the biological environment. The crosslinking reaction may be performed by, for example, a chemical reaction or heat treatment through EDAC.

The multilayer membrane may be a multilayer membrane having at least one cationic polymer electrolyte layer and one anionic polymer electrolyte layer. For example, the multilayer film may be 1 bilayer or more, and may be two or more layers, respectively.

In the present invention, the layer-by-layer (LBL) refers to a method of controlling the thickness of the polymer electrolyte multilayer film or the film by repeating as many times as desired so that the polymer electrolyte having a relative charge is adsorbed on the substrate surface. .

In addition, when the cationic polymer electrolyte layer or the anionic polymer electrolyte layer is two or more, the electrolyte layer may be laminated with the same polymer electrolyte layer or different polymer electrolyte layers, respectively. Specifically, when the anionic polymer electrolyte layer is two or more layers, specific anionic polymer electrolyte layers may be alternately stacked with the cationic polymer electrolyte layer, or different types of anionic polymer electrolyte layers may be alternately stacked with the cationic polymer electrolyte layer. Can be. The cationic polymer electrolyte layer may be the same as above.

The multilayer film of the present invention may have a gamma-cyclodextrin or a derivative thereof in the multilayer film including an alternating stack structure of the polymer electrolyte layer or include the alternating stack structure.

The gamma-cyclodextrin is a cyclic oligosaccharide in which eight glucopyranose units have α- (1,4) bonds. In the multilayer film including the gamma-cyclodextrin, a compound having a predetermined size that can be trapped in the gamma-cyclodextrin, in particular an optically inert molecule, is collected in the pore portion of the gamma-cyclodextrin, Specifically, when coordination is formed with gamma-cyclodextrin, the coordinated conjugated compound and gamma-cyclodextrin interact with each other to induce optical activity. The induced optical activity imparts a beneficial effect to the multilayer film, in particular chiral selectivity in the analysis and separation of a specific material, so that the multilayer film has a remarkable effect of performing analysis and separation of a specific material according to optical properties. .

In addition, since the diameter and volume of the pores formed in the gamma-cyclodextrin may form an inclusion complex through host-guest interaction with a large molecule such as a compound having a steroid or a macrocycle, the gamma- The multilayer membrane of the present invention containing cyclodextrin and its derivatives is characterized by having a remarkable effect in the analysis and separation of compounds having macromolecular structures such as steroids.

In addition, the gamma-cyclodextrin derivative may be a hydroxyl group (hydroxyl) bonded to each carbon constituting the glucopyranose unit is substituted with another functional group, for example, carboxylated gamma-cyclodextrin (carboxylated γ-cyclodextrin).

In the multilayer film of the present invention, preferably, at least one of the outermost layer may be an anionic polymer electrolyte layer. When the multilayer film is stacked on the colloid or the substrate, the polymer electrolyte layer contacting the colloid or the substrate is preferably a cationic polymer electrolyte layer. In this case, the outermost layer exposed to the outside is preferably an anionic polymer electrolyte layer. Can be. When gamma-cyclodextrin is included in the multilayer, when the outermost layer exposed to the outside is a cationic polymer electrolyte layer, the contact angle is increased to have a hydrophobic surface, but the outermost layer exposed to the outside is the anionic polymer electrolyte layer. When the contact angle is reduced to have a hydrophilic surface to facilitate cell contact, the outermost layer exposed to the outside may be an anionic polymer electrolyte layer. For example, when the multilayer film is laminated on a negatively charged substrate or on a negatively charged colloid, the outermost layer of the multilayer film may preferably be any one cationic polymer electrolyte layer and the other anionic polymer electrolyte layer. have.

Moreover, in one aspect of this invention, this invention relates to the manufacturing method of the said multilayer film.

In the method of manufacturing a multilayer membrane including gamma-cyclodextrin or a derivative thereof in an alternating stacked structure of an anionic polymer electrolyte layer and a cationic polymer electrolyte layer of the present invention, the anionic polymer electrolyte layer and the cationic polymer electrolyte layer are alternately stacked. Forming an alternating stacked structure; And it may be a method comprising the step of including the alternating laminated structure gamma-cyclodextrin or derivatives thereof.

Forming the alternate stacking structure may be performed by alternately stacking the anionic polymer electrolyte layer and the cationic polymer electrolyte layer.

The method of alternately stacking the anionic polymer electrolyte layer and the cationic polymer electrolyte layer may be performed by, for example, a layer-by-layer (LBL) deposition technique. The layer-by-layer (LBL) deposition technique may be performed by the method as shown in FIG. 2. In the layer-by-layer (LBL) deposition technique, the adsorption step is repeated as desired while alternately adsorbing a polymer electrolyte having a relative charge, thereby controlling the thickness of the polymer electrolyte layer, that is, the thickness of a multilayer film or a film. desirable.

As an example, the step of forming the alternating stacked structure may include a polymer electrolyte having a relative charge between a colloid having a charge (for example, a colloid having a negative charge) or a substrate (for example, a substrate having a negative charge) as in the method of FIG. 2. A process of forming a polymer electrolyte layer by immersing in a solution (positive charge polymer electrolyte solution), adsorbing a polymer electrolyte to a substrate, and a polymer electrolyte solution (negative charge) having a substrate on which the polymer electrolyte layer is formed has a relative charge with the polymer electrolyte of the polymer electrolyte solution. Or immersing the polymer electrolyte in a colloid or a substrate on which the polymer electrolyte layer is formed to form another polymer electrolyte layer on the polymer electrolyte layer, or the polymer electrolyte layer Process and another polymer electrolyte It can be carried out by repeating the process of forming the layer.

The process of forming the polymer electrolyte layer and the process of forming another polymer electrolyte layer may further include a process of washing the substrate on which the electrolyte layer is formed after each process.

The step of forming the alternate stacking structure may be performed by alternately stacking the anionic polymer electrolyte layer and the cationic polymer electrolyte layer on the colloid or the substrate.

The colloid may mean colloidal particles, and the colloidal particles may be particles having carboxylic acid functional groups on the surface thereof, for example, polystyrene particles or magnetic particles having carboxylic acid functional groups, for example Fe ₃ O ₄ .

In the process of forming the multilayer film on the colloidal particles, as shown in FIG. 3, the polymer electrolyte layer having the opposite inversion to the charged colloidal particles is laminated, and then the polymer electrolyte layer is laminated with the polymer electrolyte layer having the relative charge. The method may be performed by repeatedly stacking the multilayer film of the 1 bilayer.

The substrate is not particularly limited, but may be, for example, a glass slide made of glass, a metal substrate made of silicon or gold, or a substrate made of polystyrene. Specifically, the substrate may be a substrate that tends to be positively charged, such as mica, and may be a substrate that tends to be negatively charged, such as metal substrates such as glass, silicon, gold, and steel. It may be an organic substrate having no charge such as plastics (polystyrene, acrylic, Teflon, etc.).

In the case of the charged substrate, the polymer electrolyte layer may be formed using the polymer electrolyte of the opposite charge, and in the case of the organic substrate in the absence of the charge, the polymer electrolyte layer may be formed by using hydrophobic adsorption between the polymer materials. Can be formed.

The step of alternately stacking the anionic polymer electrolyte layer and the cationic polymer electrolyte layer may be performed by alternately immersing the polymer electrolyte solution containing the anionic polymer electrolyte and the polymer electrolyte solution containing the cationic polymer electrolyte. have.

The anionic polymer electrolyte solution used in the step of forming the electrolyte layer is pH 1.0 to pH 11.0, preferably pH 2.0 to pH 10.0, more preferably pH 3.0 to pH 5.0, even more preferably pH 3.5 to pH 4.5 PH may be adjusted to. In addition, the cationic polymer electrolyte solution used to form the electrolyte layer is preferably pH 1.0 to pH 11.0, preferably pH 2.0 to pH 10.0, more preferably pH 7.5 to pH 9.5, even more preferably The pH may be adjusted to pH 8.0 to pH 9.0. When laminating using the anionic polymer electrolyte solution of pH and the cationic polymer electrolyte solution of pH, the gamma-cyclodextrin or its derivatives can be effectively adsorbed or included in the multilayer membrane, The pH is preferably in the above pH range.

When the cationic polyelectrolyte is polyacrylamide, polyallylamine hydrochloride is immersed in a polymer electrolyte solution containing polyallylamine hydrochloride, which is a positively charged polyelectrolyte, in the case of a weak silicon substrate having a surface adhesion. After the layer is formed, an alternating layered structure can be formed using the anionic polymer electrolyte solution and polyacrylamide solution.

The step of forming the alternating stacked structure is, when the cationic polyelectrolyte is poly acrylamide, in order to give stability to the biological environment, to perform cross-linking reaction between the polymer electrolyte to form cross-linking (cross-linking) It may further comprise a step. The forming of the crosslinking may be performed by a method of forming a crosslinking reaction through chemical reaction or heat treatment through EDAC. The heat treatment may be performed by heating at 70 ° C. to 90 ° C. or 80 ° C. for 8 to 12 hours or 10 hours, and the reaction time or temperature may be adjusted by a tendency to reduce when using a vacuum oven.

The step of forming the alternating stacking structure may preferably be stacked such that the outermost layer located opposite the substrate or the colloid is an anionic polymer electrolyte layer, wherein the substrate or the colloid is a negatively charged substrate or a negatively charged colloid In this case, the lamination may be performed by laminating a multi-layered film of n bilayer. N means a natural number.

The step of including the alternating stack structure with gamma-cyclodextrin or a derivative thereof may be performed by dipping or impregnating the alternating stack structure or the substrate on which the alternating stack structure is formed into a solution containing gamma-cyclodextrin or a derivative thereof. Can be.

When the substrate having the alternating layered structure or the alternating layered structure formed is immersed or impregnated in a solution containing the gamma-cyclodextrin or a derivative thereof, the gamma-cyclo Dextrin or derivatives thereof may be adsorbed or included to finally produce a multilayer membrane comprising the cyclodextrin or derivatives thereof.

In addition, the multilayer membrane may be used as a tissue engineering material, for example, a support material for growing cells, and a device or apparatus requiring various polymer multilayer membranes including a chemical sensitive device such as a biosensing device or a chemical sensor. It can be applied to.

In one embodiment, the present invention provides a biosensing device including the multilayer film.

In the case of the multilayer membrane, the polymer electrolyte may be patterned for purposes such as selective adsorption of cells or for application of a biosensing device. In the multilayer film, at least one of the outermost layers may be an anionic polymer electrolyte layer. When the multilayer film included in the biosensing device is laminated on a colloid or a negative charge having a negative charge, the polymer electrolyte layer contacting the colloid or the substrate should be a cationic polymer electrolyte layer, and in this case, the other polymer exposed to the outside The outermost layer may preferably be an anionic polymer electrolyte layer.

In this aspect, the biosensing device of the present invention may include a colloid or negative substrate having a negative charge and the multilayer film.

A biomaterial fixing material may be combined with the multilayer pattern layer formed through the patterning. For example, the biomaterial may be any one selected from the group consisting of cells, monosaccharides, disaccharides, oligosaccharides, fatty acids, polypeptides, proteins, and polynucleotides, and preferably cells. The cell may be any cell including prokaryotic and eukaryotic cells, for example, fibroblasts, hepatocytes, neurons, cancer cells (eg HeLa cells), B cells, white blood cells (white blood cell, eg, Raw 264.7), and the like, and immune cells and embryonic cells.

The biomaterial-fixing material may be a biomaterial-fixing biomaterial. The biomaterial for fixing the biomaterial may be any oligopeptide including an oligopeptide sequence having an amino acid sequence of RGD and a protein including the oligopeptide and other receptor peptides, and the protein including the oligopeptide may be fibronectin ( fibronectin or fibrin, and the like, and the protein having the receptor peptide may be laminin, collagen, or the like.

In addition, the multilayer film may further include a thin film on the pattern layer, the thin film may include the biomaterial, preferably cells, the biomaterial is a biomaterial for fixing the biomaterial, preferably Preferably, the polypeptide may include an oligopeptide sequence having an amino acid sequence of RGD. The thin film may be attached to a polymer electrolyte pattern layer including a polymer of a polymer electrolyte or gamma-cyclodextrin or a derivative thereof. Preferably, the cell included in the thin film is a polymer electrolyte or gamma combined with a cell biomaterial. It may be attached to a pattern layer including a cyclodextrin or a derivative thereof.

When the multilayer film is used for a DNA chip, a protein chip, or a cell-based biosensor, the outermost layer not including the pattern layer may be a substrate adhesive layer bonded to a substrate. And when the multilayer film is used as a surface facing the damaged tissue for tissue regeneration inducing function, such as used for induced tissue regeneration, or as a dressing material for skin or mucosal tissue, the outermost layer not including the pattern layer Each layer may be a tissue adhesion layer that bonds to biological tissue.

In another aspect of the invention, the invention relates to a bioreactor.

The bioreactor may be a biosensing device including the multilayer film.

Specifically, the bioreactor is

It may be a biosensing device including a substrate and a multilayer film including gamma-cyclodextrin or a derivative thereof in an alternating stacked structure of the anionic polymer electrolyte layer and the cationic polymer electrolyte layer.

The biosensing device is combined with a positively charged polymer electrolyte containing an amide group in a cell-fixing biomaterial, and thus can be applied to a cell-based biosensing device because it has excellent chemical stability and can provide conditions suitable for cell culture. . The biosensing device may be, for example, a biosensor.

The substrate is a substrate that can be applied to the biosensing device, the material is not particularly limited, for example, made of glass (glass slide) or metal substrate such as silicon (silicon) or gold (gold) It may have been. Specifically, the substrate may be a substrate that tends to be positively charged, such as mica, and may be a substrate that tends to be negatively charged, such as metal substrates such as glass, silicon, gold, and steel. It may be an organic substrate having no charge such as plastics (polystyrene, acrylic, Teflon, etc.).

In the case of the charged substrate, the multilayer film may be coated using a polymer electrolyte of opposite charge, and in the case of the organic substrate in the absence of the charge, the multilayer film may be coated using hydrophobic adsorption between polymer materials. .

The multilayer membrane may be an anionic polymer electrolyte layer and a cationic polymer electrolyte layer may be prepared using a layer-by-layer (LBL) deposition technique, and may include cyclodextrin or a derivative thereof.

In the multilayer film, since the polymer electrolyte layer contacting the substrate is preferably a cationic polymer electrolyte layer, the outermost layer in the opposite direction of the substrate may be an anionic polymer electrolyte layer.

In addition, the multilayer film may be applied to a device or a device requiring various polymer multilayer films including a chemical sensitive device.

In another aspect of the invention, the invention relates to a chemical sensitive device. For example, the present invention may be a chemical sensitive device including the multilayer film.

Specifically, the chemical sensitizer may be a chemical sensitizer including a substrate and a multilayer membrane including gamma-cyclodextrin or a derivative thereof in an alternating stacked structure of the anionic polymer electrolyte layer and the cationic polymer electrolyte layer.

In the multilayer film and the biosensing device of the present invention, a gamma-cyclodextrin or a derivative thereof is included or adsorbed in a stack structure in which an existing cationic polymer layer and an anionic polymer layer are alternately stacked, and thus gamma-cyclodextrin included in the stack structure. Alternatively, the derivatives thereof may improve cell adsorption, adsorb specific substances inside the multilayered membrane, and improve optical activity of specific substances, and thus may be used in various applications including biosensors. In addition, it can be used for experiment or diagnosis, medical treatment or disease treatment in combination with biological tissue, the industrial effect will be very large.

1 is a schematic diagram showing the structure of a cyclodextrin analyzed by x-ray structure analysis according to an embodiment of the present invention.
2 is a process diagram illustrating a method of alternately stacking an anionic polymer electrolyte layer and a cationic polymer electrolyte layer by a layer-by-layer (LBL) deposition technique according to an embodiment of the present invention.
3 is a flowchart illustrating a method of alternately stacking a cationic polymer electrolyte layer and an anionic polymer electrolyte layer on colloidal particles according to an embodiment of the present invention.
4 is a flowchart illustrating a process of preparing a polymer multilayer thin film by alternately stacking a polymer electrolyte layer and an anionic polymer electrolyte layer and adsorbing gamma-cyclodextrin to the polymer multilayer thin film according to an embodiment of the present invention. to be.
FIG. 5 is a graph showing a contact angle of a polymer multilayer thin film including gamma-cyclodextrin according to an embodiment of the present invention, in which the vertical axis represents the contact angle, and the number of the horizontal axis represents the stacking degree (BL value) of the multilayer film, PAH stands for polyallylaminehydrochloride, PMA stands for polymetalacrylic acid, γCyD stands for gamma-cyclodextrin stacked, the first blue bar stands for static contact angle, and the second red bar Is the advancing contact angle, and the third green bar is the receding contact angle. FIG. 5A is a result of contact angle measurement when the opposite top surface of the substrate is a PMA layer, and FIG. 5B is a result of contact angle measurement when the outermost outer layer of the substrate is a PAH layer.
6 is a photograph confirming the degree of adsorption of kidney cells (HEK 293 kidney cells) to the polymer multilayer thin film containing gamma-cyclodextrin, according to an embodiment of the present invention, when Bare does not laminate a multilayer film on the substrate PMA Top surface refers to the case where the outermost layer opposite to the substrate is a PMA layer, PAH Top surface refers to the case where the outermost outer layer of the substrate is a PAH layer, PAH (8.5) / PMA (4.0) is laminated It refers to a multilayer having only a structure, γ-CyD Complexed PAH (8.5) / PMA (4.0) refers to a multi-layered film containing gamma-cyclodextrin.
FIG. 7 is a graph measuring UV absorbance of a multilayer film dyed with methylene blue, in order to confirm the trapping ability of a specific material of the polymer multilayer thin film including gamma-cyclodextrin according to an embodiment of the present invention. Absorbance, PAH means polyallylamine hydrochloride, PMA means polymetalacrylic acid, subscript next to the polymer indicates the degree of lamination (BL value) of the multilayer film, blue graph shows the multilayer film as methylene blue In the case of staining, the red graph means a case where the polymer multilayer membrane including gamma-cyclodextrin is dyed with methylon blue. FIG. 7A illustrates a case where an experiment is performed using a multilayer film having 10 BL laminated PAH and PMA, and FIG. 7B illustrates a case where an experiment is performed using a multilayer film obtained by laminating 10.5 BL PAH and PMA.

Hereinafter, preferred embodiments of the present invention are described. However, the following examples are only examples for illustrating the present invention, and thus the protection scope of the present invention is not limited to the following examples.

Example  1: Gamma Cyclodextrin  Containing Multilayer  Produce

Example  1-1. Materials and reagents

Poly (methacrylic acid) (PMA, Mw = 100,000) and Poly (allylamine hydrochloride) (PAH, Mw = 60,000) were purchased from Polysciences, Inc. (UK) and gamma-cyclodextrin (γ-cyclodextrin) was SUPELCO. The carboxylated γ-cyclodextrin purchased from Inc., USA was used. Methylene blue Hydrate (MeB) was used as a product of Fluka (Germany).

The polymer electrolytes (PMA, PAH and gamma-cyclodextrin) and MeB were used without further purification. Deionized water used in the preparation and washing process of the aqueous solution used in the experiment was performed using deionized water (> 18 MΩcm) produced by Millipore's Milli-Q water purifier (Millipore Co., USA).

In addition, Dulbecco's Phosphate Buffered Saline (DPBS), 0.25% Tripsin-EDTA, and Dulbecco's modified eagle medium (DMEM) used Gibco (USA) products, and also used fetal bovine serum (FBS) and Antibiotic-Antimycotic used in the preparation of DMEM. Gibco (USA) product was used.

Example  1-2. Preparation of Polymer Multilayer Thin Film

The polymer multilayer thin film was carried out by the LBL method using the solution prepared in Example 1-1 in a concentration of 0.01M in the deionized water. More specifically, the manufacturing process of the polymer multilayer film is as follows.

The PAH aqueous solution and the PMA aqueous solution were used to make the PAH and PMA in a concentration of 0.01M in the deionized water, the pH of the aqueous solution was adjusted using 0.1M HCl aqueous solution or 0.1M NaOH aqueous solution.

The substrate used in the LBL method was a glass or silicon substrate (slide glass or silicon wafer). The substrate was put together with a detergent diluted in a washing bottle, washed by ultrasonication (Ultrasonication) for 15 minutes, and then rinsed three times with the deionized water. The substrate on which the rinsing process was performed was dried using nitrogen gas.

The dried substrate was immersed in a pH-controlled cationic electrolyte (PAH) aqueous solution for 20 minutes and then taken out, followed by rinsing twice for 2 minutes using the deionized water. After the rinsing process, the solution was immersed in an anionic electrolyte (PMA) solution for 15 minutes and then taken out, and the rinsing process was performed using the deionized water in the same manner as described above. The thin film prepared by the above process was referred to as 1 bilayer, and the same procedure was repeated to increase the number of bilayers by a desired thickness.

Through the above process, a multilayer thin film of a desired thickness was prepared, and then dried in the same manner as above using nitrogen gas.

Example  1-3. Cyclodextrin  absorption

The process of preparing the polymer multilayer thin film of Example 1-2 and adsorbing gamma-cyclodextrin to the polymer multilayer thin film were performed by the method described in FIG. 4.

The process of adsorbing gamma-cyclodextrin to the polymer multilayer thin film is more specifically the gamma-cyclo- dissolve the gamma-cyclodextrin (carboxylated γ-cyclodextrin) in the deionized water of the polymer multilayer thin film prepared in Example 1-2 After dipping for 15 minutes in an aqueous solution of dextrin, it was taken out and rinsed with deionized water using the method described in Example 1-2.

Example  2: gamma Cyclodextrin  Containing Multilayer  Identify characteristics

Example  2-1: Contact angle  Measurement of wettability

The wettability (wetability, wettability) of the multilayer film including gamma-cyclodextrin prepared in Example 1-3 was performed by measuring the contact angle of water using a CA-A Contact angle analyzer of Face. The multilayer thin film was laminated using PAH aqueous solution of pH 8.5 for PAH, and the multilayer thin film was laminated using PMA aqueous solution of pH 4.0 for PMA.

In order to confirm the wettability of the multilayer film including the gamma-cyclodextrin, the multilayer thin film prepared in Example 1-2, the multilayer thin film comprising gamma-cyclodextrin prepared in Examples 1-3 and the above embodiment Experiments were performed on the multilayer thin films prepared in 1-2.

The advancing contact angle and the receding contact angle, which are the contact angles identified by the contact angle measurement, are useful for measuring the change tendency of the film with respect to moisture. Generally, the wettable film is not only low in static contact angle, Both the angle and the receding contact angle are reported to be low.

The deionized water was used as the water used for measuring the contact angle, and 15 μl of droplets were formed using a syringe, and then the film was placed on the measuring plate to slowly contact the water drop so that the contact angle was not affected by gravity. More specifically, first, the static contact angle was measured, and the advancing contact angle was measured by further adding 15 μl of water in the state of measuring the static contact angle, and measuring the contact angle again. The measurement was performed by sucking 15 μl of water from 30 μl of water droplets for measuring the advancing contact angle and measuring the contact angle again. After the measurement was repeated five times, the average value of the measured value was determined as a result value, and the result is shown in FIG. 5.

As shown in FIG. 5A, when 10 bilayers are stacked, the multilayer thin film is about 20 degrees for the static contact angle and slightly over 20 degrees for the advancing contact angle, whereas the polymer includes gamma-cyclodextrin. In the case of the multilayer thin film, the static contact angle was measured to be lower than 20 degrees, and the advancing contact angle was measured to about 20 degrees.

On the other hand, as shown in FIG. 5B, when the 10.5 bilayer is laminated, that is, when the top surface opposite to the substrate on which the contact angle is measured in the multilayer thin film is a PAH layer, the multilayer thin film is in static contact. The angle is about 40 degrees, and the advancing contact angle is slightly over 45 degrees, whereas the polymer multilayer thin film containing gamma-cyclodextrin is measured at about 60 degrees for the static contact angle and about 70 degrees for the advancing contact angle. Measured back, it was found that the surface was rather hydrophobic and the wettability was lowered.

From the above results, it was confirmed that the surface properties of the outermost layer, that is, hydrophilicity or hydrophobicity, can be enhanced by varying whether or not gamma-cyclodextrin is adsorbed depending on the type of the outermost layer of the multilayer thin film.

In addition, when the multilayer thin film is used in a biosensing device or the like, it is expected that the outermost layer may be adjusted to be an anionic polymer electrolyte layer in consideration of the ease and efficiency of cell adsorption.

Example  2-2. Check the degree of cell adsorption

The degree of cell adsorption to the multilayer membrane including gamma-cyclodextrin prepared in Example 1-3 was performed by the method of observing cell adsorption using an electron microscope. The multilayer thin film was laminated using PAH aqueous solution of pH 8.5 in case of PAH (PAH (8.5)), and the multilayer thin film was laminated using PMA aqueous solution of pH 4.0 in case of PMA (PMA (4.0)).

In order to confirm cell adsorption of the multi-layered membrane including gamma-cyclodextrin, when laminating 10 bilayers in which PMA, an anionic polymer electrolyte was stacked as a top layer, and 10.5 bilayers in which PAH, a cationic polymer electrolyte, was stacked as a top layer, were laminated. The degree of adsorption of cells was compared with the substrate surface (Bare TCPS).

The method of confirming the degree of cell adsorption was performed in the following manner.

First, in order to determine the degree of adsorption of the cells to the multilayer membrane, the rapid proliferation and easy cultivation was used kidney cells (HEK 293 kidney cells) widely used in medicine and biomaterials, and confirmed the degree of cell adsorption The substrate in which the multilayered film was laminated was used as a cell culture polystyrene dish (TCPS) having a diameter of 60 mm, and the adsorption degree of the cells was observed using an electron microscope.

More specifically, two-thirds of the TCPS having a diameter of 60 mm were laminated using the method of Example 1-2. The multilayer thin film was laminated in 10 bilayer and 10.5 bilayer, respectively, in order to change the polymer electrolyte type of the outermost layer. Culture medium was used for the culture medium (media) adjusted to 37 ℃ in a thermostat, cells were cultured in a CO ₂ incubator (CO ₂ incubator).

After removing the culture medium from the TCPS in which the cells were cultured, the process of washing the cells attached to the TCPS was repeated twice using Dulbecco's Phosphate Buffered Saline (DPBS). After the washing process, 700 μl of Tripsinf-EDTA was put in the TCPS, and wetted by tilting horizontally 2-3 times from the outermost side to remove Trypsin-EDTA using a micro-pipet. After removing the Trypsin-EDTA, the cells were incubated for 5 minutes in the CO ₂ incubator to stabilize the cells, and then 4 ml of fresh culture solution was injected into the TCPS to disperse the cells. 1/10 of the 4 ml of the culture medium in which the cells were dispersed was put together with 4 ml of the culture solution in a TCPS layered with 70% ethanol, and cultured in the CO ₂ incubator for 5 days, and then the degree of adsorption of the cells It observed, and the result is shown in FIG.

As shown in FIG. 6, the cells were not adsorbed well on the surface of the TCPS, but the cells were well stacked at the site where the multilayered films were laminated. In addition, when the top surface of the multilayer film is PAH, the cells are better in the multilayer film than when the multilayer film including gamma-cyclodextrin is laminated (γ-CyD Complexed PAH (8.5) / PMA (4.0)). On the other hand, when the top surface of the multilayer film is PMA, when the multilayer film including gamma-cyclodextrin is laminated (γ-CyD Complexed PAH (8.5) / PMA (4.0)), It was found to adhere better.

Thus, it was evaluated that the outermost layer is an anionic polymer electrolyte layer when the present invention uses a multilayer membrane containing gamma-cyclodextrin in an apparatus for carrying out the use related to cell adhesion.

Example  2-3. Host - Guest Interaction  Confirm

Gamma-cyclodextrins or derivatives thereof can form inclusion complexes through Host-Guest interactions that trap foreign substances in the pores of the gamma-cyclodextrins.

In order to confirm the trapping ability of the foreign substance of the gamma-cyclodextrin, after reacting with MeB, UV absorbance was measured.

More specifically, after laminating each of the multilayer films by the method of Example 1-2 and Example 1-3 on the glass substrate (Slide glass), MeB is dissolved in deionized water at a concentration of 0.001 M The multilayer film was impregnated with an aqueous solution and stained with MeB. The MeB stained multilayer films were washed with deionized water, and then UV of each multilayer film was measured.

The UV measurement was performed by using a UV spectrophotometer UV-visible spectrophotometer (scinco S-3100). More specifically, the glass substrate (Slide glass) was put together with a detergent diluted in a washing bottle and sonicated for 15 minutes and washed, and the base line was measured using the UV spectrophotometer. After mounting the MeB-dyed multilayer film in the holder, UV was measured using the UV spectrophotometer, and the degree of absorption was measured based on the base line. The results are shown in FIG. 7.

As shown in FIG. 7, when the PAH and the PMA are laminated, the multilayer thin film is formed both in the case where the PMA is the top surface (FIG. 7A) and in the case where the PAH is the top surface (FIG. 7B). Compared to the case of blue (bottom graph), the multilayer thin film containing gamma-cyclodextrin (red top graph) shows absorbance almost similar to that of MeB, and the alpha-cyclodextrin and MeB contained in the multilayer film react with UV Absorbance was evaluated to be increased. The increase in UV absorbance was evaluated because gamma-cyclodextrin and MeB included in the multilayer structure, that is included in the laminated structure, form an inclusion complex through the host-guest interaction.

In addition, since the MeB can be bound to an acidic substance, the difference in absorbance according to the inclusion of the gamma-cyclodextrin should be determined not as an absolute value but as the difference in absorbance between the multilayer and the multilayer including gamma-cyclodextrin. . In this aspect, when the outermost layer is the PMA layer which is the anionic polymer electrolyte layer (FIG. 7A), the difference in absorbance is remarkably shown depending on the presence or absence of gamma-cyclodextrin, and thus, the outermost layer is evaluated to be preferably anionic polymer electrolyte layer. It became.

According to the above results, considering the wettability and cell adhesion through contact angle, as a multilayer film for use in a biosensing device, a multilayer film was formed by stacking a polymer electrolyte layer as a bilayer under the conditions of PAH (8.5) / PMA (4.0). That is, it was confirmed that the case where the outermost layer is an anionic polymer electrolyte layer is preferable. In addition, the bilayer polymer electrolyte layer under the conditions of PAH (8.5) / PMA (4.0) is considered even when considering the trapping ability of the guest material with or without gamma-cyclodextrin, that is, inclusion complex formation through host-guest interaction. It was confirmed that the case where the outermost layer was an anionic polymer electrolyte layer was formed by laminating with a film.

Claims (5)

The substrate is alternately immersed in an anionic polymer electrolyte solution having a pH of 3.5 to pH 4.5 containing an anionic polymer electrolyte and a cationic polymer electrolyte solution having a pH of 8.0 to pH 9.0 containing a layer and a cationic polymer electrolyte. Forming an alternating stacked structure of the polymer electrolyte layer on the substrate; And
Impregnating the substrate having the alternating stacked structure in a solution comprising gamma-cyclodextrin or a derivative thereof to include gamma-cyclodextrin or a derivative thereof in the alternating stacked structure.
Including,
The anionic polymer is polymethacrylic acid, the cationic polymer is polyallylamine hydrochloride, and the outermost layer of the alternating laminated structure is a polymethacrylic acid layer.
A method for producing a polymer electrolyte multilayer film comprising gamma-cyclodextrin or a derivative thereof. Gamma-cyclodextrin or a derivative thereof, wherein the outermost layer comprises a gamma-cyclodextrin or a derivative thereof in an alternating lamination structure of the polymethacrylic acid layer and the polyallylamine hydrochloride layer prepared by the method of claim 1 Polymer electrolyte multilayer membrane comprising a. delete A biosensing device comprising the multilayer film of claim 2. A chemical sensitive device comprising the multilayer film of claim 2.

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