KR20090106788A - Polyelectrolyte multilayer, biosensor and method of the same - Google Patents

Abstract

PURPOSE: A biosensor is provided to easily control distance between patterns and induce cell-cell talk between the cells bound to the surface. CONSTITUTION: A biosensor comprises a substrate, multilayered film, pattern layer, and cell fixation layer. The multilayered film is formed to have anionic polymer electrolyte layer on the substrate. The pattern layer is placed on the upper layer of the multilayered film and contains cationic polymer electrolyte having amine group. The cell fixation layer contains a material for biomass fixation.

Description

Polymer Electrolyte Multilayer Membrane, Biosensing Device and Manufacturing Method Thereof {POLYELECTROLYTE MULTILAYER, BIOSENSOR AND METHOD OF THE SAME}

The present invention relates to a polymer electrolyte multilayer membrane, a biosensing device and a method of manufacturing the same.

The study of portable biosensors is a field of biological microdevices, which enables the patient to self-diagnose complex and long-term measurement processes in a short time in a convenient way in a medical institution that a patient usually needs to go through. This study is proposed to aim at improving the early and accurate medical diagnosis.

There are various types of biosensors depending on the application field, but until recently, mainly reported devices such as a glucose meter for measuring glucose (sugar) to capture by-products or biologically active substances for detecting by-products of a kind of catalytic reaction such as enzymatic action Cell-based biosensors that can be divided into capture-based sensing devices and that use cells or tissues as sensory materials have recently been proposed. .

Cell-based biosensors are cell-based biosensors that have superior sensitivity to previously developed biosensors, and have a receptor or ion-channel in the cell. Materials to be used for detection, such as enzymes are kept in a biological state, and there is an advantage that can be used as a substitute for animal testing in connection with toxicity tests. In relation to the biosensor described above, a single neuron capable of detecting a single stimulatory molecule, an immune cell capable of acting by a single antigenic peptide, and a cell that is sensitive to various types of environmental toxic samples may be sensitive. Biosensors using hepatocytes have been proposed. However, at present, research on the cell-based biosensors is still limited.

The cell based biosensor generally consists of two transducers. More specifically, the cell is a primary converter that detects the presence of a bioactive material and converts it into a cellular signal, and a secondary converter that detects and processes the cellular signal and sends it out as an electrical or optical signal that can be analyzed. It is composed. The primary transducer has been mainly used microbial cells such as bacteria or yeast, and recently, mammalian cells that can give medically more meaningful information for medical screening, toxic material detection or diagnostics has been of interest. In addition, the secondary converter may use a microelectrode, a piezoelectric detection device, and a fluorescence or luminescence material like other types of biosensors.

In the case of the cell-based biosensors, various secondary transducers may be used as detectors, but since the primary transducers supplying the primary signal should be cells in any case, the cell itself may be appropriately arranged (cell array) to construct the cell-based biosensors. Technology is essential.

Cell array technology is largely divided into flat array method and suspension array method. The flat array method is introduced in a semiconductor device technology, such as a DNA chip, and is used in a cell chip or the like that arranges the cells themselves on the surface as particles. In addition, the suspension array method is a technique used for flow cytometry for measuring the number of cells in a certain volume, ie density, so as to attach to the microparticles and detect a signal in a suspended state in a liquid channel. In the manufacture of cell-based biosensors, technology based on electrical signals is currently being researched more intensively, and thus, the flat array method is mainly used.

The key to any cell alignment technique is to control the cell's interaction with the surface. This is because most of the cell's biological responses are absolutely surface affected. Specifically, cell attachment, proliferation, migration, differentiation, and even apoptosis have been reported to be affected by surface properties. This is because technology that regulates properties can be linked to technology that regulates the function of cells.

In this regard, there are some limitations that existing research must overcome.

First, because cells are living organisms, they must breathe for life, require nutrition, and be protected from contamination and infection, so they must build a surface that provides a biological environment in which cells can grow. In this regard, the existing studies only consider the adsorption of cells, but rather induce excessive adsorption of cells to form giant cells rather than normal cell proliferation, or there is a limit to overwhelm immune response. Formation or hyperimmune reaction of such giant cells may be fatal enough to threaten the life of the patient in the case of a living implant as well as paralysis of the device.

Secondly, with regard to portable biosensors, recent research trends regarding the miniaturization of elements of secondary converters to ensure portability are closely related to the construction of microfluidic systems. Recently, the size of the cultivation chamber is based on a microfluidic device using a microchannel method, or specifically, a chamber part containing cells is made of PDMS (polydimethylsiloxane) and then attached to an electronic substrate. A study on portable cell-based biosensors that can be reduced has been reported. However, there is a problem that only the processing technology for designing and fabricating the structure of the microchannel may not properly implement the functions of the biosensors involved in biological activity. The chemical and structural properties of the internal and internal components of the microchannel, such as wettability, surface charge, chemical affinity, and response to temperature or pressure, influence fluid flow, separation of analytes, and analytical functions. Because.

In order to solve this problem, the adsorption of cells or proteins that help the adsorption of cells is not unconditionally good, but rather, the development of a base material to control such adsorption is required, and as a base material, a selective adsorption reaction can be generated thereon. There is a need for the development of techniques to enable the relocation of materials present. In addition, it is required to develop a material suitable for freely implementing chemical, mechanical, and geometrical characteristics by controlling the chemical structure of a monomer or copolymerizing different monomers.

In order to meet the above requirements, an object of the present invention is to provide a polymer electrolyte multilayer membrane having excellent biocompatibility and capable of selective adsorption reaction, a biosensing device including the multilayer membrane, and a method of manufacturing the same.

In order to achieve the above object, the present invention includes an alternating layered structure of an anionic polymer electrolyte layer and a cationic polymer electrolyte layer, wherein any one or both layers of the outermost layer of the alternating layered structure is an anionic polymer electrolyte layer A multilayer film is provided. The anionic polymer electrolyte layer may be preferably any one selected from the group consisting of hyaluronic acid and polyacrylic acid, the cationic polymer electrolyte layer is preferably a group consisting of poly allylamine hydrochloride and poly acrylamide It may be made of any one selected from.

In addition, the present invention, in order to achieve the above object; The multilayer film formed on the substrate such that the anionic polymer electrolyte layer of the outermost layer becomes the uppermost layer; A pattern layer disposed on an uppermost layer of the multilayer film and including a cationic polymer electrolyte having an amine group; And it provides a biosensing device comprising a cell fixing layer comprising a cell-fixing biomaterial coupled to the cationic polymer electrolyte of the pattern layer.

In addition, the present invention, in order to achieve the above object, the anionic polymer electrolyte layer so that the anionic polymer electrolyte layer is the uppermost layer, by alternately laminating an anionic polymer electrolyte layer and a cationic polymer electrolyte layer on the substrate to form the multilayer film; Forming a patterned layer by patterning the inkjet printing method using an ink including a cationic polymer electrolyte having an amine group on an uppermost layer of the multilayer film; And it provides a biosensing device manufacturing method comprising the step of combining the cationic polymer electrolyte having an amine group of the patterned layer and the cell-fixing biomaterial to form a cell fixing layer.

In addition, the present invention, in order to achieve the above object, an anionic polymer electrolyte layer made of any one selected from the group consisting of hyaluronic acid and polyacrylic acid and a cation consisting of any one selected from the group consisting of polyallylamine hydrochloride and poly acrylamide Forming a multilayer film comprising an alternating stack structure of the polymer electrolyte layer, wherein one or both layers of the outermost layer of the alternating stack structure are anionic polymer electrolyte layers; And patterning by inkjet printing using an ink including a cationic polymer electrolyte having an amine group on any one of the outermost layers of the anionic polymer electrolyte layer of the multilayer film.

The inventors of the present invention are layer-by-layer (LBL) deposition using hyaluronic acid, polyacrylic acid, polyallylamine hydrochloride, or polyacrylamide, which is safest in terms of immunity and toxicity, and which can secure antimicrobial and antibacterial properties. When a multi-layered film was prepared by the technique, and patterned by inkjet printing with an ink containing a cationic polymer electrolyte having an amine group on the substrate coated with the multi-layered film, it is suitable for cell growth and has excellent hygroscopicity. The present invention was completed by confirming that non-selective adsorption of the nanoparticles on the surface can be easily controlled in a controlled distance and form according to application conditions.

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

In the present invention, the polymer electrolyte refers to a polymer capable of dissociating in a solvent, and the polymer electrolyte may include a cationic polymer electrolyte and an anionic polymer electrolyte.

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 the various weakly electrolytic cationic polymers, neutral water-soluble polymers include neutral water-soluble polymers such as polyacrylamide, which are not ionized well in the general pH range of 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 invention, 1 bilayer means a multilayer film in which one layer of each of two polymer electrolyte layers having different kinds is stacked.

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

The multilayer film may be a polymer electrolyte multilayer film, and more specifically, an alternating stack structure of an anionic polymer electrolyte layer and a cationic polymer electrolyte layer, wherein one or both layers of the outermost layer of the alternating stack structure are anionic It may be a multilayer film which is a polymer electrolyte layer.

The anionic polymer electrolyte layer may be preferably made of any one selected from the group consisting of hyaluronic acid (HA) and polyacrylic acid (PAC), and the cationic polymer electrolyte layer is preferably It may be made of any one selected from the group consisting of poly allylamine hydrochloride (poly (allylamine hydrochloride), PAH) and poly acrylamide (PAAm).

The multilayer membrane may be a multilayer membrane in which a cationic polymer electrolyte layer and an anionic polymer electrolyte layer are each one or more layers. For example, the multilayer film may be 1 bilayer or more, and may be two or more layers, respectively.

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 outermost layer refers to the outermost layer of the multilayer film, that is, the outermost layer, that is, the polymer electrolyte layer located at the outermost layer.

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 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 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 film may further include a pattern layer including a cationic polymer electrolyte having an amine group in a layer that is an anionic polymer electrolyte layer in the outermost layer. When both of the outermost layers are anionic polymer electrolyte layers, the pattern layer may further include a cationic polymer electrolyte having an amine group on only one of the outermost layers.

The pattern layer may be patterned by an inkjet printing method using an ink containing a cationic polymer electrolyte having the amine group. The cationic polymer electrolyte having an amine group may be polyallylamine hydrochloride.

The multilayer film may further include a material for fixing a biomaterial coupled to the cationic polymer electrolyte of the pattern layer. 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, leukocyte cells ( white blood cells such as Raw264.7), and the like, including 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), fibrin, or 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 pattern layer including a cationic polymer electrolyte, and preferably, cells included in the thin film may be attached to a pattern layer including a cationic polymer electrolyte to which a biomaterial for cell tablets is bound. have.

When the multilayer film is used for a DNA chip, protein chip, or cell-based biosensor, the outermost layer that does not include 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 is a multilayer membrane in which an anionic polymer electrolyte layer and a cationic polymer electrolyte layer are alternately stacked, and the anionic polymer electrolyte layer is made of any one selected from the group consisting of hyaluronic acid and polyacrylic acid. The polymer electrolyte layer is one selected from the group consisting of polyallylamine hydrochloride and poly acrylamide, and any one or both layers of the outermost layer of the multilayer film may be a biosensing device including a multilayer film which is an anionic polymer electrolyte layer. Can be.

Specifically, the bioreactor is

Board; The multilayer film formed on the substrate such that the anionic polymer electrolyte layer of the outermost layer becomes the uppermost layer; A pattern layer disposed on an uppermost layer of the multilayer film and including a cationic polymer electrolyte having an amine group; And a fixed layer including a material for fixing a biomaterial coupled to the cationic polymer electrolyte of the pattern 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 film includes an alternating stacked structure of an anionic polymer electrolyte layer and a cationic polymer electrolyte layer, and may be formed on the substrate such that the anionic polymer electrolyte layer of the outermost layer of the multilayer film becomes the uppermost layer.

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 film may be an anionic polymer electrolyte layer and a cationic polymer electrolyte layer manufactured using a layer-by-layer (LBL) deposition technique.

The pattern layer may be disposed on the uppermost layer of the multilayer and include a cationic polymer electrolyte having an amine group. Since the uppermost layer of the multilayer film is an anionic polymer electrolyte layer, the pattern layer including the cationic polymer electrolyte having the amine group may be stably combined with the uppermost layer of the multilayer film.

The cationic polymer electrolyte having an amine group may be polyallylamine hydrochloride. The pattern layer may be patterned by an inkjet printing method using an ink containing a cationic polymer electrolyte having the amine group.

The biological material means an organic material and a material existing in living organisms or organisms. For example, the biological material may be any one selected from the group consisting of cells, monosaccharides, disaccharides, oligosaccharides, fatty acids, polypeptides, proteins, and polynucleotides. May be a cell. The cell may be any cell including prokaryotic and eukaryotic cells, for example fibroblasts, hepatocytes, neurons, cancer cells (eg HeLa cells), B cells, leukocyte cells ( white blood cells such as Raw264.7), and the like, including immune cells and embryonic cells.

The material for fixing the biomaterial may be reacted with the amine group of the cationic polymer electrolyte including the amine group, and the material for fixing the biomaterial is not particularly limited, and the type is not particularly limited. 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), fibrin, or the like, and the protein having the receptor peptide may be laminin, collagen, or the like. The biomaterial for fixing the biomaterial may include an oligopeptide including an oligopeptide sequence having an amino acid sequence of RGD and one or more RGD repeats in the amino acid sequence of the oligopeptide.

When the biomaterial is a cell and the biomaterial fixing material is a biomaterial fixing biomaterial, preferably, the biomaterial fixing material may be a cell fixing biomaterial.

Preferably, the cell-fixing biomaterial bound to the cationic polymer electrolyte of the pattern layer may be a polypeptide including an oligopeptide having an amino acid sequence of RGD or a polypeptide comprising an oligopeptide sequence having an amino acid sequence of RGD, and more. Preferably, the polypeptide may be a GRGDSPC amino acid sequence.

The biosensing device may further include cells attached to a pattern layer including a cationic polymer electrolyte to which a cell-fixing biomaterial is bound. The attached cells may be attached in a thin film form.

In addition, another bioreactor of the present invention

Board; The multilayer film formed on the substrate such that the anionic polymer electrolyte layer of the outermost layer becomes the uppermost layer; A pattern layer disposed on an uppermost layer of the multilayer film and including a cationic polymer electrolyte having an amine group; And a biomaterial bonding layer including a biomaterial coupled to the pattern layer.

When one aspect of the biomaterial is a protein chip, the biomaterial may be any one selected from the group consisting of a polypeptide, an antibody, or a protein, and one aspect of the biomaterial is a DNA chip ( DNA chip) may be any one selected from the group consisting of polynucleotides and antibodies.

The biomaterial binding layer may include a biomaterial fixed to the pattern material or a biomaterial fixing material used to induce or stabilize the coupling of the pattern layer and the biomaterial and the biomaterial.

In another aspect of the present invention, the present invention relates to a method for producing a biosensing device comprising the step of patterning by using an inkjet printing method using poly allylamine hydrochloride as an ink.

Specifically, the biosensing device manufacturing method

Alternately stacking an anionic polymer electrolyte layer and a cationic polymer electrolyte layer on a substrate such that the anionic polymer electrolyte layer is a top layer to form the multilayer film; Forming a patterned layer by patterning the inkjet printing method using an ink including a cationic polymer electrolyte having an amine group on an uppermost layer of the multilayer film; And a method of manufacturing a biosensing device comprising combining a cationic polymer electrolyte having an amine group of the pattern layer with a cell fixing biomaterial to form a cell fixing layer.

Forming the multilayer film on the substrate may be performed by a layer-by-layer (LBL) deposition technique in which an anionic polymer electrolyte layer and a cationic polymer electrolyte layer are alternately stacked on the substrate.

More specifically, the substrate, for example, a glass slide made of glass, a substrate made of silicon, a substrate made of polystyrene includes a polymer electrolyte solution containing an anionic polymer electrolyte and a cationic polymer electrolyte. It may be performed by a method of alternately immersing in a polymer electrolyte solution, and when the solution is prepared by alternately immersing each solution may be defined as a bilayer 1 bilayer. For example, when the multilayer film is a 3 bilayer, the step of alternately immersing the substrate (silicon) in a polymer electrolyte solution containing an anionic polymer electrolyte and a polymer electrolyte solution containing a cationic polymer electrolyte three times Can be.

The polymer electrolyte solution including the anionic polymer electrolyte and the polymer electrolyte solution including the cationic polymer electrolyte used in the step of forming the multilayer film are preferably at pH 2.0 to pH 5.0, more preferably at pH 2.5 to pH 4.5. The pH may be adjusted. In addition, when the cationic polymer electrolyte is poly acrylamide, the pH may be pH 2.75 to pH 3.5.

In addition, when the cationic polymer electrolyte is polyacrylamide, in the case of the substrate, in particular, in the case of a weak silicon substrate having a surface adhesion, the polymer electrolyte solution containing polyallylamine hydrochloride, which is a positively charged polymer electrolyte, is coated once. , By using a polymer electrolyte solution containing an anionic polymer electrolyte and poly acrylamide, respectively, to form a multilayer membrane, and to keep the pH of both the polyelectrolyte solution and the rinse solution low to prevent deprotonation of the multilayer membrane using hydrogen bonding. It is important to adjust the pH, preferably pH 2.75 to pH 3.5.

In the forming of the multilayer film, when the cationic polymer electrolyte is polyacrylamide, in order to give stability to the biological environment, crosslinking reaction is performed between the polymer electrolytes to form cross-linking. It may further comprise. The forming of the crosslinking may be performed by a chemical reaction through EDAC or a crosslinking reaction through heat treatment. The heat treatment may be performed by heating at 80 ° C. for 8 to 12 hours, and the reaction time or temperature may be controlled by reducing the method using a vacuum oven.

The forming of the pattern layer may be performed by a method of inkjet printing using an ink including a cationic polymer electrolyte having an amine on the substrate on which the multilayer film is formed. The cationic polymer electrolyte having an amine group may be polyallylamine hydrochloride.

The inkjet printer used in the inkjet printing may be a printer including a piezo injector-type nozzle using a property of deforming or vibrating when electricity is applied to a crystal, and the size of the nozzle of the preturn may be 20 to 50, preferably 25 to 40.

The ink including the cationic polymer electrolyte having an amine group may further include one or more selected from the group consisting of ethylene glycol and glycerol. The ink may be adjusted by adding one or more selected from the group consisting of ethylene glycol and glycerol. In addition, the viscosity of the ink used in the inkjet printing method may be 1cP to 20cP, preferably 5cP to 15cP, more preferably 7cP to 12cP.

Since the patterned polymer electrolyte ink is fixed to the surface, that is, the polymer electrolyte multilayer film through strong interaction with the outermost layer, which is an anionic polymer electrolyte layer, electrostatic attraction is applied to the film-coated substrate after the inkjet printing method. Excess ink other than the ink adsorbed through may further comprise the step of removing with PBS.

The step of forming a cell-fixing layer by combining the cationic polymer electrolyte having an amine group of the pattern layer and the cell-fixing biomaterial is performed by combining the amine group and the cell-fixing material of the cationic polymer electrolyte having an amine group used as a patterning ink. can do.

In addition, the forming of the cell-fixing layer is immersed in the solution containing the cell-fixing biomaterial and the aqueous solution containing a binding material used for the combination of the cell-fixing material and the cationic polymer electrolyte having an amine group sequentially Can be done.

The material for fixing the biomaterial may react with the amine group of the cationic polymer electrolyte including the amine group, and the material for fixing the biomaterial is not particularly limited as long as it has a reactor capable of fixing the biomaterial. The biomaterial fixing material may be a biomaterial fixing biomaterial. The biomaterial for fixing the biomaterial may be any oligopeptide including the oligopeptide sequence having an amino acid sequence of RGD and a protein including the oligopeptide and other receptor peptides, and the protein comprising the oligopeptide Fibronectin, fibrin, or the like, and the protein having the receptor peptide may be laminin, collagen, or the like.

When the biomaterial is a cell and the biomaterial fixing material is a biomaterial fixing biomaterial, preferably, the biomaterial fixing material may be a cell fixing biomaterial.

Preferably, the cell-fixing biomaterial bound to the cationic polymer electrolyte of the pattern layer may be a polypeptide including an oligopeptide having an amino acid sequence of RGD or a polypeptide comprising an oligopeptide sequence having an amino acid sequence of RGD, and more. Preferably, the polypeptide may be a GRGDSPC amino acid sequence.

The binding material used for binding the cell-fixing material and the cationic polymer electrolyte having an amine group is DCC (dicyclohexylcarboimide), EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbo-diimide Hydrochloride), N-hydroxysuccinimide ester It may include a compound and similar compounds thereof, the compound containing the N-hydroxysuccinimide ester may be sulfosuccinimidyl 6- [3 ′-(2-pyridyldithio) propionamido] hexanoate (Sulfo-LC-SPDP).

The concentration of the solution containing the cell-fixing biomaterial may be a concentration of 0.1 to 10, preferably 0.25 to 0.75.

For example, the combination of the amine group of polyallylamine hydrochloride and the cell-fixing biomaterial used as the patterning ink may be used to form the patterned substrate by sulfosuccinimidyl 6- [3`- (2-pyridyldithio) propionamido] hexanoate (Sulfo-LC-). After immersion in SPDP), it may be carried out by a method of immersion in the oligopeptide solution.

In addition, the biosensing device manufacturing method of the present invention further comprises the step of attaching the cell to the pattern layer comprising a cationic polymer electrolyte to which the cell-fixing biomaterial is bonded by reacting the cell to the cell-fixing biomaterial of the cell fixing layer. It can be included as.

Reacting the cell to the cell-fixing biomaterial of the cell-fixing layer may be performed by immersing the substrate with the cell-fixing layer attached to the cell culture medium of the target cell. It may be a cell which is a transducer capable of sending.

The cell may be any cell including prokaryotic and eukaryotic cells, for example fibroblasts, hepatocytes, neurons, cancer cells (eg HeLa cells), B cells, leukocyte cells ( white blood cells such as Raw264.7), and the like, including immune cells and embryonic cells.

In another aspect of the invention, the invention is an anionic polymer electrolyte layer consisting of any one selected from the group consisting of hyaluronic acid and polyacrylic acid and a cation consisting of any one selected from the group consisting of polyallylamine hydrochloride and poly acrylamide Forming a multilayer film comprising an alternating stack structure of the polymer electrolyte layer, wherein one or both layers of the outermost layer of the alternating stack structure are anionic polymer electrolyte layers; And patterning by inkjet printing using an ink including a cationic polymer electrolyte having an amine group on any one of the outermost layers of the anionic polymer electrolyte layer of the multilayer film.

The cationic polyelectrolyte having the amine group may be polyallylamine hydrochloride, and the ink including the cationic polyelectrolyte having the amine group may have a viscosity of 1 cP to 20 cP.

The patterning method may be applied to a biosensing device or a biological tissue, preferably a human tissue adhesive, capable of binding to a living tissue for use in experiments, diagnosis or disease treatment.

The biosensing device of the present invention and its manufacturing method can be used to control non-selective adsorption of cells, antimicrobial and antibacterial surface treatment, biologically inert coating, and the distance between patterns. It can be easily adjusted to induce cell-cell talk between cells bound to the surface at a controlled distance and shape according to the application conditions, so that not only a biosensor, but also neurons or embryonic cells It can contribute to research such as development, and can be used for medical or disease treatment as well as experimental or diagnostic in combination with biological tissues, the industrial effect will be very large.

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: Preparation of Polymer Multi-Layer

Example 1-1 Reagents

Poly (acrylic acid) (PAA, Mw = 90,000), Poly (allylamine hydrochloride) (PAH, Mw = 60,000), Polyacrylamide (PAAm), and Hyaluronic acid (HA) were used as reagents from Polysciences, and the Potassium salt was removed from the substituents. Osmotic permeation (dialysis method) was used.

Specifically, the salt removal process of hyaluronic acid using the dialysis method is as follows.

Since hyaluronic acid purchased from Polysciences may interfere with hydrogen bonding due to the presence of a Potassium salt in the substituent, salt was removed through osmotic permeation. The permeable membrane was composed of Cellulose ester (CE) manufactured by Spectrum, and a membrane having a molecular weight cut off (MWCO) of 1,000 was used. The water used to prepare the solution used in the experiment was a device (Milli-Q, Model: Biocel 18 MΩ) to generate both the deionized water and Mill 3 3 purified water at the same time. 1N HCl was added to 10 ml of a 0.01 M hyaluronic acid solution (about 1: 1.1), and then placed in a permeable membrane and placed in a beaker containing 1 L of purified water, and gently stirred on a magnetic stirrer. Permeation was continued for 48 hours while water was replaced with fresh purified water every 4 hours to increase permeability. After the permeation reaction, the collected solution was lyophilized to obtain pure hyaluronic acid.

In addition, Dulbecco's Phosphate Buffered Saline (DPBS), 0.25% Tripsin-EDTA, and Dulbecco's modified eagle medium (DMEM) used Gibco products, and Gibco's fetal bovine serum (FBS) and Antibiotic-Antimycotic were used to make DMEM. . In addition, RGD peptide was used as a product of Peptron inc.

Example 1-2 Preparation of Polymer Multilayer Membrane

The process of making and washing the polyelectrolyte aqueous solution was performed using deionized water (> 18 MΩ) produced using Millipore's Milli-Q water purifier.

Among the polymer electrolytes, PAA, PAH and PAAm aqueous solutions were prepared at a concentration of 0.01 M, HA aqueous solutions were prepared at a concentration of 0.002 M (based on the repeat unit molecular weight), and filtered through a filter having a pore size of 0.45 μm to remove dust or microorganisms. The pH was adjusted to 3.0 or less with 0.1 M HCl or NaOH aqueous solution. In the present invention, the polymer electrolyte multilayer membrane was formed of a polymer electrolyte multilayer membrane using ionic bonds using positive and negative charges of the polymer electrolyte, and in the case of PAAm, a hydrogen-bonded polymer electrolyte multilayer membrane was formed using hydrogen bonds of HA and PAAm. In this experiment, a glass or silicon substrate was used as the substrate for measuring the properties of the multilayer film. Since the surface adhesion of the silicon substrate was weak, the PAH, a positively charged polymer electrolyte after plasma treatment, was used. After coating once, HA and PAAm were alternately laminated. In order to prevent deprotonation in the production of a multilayer using hydrogen bonds, it is important to keep both the pH of the polymer electrolyte solution and the rinse solution low. In this experiment, the pHs of the polymer electrolyte solution and the washing solution were fixed at pH 3.0. On the other hand, in the preparation of the multilayer membrane using the ion bond was performed by adjusting the pH conditions to pH 4.5, pH 7.5 or pH 10.0 in addition to pH 3.0.

The substrates used in the experiment were all washed with dilute detergent solution and washed with deionized water several times and dried with nitrogen gas.

The production of the polymer multilayer film is alternately adsorbed at various pH conditions using the opposite charges of the positively charged polymer PAH and the negatively charged polymer PAA and HA, or the PAA-coated substrate is separated from the positively charged polymer, specifically, the neutral water-soluble polymer. It was carried out by a layer-by-layer (LBL) deposition technique in which the negatively charged polymers were alternately sucked using hydrogen bonds with PAA and HA.

Specifically, when the LBL deposition technique is performed by ion bonding, the substrate is put together with a detergent diluted in a washing bottle and subjected to ultrasonic treatment for 15 minutes, and then washed with deionized water and dried with nitrogen gas. I was. The dried substrate was immersed in an aqueous PAH solution for 15 minutes, washed two times or more with deionized water for 2 minutes, and then immersed in an HA solution or PAA solution for 15 minutes, and then prepared through the same washing process as 1 bilayer. The process was repeated to increase the number of bilayers as desired, and 3 bilayers were performed in this experiment.

In addition, when performing the LBL deposition technique by hydrogen bonding, the substrate is immersed in PAH aqueous solution for 15 minutes and washed twice with deionized water for 2 minutes or more to form an initial coating film, and then immersed in HA solution for 15 minutes and the same After the washing process, it was again immersed in the PAAm solution for 15 minutes and through the same washing process to form a coating film combined with the two polymer electrolytes. The coating film formed by the above process was called 1bilayer, and the process was repeated until the desired thickness was formed.

The polymer multilayer film prepared by the above process was dried using nitrogen gas, and then stored until the next step.

Example 1-3 Crosslinking Reaction of Hydrogen Bonded Polymer Multi-Layer

The hydrogen-bonded polymer electrolyte multilayer film formed by using the hydrogen bonds is formed by hydrogen bonds and may be eluted when it enters the physiological saline of pH 7.2 to 7.4 present in the biological environment, and thus crosslinked to give stability to the biological environment. The reaction was further performed.

Specifically, in order to apply the polymer multilayer membrane biologically or biochemically, cross-linking was weakly performed by performing a crosslinking reaction between the polymer electrolytes.

The crosslinking reaction was performed by heat treatment, and more specifically, the polymer multilayer film was heated by heating at 80 ° C. for about 10 hours.

Example 2: Checking the properties of the polymer multilayer

Example 2-1: Measurement of Wetting through Measurement of Contact Angle

In order to measure the wettability (wetability, wettability) of the multilayer films prepared in Examples 1-2 and 1-3, the contact angle of water was measured using a CA-A Contact angle analyzer of Face. Advancing contact angle and receding contact angle, which are the contact angles identified by the contact angle measurement, are indices that help to measure the change tendency of the film with respect to moisture. In general, the wettable film is not only low in static contact angle but also reported in low advancing contact angle and receding contact angle.

The water used for measuring the contact angle was used after filtering the purified water, using a syringe to form a drop of 10 ul and then slowly raise the measuring plate on which the film is placed so as to contact the water drop so that the influence of gravity on the contact angle measurement It was. More specifically, as shown in FIG. 2, the static contact angle is measured, and the advancing contact angle is measured by further adding 10 ul of water in the state of measuring the static contact angle, and measuring the contact angle again. The receding contact angle was measured by sucking 10 ul of water from 20 ul of water droplets for measuring the advancing contact angle and measuring the contact angle again. The results are shown in FIG. 3.

As shown in FIG. 3, all of the polymer electrolyte multilayer films prepared in the present invention were found to have excellent wettability. Specifically, in the case of PAA / PAAm and HA / PAAm multilayer membranes produced by hydrogen bonding, the average value of all contact angles was less than 20 degrees or less than 30 degrees, and in the case of PAH / HA and PAH / PAA multilayer membranes prepared by ion bonding. Most contact angles were also found to be excellent in wettability of less than 30 degrees. In addition, the receding contact angle value was confirmed to be low, and was expected to have a very good affinity for moisture. In the case of the PAA / PAAm multilayer film, all contact angles were found to be less than 20 degrees on average, and it was confirmed that the wettability was excellent.

In order to increase reactivity with PAH ink in the subsequent patterning process, the measured values were measured when the multilayer film was dried to form a weak anhydride group. It was expected.

Example 2-2: Measurement of Surface Morphological Properties Using Atomic Force Microscopy (AFM)

In order to examine the properties of the multilayer films prepared in Examples 1-2 and 1-3, surface morphological properties were confirmed using AFM. In general, as confirmed by the surface morphological properties, it is reported that the surfaces exhibiting the effect of preventing the non-selective adsorption of the cells exhibits a shape as if the fine particles are distributed on the measured surface as a whole.

Specifically, the microscope used in this experiment was using Digital Instrument's Dimension 3100 AFM, and the surface morphological properties were confirmed by taking a height image of AFM in a tapping mode using the AFM. It was performed by the method of checking the phase difference of the surface, and the results are shown in FIGS. 4 and 5.

As shown in FIG. 4 and FIG. 5, the polymer electrolyte multilayer membrane of the present invention was confirmed that all the fine particles are distributed, it can be seen that the effect of preventing the non-selective adsorption of cells.

Specifically, as shown in FIG. 4, it was confirmed that fine particles are distributed in the polymer electrolyte multilayer membrane prepared by using the ionic bond of the present invention and the polymer electrolyte multilayer membrane prepared by using the hydrogen bond.

In addition, as shown in FIG. 5, in the polymer electrolyte multilayer membrane prepared by using ionic bonds, it was confirmed that the fine particles were distributed under acidic conditions, that is, under low pH conditions. It was found to have a non-selective adsorption effect

Example 2 -3: polymer electrolyte Multilayer film  Cell Adsorption Experiment of Film

Cell adsorption experiments of the polymer electrolyte multilayer membrane films were performed using the HA / PAAm multilayer membranes prepared in Examples 1-2 and 1-3.

In order to examine the interaction between the multilayered membrane and the cells, experiments were performed using kidney cells (HEK 293 kidney cells), and the results are shown in FIG.

As shown in FIG. 6, when the multilayer film was coated on a glass substrate, cell adsorption was significantly reduced compared to the glass substrate without the multilayer film, and it was confirmed that non-selective adsorption could be prevented. In addition, the degree of cell adsorption was remarkably reduced compared to FIG. 6B in which HA is the outermost layer, and that in which the outermost layer of HA is able to inhibit non-selective adsorption of cells. It was confirmed that the selective adsorption of the cells can be secured.

Example  3: Inkjet Printing  Used Patterning

Based on the properties of the multilayer films prepared in Examples 1-2 and 1-3, in particular, the ability to prevent non-selective adsorption of cells, inkjet fritting is performed to fix the bioactive material to the surface in the form of a micropattern. Was performed.

The inkjet printer used in the inkjet printing is UJ-2100 of UNIJET Inc. including a piezo injector nozzle using a property that deforms or vibrates when electricity is applied to a crystal, and the nozzle uses a size of 30 did. In addition, the viscosity of the ink used for inkjet printing was adjusted to 10 cP.

Polymer electrolyte multilayer membrane prepared from PAH / PAA (pH4.5 / pH3.0) 3 bilayer prepared in Example 1-2 The ionic bond with PAA forming the outermost layer of the polymer electrolyte multilayer membrane of the substrate coated with 3bilayer film Inkjet printing was performed using PAH, a polymer electrolyte capable of reacting, as an ink. The inkjet printing was performed to complete PAH molecular electrolyte patterning using a bitmap picture file. The ink was prepared in 0.05M PAH solution adjusted to pH 7.5.

Since the patterned polymer electrolyte ink is fixed to the surface, that is, the polymer multilayer through strong interaction, the remaining ink except for the ink adsorbed through the electrostatic attraction on the film-coated substrate was removed using PBS.

In order to investigate the completeness of the polymer pattern formed by inkjet printing using the PAH aqueous solution as an ink, the fluorescent material was labeled.

Specifically, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC), which is a coupling agent coupled with PAH, a polymer electrolyte used as an ink on a substrate patterned through inkjet printing, and 6- which is a fluorescent substance After binding to the carboxyfluorescein-N-hydroxysuccinimide ester, it was confirmed by fluorescence microscopy, and the results are shown in FIG.

FIG. 7 is a photomicrograph of a patterned substrate obtained by introducing a fluorescent material by performing inkjet printing using PAH as an ink on a substrate coated with a 3 bilayer polymer electrolyte multilayer film prepared at pH 3.0 and pH 4.5. As shown in FIG. 7, it was confirmed that the patterning technique of the present invention using the polymer electrolyte as an ink can be patterned in a desired shape within a micrometer range.

Example  4: Patterned  Fabrication of Biosensitizer with Cell Array on Substrate

The biosensing device was manufactured by performing a cell array on the substrate on which the polymer electrolyte ink prepared in Example 3 was patterned.

Specifically, using an amine functional group of PAH used as the ink for patterning, a coupling reaction of an oligopeptide containing an RGD amino acid sequence known to be recognized as a receptor when the cell is adsorbed to the surface. Combined through. The sequence of the oligopeptide used in the present invention was GRGDSPC.

The aqueous solution containing the oligopeptide at a concentration of 0.5 was prepared on the patterned substrate, and a coupling reaction was performed using sulfosuccinimidyl 6- [3`- (2-pyridyldithio) propionamido] hexanoate (Sulfo-LC-SPDP) as a coupling agent. Was performed. In other words, the patterned substrate of Example 3 was immersed in a Sulfo-LC-SPDP solution for 30 minutes, and then washed twice in PBS (phosphate buffered saline solution) solution for 5 minutes, and the oligopeptide containing the RGD amino acid sequence. The reaction was performed by soaking in the solution for 8 hours. After the reaction was carried out, washed with PBS, and then dried.

In order to confirm whether the pattern of the peptide is well formed and as a result, the cell arrangement can be uniformly performed, a cell adsorption experiment was performed. The substrate subjected to the binding reaction with the oligopeptide was put in a cell culture vessel (Cell culture petri-dish), a cell culture solution containing DMEM was added, and observed while incubating in a CO ₂ incubator at a temperature of 37 ° C. suitable for cell culture. It was. Substrates subjected to the binding reaction with the oligopeptide were washed with 70% alcohol, dried and sterilized before cell experiments, and the cells were human kidney cells known to bind to oligopeptides containing the RGD amino acid sequence. Embryonic Kidney cell) 293 cells were used.

Observation of the cell culture observed how the cells adsorb onto the patterned polymer electrolyte coating membrane every 6 hours to 2 hours after the basic adsorption of the cells is almost complete, and how they move according to the pattern (cell migration and movement, etc.). The results are shown in FIGS. 8 and 9.

As shown in FIG. 8, 293 cells were found to be arranged at regular intervals on the separator electrolyte coating film patterned according to the ink pattern of the micron unit. That is, when the patterning method of the present invention is used, the cells can be uniformly arranged on the substrate. The present invention provides a method for producing a bioreactor in which cells are selectively adsorbed by a method capable of controlling adsorption using a polymer having antimicrobial properties. It was confirmed that it can.

In addition, as shown in FIG. 9, the 293 cells were confirmed to be adsorbed in a circular shape having a diameter of about 50 μm according to the ink pattern of the micron unit, and cultured for 24 hours, the cells grew well while maintaining the pattern. It was confirmed that.

From the above results, the polymer electrolyte multilayer membrane of the present invention can control the non-selective adsorption of cells, the patterning method of the present invention can easily control the distance between the patterns, the cells or according to the pattern formed by the patterning method Since the peptide can be bound, the polymer electrolyte multilayer film and the patterning technique of the present invention can also induce cell-cell talk between cells bound to the surface at a controlled distance and shape according to the application conditions. In addition to the device (Biosensor), it can be contributing to the study of the development of neurons or embryonic cells, it was confirmed that it can be used for medical or disease treatment as well as experimental or diagnostic in combination with living tissue.

1 is a schematic view showing a method of manufacturing a polymer electrolyte multilayer film using a layer-by-layer (LBL) deposition technique according to an embodiment of the present invention.

Figure 2 is a photograph of measuring the contact angle in order to measure the wettability of the coated substrate using a polymer electrolyte multilayer according to an embodiment of the present invention, Figure 3a is prepared by using a poly allylamine hydrochloride and hyaluronic acid 3 is a photograph of a multi-layer coated substrate prepared using polyallylamine hydrochloride and poly acrylic acid.

3 is a graph showing a contact angle test result showing the wettability of the polymer electrolyte multilayer according to an embodiment of the present invention.

FIG. 4 is a photograph showing an AFM image of a multi-membrane prepared using ionic bonds and hydrogen bonds using hyaluronic acid or polyacrylic acid, polyallylamine hydrochloride or polyacrylamide according to an embodiment of the present invention. Is a polyacrylic acid and polyallylamine hydrochloride multilayered membrane prepared under the condition of pH 3.0 / 3.0, and FIG. 4B is a case of hyaluronic acid and polyallylamine hydrochloride prepared under the condition of pH 4.5 / 3.0, FIGS. 4C and 4D Examples of hydrogen bonding are polyacrylic acid and polyacrylamide, hyaluronic acid and polyacrylamide prepared under the conditions of pH 3.0 / 3.0, respectively. The size of the picture taken in FIG. 4 is 10 μm × 10 μm.

FIG. 5 is a photograph showing an AFM image of a multi-layer prepared using polyallylamine hydrochloride and hyaluronic acid formed at various pH conditions according to an embodiment of the present invention. FIG. 5A is prepared at pH 3.0. 5b is prepared under the condition of pH 7.5 for polyallylamine hydrochloride and pH 3.0 for hyaluronic acid, and FIG. 5c is prepared at pH 10.0.

FIG. 6 is a micrograph showing the interaction between HEK 293 cells and a multilayer membrane prepared using hyaluronic acid and polyacrylamide to confirm non-selective cell adsorption of the multilayer membrane according to an embodiment of the present invention. FIG. 6A Is a photograph measured using a glass substrate coated with hyaluronic acid and poly acrylamide whose outermost layer is polyacrylamide, and FIG. 6B is a glass substrate coated with hyaluronic acid and polyacrylamide whose outermost layer is hyaluronic acid. It is a photograph measured and FIG. 6C is a photograph measured using the glass substrate which is not coated by a multilayer film. The size of the scale bar shown in the figure is 200 μm.

FIG. 7 is a micrograph of a substrate patterned by inkjet printing using polyallylamine hydrochloride ink on a substrate coated with polyallylamine hydrochloride and hyaluronic acid according to an embodiment of the present invention. to be.

FIG. 8 is a photograph of a biosensing apparatus in which cells are cultured on a substrate patterned by micron by performing inkjet printing using polyallylamine hydrochloride ink according to an embodiment of the present invention. The scale bar shown in this figure is 100 μm.

FIG. 9 is a micrograph of HEK 293 cells cultured on a patterned substrate using polyallylamine hydrochloride as an ink in order to confirm non-selective cell adsorption of a multilayer membrane according to an embodiment of the present invention. HEK 293 cells were cultured for 14 hours, and FIG. 9B is HEK 293 cells cultured for 24 hours. The scale bar shown in this figure is 50 μm.

Claims (35)

An alternating stack structure of an anionic polymer electrolyte layer and a cationic polymer electrolyte layer, The anionic polymer electrolyte layer is made of any one selected from the group consisting of hyaluronic acid and polyacrylic acid, the cationic polymer electrolyte layer is made of any one selected from the group consisting of poly allylamine hydrochloride and poly acrylamide, One or both layers of the outermost layer of the alternating laminated structure is an anionic polymer electrolyte layer. The method of claim 1, A multilayer membrane having at least two anionic polymer electrolyte layers and a cationic polymer electrolyte layer. The method of claim 2, The anionic polymer electrolyte layer is made of the same or different polymers, the cationic polymer electrolyte layer is made of the same or different polymers. The method of claim 1, A multilayer film further comprising a pattern layer comprising a cationic polymer electrolyte having an amine group on any one of the outermost layers, which is an anionic polymer electrolyte layer. The method of claim 4, wherein The pattern layer is a multilayer film patterned by the inkjet printing method using an ink containing a cationic polymer electrolyte having the amine group. The method of claim 4, wherein The cationic polymer electrolyte having an amine group is polyallylamine hydrochloride. The method of claim 4, wherein Multilayer membrane further comprising a cell-fixing biomaterial coupled to the cationic polymer electrolyte of the pattern layer. The method of claim 7, wherein A multilayer film further formed with a thin film on the pattern layer. The method of claim 8, The thin film is a multilayer film containing cells. The method of claim 9, The cell-fixing biomaterial is a polypeptide including an oligopeptide having an amino acid sequence of RGD, and the cell is attached to a pattern layer including a cationic polymer electrolyte to which the cell-fixing biomaterial is bound. A multilayer film in which an anionic polymer electrolyte layer and a cationic polymer electrolyte layer are alternately stacked, The anionic polymer electrolyte layer is made of any one selected from the group consisting of hyaluronic acid and polyacrylic acid, the cationic polymer electrolyte layer is made of any one selected from the group consisting of poly allylamine hydrochloride and poly acrylamide, Any one or both layers of the outermost layer of the multilayer film comprises a multilayer film which is an anionic polymer electrolyte layer. Board; The multilayer film of claim 1 formed on the substrate such that the anionic polymer electrolyte layer of the outermost layer becomes the uppermost layer; A pattern layer disposed on an uppermost layer of the multilayer film and including a cationic polymer electrolyte having an amine group; And Cell fixing layer comprising a material for fixing a biological material bound to the cationic polymer electrolyte of the pattern layer Biosensing device comprising a. The method of claim 12, A biosensing device comprising at least two anionic polymer electrolyte layers and a cationic polymer electrolyte layer. The method of claim 13, Anionic polymer electrolyte layer is made of the same or different polymers, the cationic polymer electrolyte layer is made of the same or different polymers. The method of claim 12, The cationic polymer electrolyte having an amine group is polyallylamine hydrochloride. The method of claim 12, The pattern layer is a biosensing device that is patterned by an inkjet printing method using an ink containing a cationic polymer electrolyte having an amine group. The method of claim 12, The cell-fixing biomaterial is a polypeptide comprising a oligopeptide having an amino acid sequence of RGD. The method of claim 12, A biosensing device in which cells are further attached to a pattern layer including a cationic polymer electrolyte to which a cell-fixing biomaterial is bound. Board; The multilayer film of claim 1 formed on the substrate such that the anionic polymer electrolyte layer of the outermost layer becomes the uppermost layer; A pattern layer disposed on an uppermost layer of the multilayer film and including a cationic polymer electrolyte having an amine group; And Biomaterial bonding layer comprising a biomaterial bonded to the pattern layer Biosensing device comprising a. The method of claim 19, The biomaterial is any one selected from the group consisting of polypeptides, antibodies and proteins. Alternately stacking the anionic polymer electrolyte layer and the cationic polymer electrolyte layer on the substrate so that the anionic polymer electrolyte layer becomes the uppermost layer to form the multilayer film of claim 1; Forming a patterned layer by patterning the inkjet printing method using an ink including a cationic polymer electrolyte having an amine group on an uppermost layer of the multilayer film; And Forming a cell-fixing layer by combining a cationic polymer electrolyte having an amine group of the patterned layer with a cell-fixing biomaterial Biosensing device manufacturing method comprising a. The method of claim 21, A biosensing device comprising at least two anionic polymer electrolyte layers and a cationic polymer electrolyte layer. The method of claim 22, The anionic polymer electrolyte layer is a polymer electrolyte layer made of the same or different polymers, and the cationic polymer electrolyte layer is a polymer electrolyte layer made of the same or different polymers. The method of claim 23, wherein A cationic polymer electrolyte having an amine group is polyallylamine hydrochloride. The method of claim 23, wherein Forming the multilayer film is a biosensing device manufacturing method that is performed by a layer-by-layer (LBL) deposition technique to alternately adsorb the polymer electrolyte layer on the substrate. The method of claim 25, The layer-by-layer (LBL) deposition technique is a method of manufacturing a biosensing device in which a substrate is alternately immersed in a polymer electrolyte solution containing an anionic polymer electrolyte and a polymer electrolyte solution containing a cationic polymer electrolyte. . The method of claim 26, At least one selected from the group consisting of a polymer electrolyte solution containing an anionic polymer electrolyte and a polymer electrolyte solution containing a cationic polymer electrolyte, wherein the pH of the solution is adjusted to pH 2.0 to pH 5.0. The method of claim 21, The step of forming the pattern layer is a method of manufacturing a biosensitizer by patterning by inkjet printing method using a printer comprising a nozzle of the piezo injector (piezo injector) method. The method of claim 21, Ink containing a cationic polymer electrolyte having an amine group has a viscosity of 1cP to 20cP biosensing device manufacturing method. The method of claim 21, Ink comprising a cationic polymer electrolyte having an amine group further comprises at least one member selected from the group consisting of ethylene glycol and glycerol. The method of claim 21, Forming the cell-fixing layer may be performed by sequentially immersing the patterned substrate in a polypeptide solution containing an aqueous solution of sulfosuccinimidyl 6- [3`- (2-pyridyldithio) propionamido] hexanoate (Sulfo-LC-SPDP) and oligopeptides RGD. Method of producing a biosensing device. The method of claim 21, Reacting the cell to the cell-fixing biomaterial of the cell-fixing layer further comprising the step of attaching the cell to the pattern layer comprising the cationic polymer electrolyte to which the cell-fixing biomaterial is coupled. An alternating stacked structure of an anionic polymer electrolyte layer made of any one selected from the group consisting of hyaluronic acid and polyacrylic acid and a cationic polymer electrolyte layer made of any one selected from the group consisting of polyallylamine hydrochloride and polyacrylamide, Forming a multilayer film in which one or both layers of the outermost layer of the alternate stacked structure is an anionic polymer electrolyte layer; And Patterning by inkjet printing using an ink containing a cationic polymer electrolyte having an amine group on any one of the outermost layers of the anionic polymer electrolyte layer of the multilayer film Patterning method comprising a. The method of claim 33, wherein The cationic polymer electrolyte having an amine group is polyallylamine hydrochloride. The method of claim 33, wherein Ink containing the cationic polymer electrolyte having an amine group has a viscosity of 1cP to 20cP.

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