• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
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    • 专利摘要:
    A biochip which is capable of detecting and analyzing multivalent bonds between target protein and binding mediator of monovalent bonds and a method for producing the same are disclosed. A biochip according to one embodiment comprises: a hydrogel functional layer on which a binding mediator is formed and whose physical properties are changed by a reaction between the target protein to be introduced and the binding mediator; and a transducer which is designed to deliver a shift signal, which corresponds to a change in the physical properties of the hydrogel functional layer, to an analytical instrument, the reaction being multivalent bonds between the target protein and the binding mediator and a deswelling in at least part of the hydrogel functional layer takes place through the multivalent bonds.
    公开号:CH713472B1
    申请号:CH00810/18
    申请日:2016-12-23
    公开日:2021-04-30
    发明作者:Sung Hyuk-Kee；Seong Kim Jong
    申请人:Scholar Foxtrot Co Ltd；
    IPC主号:
    专利说明:

    Technical area
    Disclosed is a biochip that can detect and analyze multiple bonds of a target protein separately from single bonds, and a method for producing the same.
    State of the art
    Proteins play an important role in the body as regulators that mediate intracellular responses. Mechanisms brought about by proteins are regulated by multiple bonds between heterologous or homologous proteins. Multiple bonds of protein can work in the body as follows: (1) Creation of structures through protein polymerization (e.g. thrombogenesis and actin polymerization), (2) (glyco) proteins between heterologous or homologous cell membrane proteins bind to ligands (glyco) proteins to form dimers or multimers for intracellular transmission of signals, (3) expressing a gene through an interaction and binding between intracellular proteins.
    As described above, multiple bonds between proteins in the body lead to significant bioregulatory processes such as thrombus formation, cancer formation and immune reactions in vivo. It is therefore necessary to detect and analyze the multiple bonds between the proteins in the body. In order to detect multiple bonds of the proteins, a pretreatment process, such as fluorescent labeling, is required together with a purification and concentration of the protein. Without such a series of pretreatment processes (label-free, unlabeled) it is very difficult to distinguish and detect multiple bonds between target protein and binding mediators from simple bonds. For example, even with prior art refractive index converter technology, it is difficult for the refractive index converter to provide sufficient sensitivity and selectivity for multiple bonds to detect multiple bonds of the protein.
    Disclosure of the invention
    Technical problem
    A biochip that can detect and analyze multiple bindings of a target protein separately from single bindings, and a method for making the same, are provided.
    The problem to be solved is not limited to the above-mentioned problem, and other objects that are not mentioned will be clearly understood by those skilled in the art from the following description.
    Technical solutions
    According to the invention, a biochip can include: a hydrogel functional layer in which a binding mediator is formed and a physical property is changed by a reaction between the target protein to be introduced and the binding mediator; and a transducer for supplying a displacement signal, which corresponds to the change in the physical property of the hydrogel functional layer, to an analytical instrument, the reaction being multiple bonds between the target protein and the binding mediator. Swelling takes place in at least one part of the hydrogel functional layer due to the multiple bonds.
    According to the invention, the reaction is a multiple bond between the target protein and the binding mediator, and swelling can occur in at least part of the hydrogel functional layer as a result of the multiple bond.
    The physical property can be a refractive index of at least a part of the hydrogel functional layer, the transducer can include a waveguide, and the displacement signal can be an output signal of the waveguide.
    The physical property is a refractive index of at least a portion of the hydrogel functional layer, the transducer includes a gold thin film and the displacement signal is an output signal that corresponds to a surface plasmon resonance (SPR) that occurs in the gold thin film .
    The physical property can be a volume of at least a part of the hydrogel functional layer, the transducer can include a piezoelectric element, and the displacement signal can be an output signal of the piezoelectric element.
    According to one embodiment, the hydrogel functional layer can contain a copolymer which consists of a main monomer and a comonomer. The main monomer here is selected from the group consisting of N-isopropylacrylamide, acryloylglycine amide, vinyl caprolactam and vinyl methyl ether, and the comonomer is allylamine (AA), dimethylaminoethyl methacrylate (DMAEMA), dimethylaminoethyl acrylate (DMAEA), acrylic acid (AAc) and methacrylic acid (MAA) .
    The hydrogel functional layer can also contain a crosslinking agent and the hydrogel functional layer can contain 55 to 98% of the main monomer, 2 to 40% of the comonomer and 0.1 to 5% of the crosslinking agent.
    According to one embodiment, the hydrogel functional layer is at least one copolymer selected from the group consisting of poly (N-isopropylacrylamide-co-allylamine) (poly (NIPAM-co-AA)), poly (N- isopropylacrylamide-co-2- (dimethylamino) ethyl methacrylate) (poly (NIPAM-co-DMAEMA)), poly (N-isopropylacrylamide-co-2- (dimethylamino) ethyl acrylate) (poly (NIPAM-co-DMAEA)), poly ( N-isopropylacrylamide-co-acrylic acid) (poly (NIPAM-co-AAc)), poly (N-isopropylacrylamide-co-polyethylene glycol-acrylic acid) (poly (NIPAM-co-PEG-AAc)), [oly (N-isopropylacrylamide -co-methacrylic acid) (poly (NIPAM-co-MAAc)), and hydroxypropyl cellulose.
    According to one embodiment, the binding mediator is a. Ligands, a receptor, a deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA) and the surface of the hydrogel functional layer can be modified by forming the binding mediator.
    According to one embodiment, the binding mediator is ligand, a receptor, a deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA), and the surface of the hydrogel functional layer is made of nanoparticles to form the binding mediator, or can using at least one of the proteins can be modified as a coupling fragment.
    The ligand or the receptor can be linked to the hydrogel functional layer by at least one of carbodiimide crosslinkers, crosslinkers in the form of a Schiff base, azlactone crosslinkers, carbonyldiimidazole (CDI) crosslinkers, iodoacetyl crosslinkers, hydrazide crosslinkers, Mannich- Crosslinker and maleimide crosslinker be linked.
    According to one embodiment, the hydrogel functional layer is divided into two or more regions for reaction with the target protein.
    The method for producing the hydrogel functional layer contained in the biochip described above includes: Mixing 55 to 98% of the main monomer, 2 to 40% of the comonomer and 0.1 to 5% of the crosslinking agent, so that the sum of the monomers 100% is; Heating the aqueous solution containing the monomer; Initiating a reaction by adding an initiator; and obtaining an aqueous hydrogel solution generated by the reaction.
    Maintaining the aqueous hydrogel solution may include maintaining an oxygen-free environment while heating the aqueous solution.
    In one embodiment, the main monomer is selected from the group consisting of N-isopropyl acrylamide, poly (N-acryloylglycine amide), hydroxypropyl cellulose, poly (vinyl caprolactam) and polyvinyl methyl ether. The comonomers can consist of at least one of allylamine (AA), dimethylaminoethyl methacrylate (DMAEMA), dimethylaminoethyl acrylate (DMAEA), acrylic acid (AAc), polyethylene glycol (PEG), and methacrylic acid (MAAc).
    The method for manufacturing the biochip described above may include: synthesizing a hydrogel in a form of nanoparticles; Activating the surface of the transducer with at least one of a positive charge, a negative charge, an epoxy, or a mercapto; and applying the hydrogel to the surface of the transducer to form the hydrogel functional layer.
    According to one embodiment, synthesizing the hydrogel includes the step of mixing 55 to 98% of the main monomer, 2 to 40% of the comonomer and 0.1 to 5% of the crosslinking agent so that the sum is 100%; Heating the aqueous solution containing the monomer; and initiating a reaction by adding an initiator; and it may include the step of obtaining an aqueous hydrogel solution generated by the reaction. The main monomer here is selected from the group consisting of N-isopropyl acrylamide, poly (TV-acryloylglycine amide), hydroxypropyl cellulose, poly (vinyl caprolactam), and polyvinyl methyl ether, and the comonomer is allylamine (AA), dimethylaminoethyl methacrylate (DMAEMA), dimethylaminoethyl acrylate (DMAEMA) , Acrylic acid (AAc), polyethylene glycol (PEG) and methacrylic acid (MAAc).
    According to one aspect, the hydrogel functional layer is poly (N-isopropylacrylamide-co-allylamine) (poly (NIPAM-co-AA)), poly (N-isopropylacrylamide-co-2- (dimethylamino) ethyl methacrylate) (poly ( NIPAM-co-DMAEMA)), poly (N-isopropylacrylamide-co-2- (dimethylamino) ethyl acrylate) (poly (NIPAM-co-DMAEA)), poly (N-isopropylacrylamide-co-acrylic acid) (poly (NIPAM-co -AAc)), poly (N-isopropylacrylamide-co-polyethylene glycol-acrylic acid) (poly (NIPAM-co-PEG-AAc)) and poly (N-isopropylacrylamide-co-methacrylic acid) (poly (NIPAM-co-MAAc)) , N-isopropyl acrylamide, poly (N-acryloylglycine amide), hydroxypropyl cellulose, poly (vinyl caprolactam) and polyvinyl methyl ether.
    According to the invention, the surface of the transducer is activated with positive charge, negative charge, epoxy and / or mercapto using at least one of aminosilane, carboxysilane, epoxysilane and mercaptosilane.
    According to one embodiment, the step of modifying the surface of the hydrogel functional layer includes forming at least one of a ligand, a receptor, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) on the surface of the hydrogel functional layer.
    According to one embodiment, the method further includes modifying the surface using at least one of nanoparticles or proteins as a coupling fragment in order to add at least one of a ligand, receptor, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA ) to build.
    The step of modifying the surface is linking at least one of the ligand, the receptor, the deoxyribonucleic acid or the ribonucleic acid to the surface of the hydrogel functional layer, using at least one crosslinking agent selected from the group consisting of from 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) hydrochloride, dicyclohexylcarbodiimide (DCC), sodium cyanoborohydride (NaCNBH3), azlactone, carbonyldiimidazole (CDI), iodoacetyl, hydrazide, diaminodipropylamine (N-Doxuccylamine (N-Doxuccide) and diaminodipropylamine ) -Estern exists.
    The method of making the biochip described above provided may include: activating a surface of the transducer with at least one of a positive charge, a negative charge, an epoxy, or a mercapto; Applying an aqueous hydrogel solution to the surface of the transducer; and adding an initiator to the aqueous hydrogel solution to form the hydrogel functional layer in the form of a gel mass.
    According to the invention, the aqueous hydrogel solution contains 55 to 98% of a main monomer, 2 to 40% of the comonomer and 0.1 to 5% of the crosslinking agent.
    According to one embodiment, the hydrogel functional layer includes at least one selected from the group consisting of poly (N-isopropylacrylamide-co-allylamine), (poly (NIPAM-co-AA)), poly (N- isopropylacrylamide-co-2- (dimethylamino) ethyl methacrylate), (poly (NIPAM-co-DMAEMA)), poly (N-isopropylacrylamide-co-2- (dimethylamino) ethyl acrylate), (poly (NIPAM-co-DMAEA)), Poly (N-isopropylacrylamide-co-acrylic acid), (poly (NIPAM-co-AAc)), poly (N-isopropylacrylamide-co-polyethylene glycol-acrylic acid), (poly (NIPAM-co-PEG-AAc)), poly ( N-isopropyl acrylamide-co-methacrylic acid), (poly (NIPAM-co-MAAc)), N-isopropyl acrylamide, poly (N-acryloylglycine amide), hydroxypropyl cellulose, poly (vinyl caprolactam) and polyvinyl methyl ether.
    According to one embodiment, the step of modifying the surface of the hydrogel functional layer includes the formation of a ligand, a receptor, a deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA) on the surface of the hydrogel functional layer.
    According to one embodiment, the method further includes modifying a surface of the hydrogel functional layer using at least one of nanoparticles and a protein as a coupling fragment to a ligand, a receptor, deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA) the surface of the hydrogel functional layer.
    Effect of the invention
    The biochip disclosed herein can detect multiple bonds between the target protein and the binding mediator, which are distinguishable from simple bonds, even without a pretreatment process, such as fluorescent labeling (= label-free).
    Brief description of the drawings
    FIG. 1 is a structural diagram of a biochip according to an embodiment. FIG. 2 is a diagram illustrating an operation of a biochip according to an embodiment. FIG. 3 (a) illustrates differential interference contrast (DIC) microscopic images and characteristics of change in temperature and change in pH for characterizing and comparing changes in a refractive index of a hydrogel functional layer due to single bonds and multiple bonds of the target protein. FIG. 3 (b) illustrates a schematic diagram of a hydrogel functional layer, the surface of which is modified by carbodiimide crosslinking between target protein and a ligand or receptor, and the analysis of multiple bonds using an optical microscope. FIG. 4 illustrates an example of a carbodiimide crosslink or maleimide crosslink. FIG. 5 illustrates an example of a biochip that is designed to detect a change in the refractive index of a hydrogel functional layer. FIG. 6 illustrates another example of a biochip configured to detect a change in a refractive index of a hydrogel functional layer. FIG. 7 illustrates an example of a biochip employing a multi-channel hydrogel functional layer. FIG. 8 illustrates an example of a method of synthesis of hydrogel used to form the hydrogel functional layer. FIG. 9 illustrates an example of a method of manufacturing a biochip to form a hydrogel functional layer using hydrogel produced by the synthesis method of FIG. 7 is synthesized to form. FIG. 10 illustrates an example of a method of making a biochip using a hydrogel in a form of a gel mass. FIG. 11 illustrates experimental performance results of a biochip according to an embodiment.
    Best Mode For Carrying Out The Invention
    In the following, exemplary embodiments are described in detail with reference to the accompanying drawings. Like numerals in the drawings refer to like elements.
    Various modifications can be made to the embodiments described below. The examples described below are not intended to be construed as limiting the embodiments, and they should be understood to include all changes in, and all equivalents or alternatives to, them.
    The terminology used herein is intended for the purpose of describing particular exemplary embodiments only and is not intended to be limiting of the examples. Singular forms include the plural forms, unless the context clearly dictates otherwise. In this specification it is intended that the terms “comprise” or “have” the presence of a feature, an integer, a step, procedure, element, a component, or a combination thereof as indicated in the specification, and one or more others Specify features. It is to be understood that the present disclosure does not preclude the possibility of the presence or addition of integers, steps, operations, elements, components, or combinations thereof.
    Unless otherwise defined, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms, such as those in commonly used dictionaries, should be construed to have meanings consistent with their meaning in the context of the relevant field, and should not be construed in an idealized or overly formal sense, except in this application is not expressly defined as such.
    In addition, in the description accompanying the accompanying drawings, the same reference numerals are given to the same components regardless of the reference numeral, and redundant description thereof will be omitted. In the following description of the embodiment, the detailed description of well-known related technologies is omitted if it is determined that the detailed description thereof may unnecessarily obscure the essence of the embodiment.
    <Overview of biochips>
    FIG. 1 is a structural diagram of a biochip.
    Referring to FIG. 1, the biochip according to one embodiment contains a hydrogel functional layer 110, on which a binding mediator is formed and whose physical properties are changed by a reaction between the target protein to be introduced and the binding mediator, and a transducer 120 which is designed to generate a shift signal in accordance with a change the physical property of the hydrogel functional layer to be transferred to an analytical instrument.
    The physical properties described above are usually used as mechanical properties such as strength, hardness and elongation of the material, as electrical properties such as electrical conductivity, specific resistivity, permeability, refractive index, as thermal properties such as thermal conductivity, coefficient of thermal expansion , specific heat, and defined as temperature properties such as melting point and boiling point.
    The biochip 100 can be mounted in an analytical instrument (not shown in the drawings), for example a biosensor, and can further include a physical interface (wired or wireless) to transmit a displacement signal to the analytical instrument.
    The analytical instrument is equipment that provides a user with visual, audible and haptic analysis results associated with multiple bonds between the target protein and the binding mediator. The analytical instrument can be, for example, a Reichert® surface plasmon resonance (SPR) instrument. In addition, depending on the implementation, the analysis instrument can be implemented as a mobile device. The biochip 100 can be designed such that it can be detachably attached to various analytical instruments, and it can be implemented interchangeably.
    The hydrogel functional layer 110 provides a resolution capable of distinguishing between multiple bonds and single bonds between a target protein and a binding mediator introduced into the biochip 100. If the hydrogel functional layer 110 is used, it is possible to differentiate between single bonds and multiple bonds of a target protein and the binding mediator without a fluorescence-marking molecule (label-free).
    A binding mediator is formed on the surface of the hydrogel functional layer 110 for reaction with the target protein. The binding mediator can include at least one of a receptor, a ligand, DNA, and RNA. Depending on the embodiment, the binding mediator can be a mixture of at least two of the receptor, the ligand, the DNA, and the RNA. The binding mediator formed on the hydrogel functional layer 110 can be specifically designed to detect multiple bindings with a specific target protein.
    The multiple bonds (or dimerization) of the target protein with the binding mediator cause swelling in at least one part of the hydrogel functional layer 110, which swelling changes the physical properties of the hydrogel functional layer 110. Since the hydrogel functional layer 110 hardly changes the physical properties under a single bond between the target protein and the binding mediator, the hydrogel functional layer 110 can distinguish the single and multiple bonds between the target protein and the binding mediator from one another.
    According to one aspect, the physical property of the hydrogel functional layer 110 can be a refractive index.
    According to another aspect, the physical property of the hydrogel functional layer 110 can be a volume of the hydrogel functional layer 110.
    The transducer 120 outputs a change signal of the physical property of the hydrogel functional layer 110, which is generated by the multiple bonds between the target protein and the binding mediator introduced into the biochip 100. In some embodiments, the transducer 120 may further include a physical interface unit (not shown) configured to provide the displacement signal to an external analytical instrument. According to another implementation, the converter 120 may further include a signal processor (not shown) configured to process the displacement signal and a physical interface unit configured to transmit the result of the processing to an external device. It will be apparent to one of ordinary skill in the art that the structure and function of transducer 120 can be appropriately modified depending on the application of biochip 100 described herein.
    If the physical property that is changed due to the deswelling caused by multiple bonds between the target protein and the binding mediator in at least a part of the hydrogel functional layer 110 is a refractive index, the transducer 120 formed on the hydrogel functional layer 110 can be a Waveguide for outputting a displacement signal, which is either an optical signal or an electrical signal corresponding to the change in the refractive index, to the analysis instrument. Waveguides include surface plasmon resonance (SPR) waveguides, ring resonator waveguides, long period fiber grating waveguides, grating couplers, and grating waveguides.
    If the physical property that is changed due to deswelling due to multiple bonds between the target protein and the binding mediator in at least a part of the hydrogel functional layer 110 is a volume, the transducer 120 formed on the hydrogel functional layer 110 can be a Include a piezoelectric element for outputting a displacement signal corresponding to the change in volume to the analysis instrument. In this case, a quartz crystal microbalance (QCM) can be used as the analytical instrument.
    Depending on how often or how concentrated the multiple bonds occur between the target protein and the binding mediator, the change in the physical properties of the hydrogel functional layer 110 becomes greater. Using this relationship, multiple bonds between the target protein and the binding mediator can be determined and detected using a lookup table (not shown) generated by comparing the amount of multiple bonds with changes in the physical properties of the hydrogel functional layer 110 and the amount of multiple bonds can be measured. The lookup table can be recorded in a memory that is connected to an external analysis instrument, such as a biosensor, which is connected to the biochip 100 via a physical interface.
    FIG. 2 is a schematic diagram of a target protein reacting with a biochip 200 that includes a hydrogel functional layer 210 and a transducer 220 in accordance with one aspect.
    Referring to FIG. 2, when the target protein is bound to the binding mediators 211 and 222 formed on the hydrogel functional layer 210, the target protein is bound to the binding mediators 211 and 222 depending on the type of the target protein. Multiple bonds (dimerization) 221 or single bonds 222 occur. If multiple bonds occur, swelling occurs in the region 231 of the hydrogel functional layer 210 in which the multiple bonds occur, whereby the physical properties of the hydrogel functional layer 210 are changed. In the region in which simple bonds occur, the physical properties of the hydrogel functional layer 210 hardly change.
    In one example, the physical property may be a refractive index, and the transducer 220 transmits a displacement signal, which corresponds to a change in the refractive index due to the region 231 of the hydrogel functional layer 210, to the analytical instrument. In this case, the transducer 220 can be a waveguide. Due to the change in the refractive index of the hydrogel functional layer 210 caused by the region 231, the effective refractive index of at least one portion of the waveguide, which is the transducer 220, changes. The optical input signal to the biochip 200 is through the light source of the analysis instrument (not shown), the resonance frequency shifts due to the change in the effective refractive index of the transducer 220, and the output light signal with the shifted resonance frequency or the corresponding electrical signal (displacement signal) is used to detect the Transferring analysis instruments. The analytical instrument can analyze the displacement signal to determine the amount of multiple bindings between the target protein and the binding mediator.
    In another example, the physical property can be a refractive index, and the transducer 220 transmits a displacement signal, which corresponds to a change in the refractive index due to the region 231 of the hydrogel functional layer 210, to the analysis instrument. In this case, the transducer 220 may be a combination of a gold thin film and glass. Due to the change in the refractive index of the hydrogel functional layer 210 caused by the region 231, a surface plasmon resonance (SPR) occurs in the gold thin film, which is the transducer 220. More specifically, an input signal to the biochip 200 through an analytical instrument (not shown) may change its path due to the surface plasmon resonance generated in the gold thin film, or may generate a leak signal on the gold thin film. The displacement signals themselves (output light signals or corresponding electrical signals), such as deflected signals or leak signals, are transmitted to the detector of the analysis instrument.
    In yet another example, the physical property can be a volume, and the transducer 220, in which a piezoelectric element is included, can provide an electrical signal (i.e., a displacement signal) that is indicative of a change in a volume of the region 231 of the Hydrogel functional layer 110 corresponds, transferred to an analytical instrument. The analytical instrument can analyze the displacement signal to measure the amount of multiple bindings between the target protein and the binding mediator that corresponds to the change in volume of region 231.
    <Illustration of the mechanism of the hydrogel functional layer>
    FIG. 3 illustrates the change in the physical properties (refractive index) of the hydrogel functional layer due to multivalent and monovalent bonds by means of the change in the image in an optical microscope.
    Referring to FIG. 3 it can be seen that the physical properties (refractive index) of the hydrogel functional layer are changed due to multiple bonds between target protein and binding mediator that have occurred on the hydrogel functional layer. More specifically, when multiple bonds occur between the target protein and the binding mediator on the hydrogel functional layer, swelling occurs in at least a part of the hydrogel functional layer due to dimerization or multimerization due to multiple bonds. As described above, the binding mediator can be at least one of a receptor, a ligand, a DNA, and an RNA. Depending on the embodiment, the binding mediator can be a mixture of two or more of the receptor, ligand, DNA and RNA. Various combinations of binding mediators can be designed to detect multiple bindings between specific target proteins and binding mediators.
    The biochip described herein can effectively detect multiple bonds between target protein and binding mediator using a hydrogel functional layer. For example, if the bonds between the target protein and the binding mediator are not multiple, the physical properties of the hydrogel functional layer hardly change. When the bonds between the target protein and the binding mediator are multiple bonds, the physical properties of the hydrogel functional layer change significantly due to dewelling of the hydrogel functional layer. Thus, it is possible to distinguish single bonds from multiple bonds between the target protein and the binding medium using the hydrogel functional layer, and it is possible to determine the amount of multiple bonds between the target protein and the binding mediator based on a change in the physical properties of the hydrogel -Functional layer easy to detect.
    The physical properties of the hydrogel functional layer can change locally or altogether as a function of the method for producing the hydrogel functional layer or of the reactant.
    According to one aspect, the hydrogel functional layer can be produced in a multi-channel process. By dividing the physical surface of the hydrogel functional layer two or more times, it is possible to detect various multiple bonds between the target protein and the binding mediators in a biochip. According to one aspect, the area of a single hydrogel functional layer can be subdivided two or more times and the binding medium that is formed in the corresponding area can be different. According to another aspect, a transducer can be provided for each of the channels of the hydrogel functional layer, and a single transducer can pick up a change in the physical properties of the hydrogel functional layer that corresponds to two or more channels. This is explained below with reference to FIG. 6 described in more detail.
    According to one aspect, the hydrogel functional layer can contain a copolymer which consists of a main monomer and a comonomer. According to one embodiment, the hydrogel functional layer can be formed by polymerizing the main monomer and the comonomer using a crosslinking agent. Preferably, monomers can be used which are capable of forming a hydrogel which is sensitive to heat, ionic strength or pH.
    In one aspect, the major monomer can be selected from the group consisting of N-isopropyl acrylamide, poly (iV-acryloylglycine amide), hydroxypropyl cellulose, poly (vinyl caprolactam), and polyvinyl methyl ether. The comonomer can be selected from the group consisting of allylamine (AA), dimethylaminoethyl methacrylate (DMAEMA), dimethylaminoethyl acrylate (DMAEA), acrylic acid (AAc), polyethylene glycol (PEG), and methacrylic acid (MAAc).
    According to one aspect, the hydrogel functional layer can contain 55 to 98% of the main monomer, 2 to 40% of the comonomer and 0.1 to 5% of the crosslinking agent. In one aspect, when the amount of the main monomer is less than 55%, the reactivity may decrease, when it exceeds 98%, the ability to detect multiple bonds of the target protein may decrease. In another aspect, when the amount of the comonomer is less than 2% or exceeds 40%, the ability to detect multiple bonds of the target protein may decrease. In addition, when the content of the crosslinking agent is less than 0.1%, it may be difficult to form the hydrogel functional layer, and when the content of the crosslinking agent exceeds 5%, the ability to detect multiple bonds of the target protein may decrease.
    In one aspect, the hydrogel functional layer can include at least one selected from the group consisting of poly (N-isopropyl acrylamide-co-allylamine) (poly (NIPAM-co-AA)), poly (N-isopropyl acrylamide -co-2- (dimethylamino) ethyl methacrylate) (poly (NIPAM-co-DMAEMA)), poly (N-isopropylacrylamide-co-2- (dimethylamino) ethyl acrylate) (poly (NIPAM-co-DMAEA)), poly (N -isopropylacrylamide-co-acrylic acid) (poly (NIPAM-co-AAc)), poly (N-isopropylacrylamide-co-polyethylene glycol acrylic acid) (poly (NIPAM-co-PEG-AAc)), [oly (N-isopropylacrylamide- co-methacrylic acid) (poly (NIPAM-co-MAAc)), N-isopropyl acrylamide, poly (N-acryloylglycine amide), hydroxypropyl cellulose, poly (vinyl caprolactam) and polyvinyl methyl ether.
    Preferably, the major monomer can be N-isopropyl acrylamide (temperature sensitive hydrogel) and the comonomer can be acrylic acid (pH sensitive hydrogel). In this case the crosslinking agent can be MBA (N, N'-methylene-bis-acrylamide).
    According to one aspect, the hydrogel functional layer can further contain an initiator as a factor in order to initiate the polymerization reaction. Ammonium persulfate (APS) can be used as the initiator.
    According to one aspect, the hydrogel functional layer can contain 55 to 98% of N-isopropyl acrylamide, 2 to 40% of acrylic acid and 0.1 to 5% of a BIS crosslinking agent.
    In particular, when acrylic acid is used as the comonomer, it is preferably incorporated in at least 2% of the materials constituting the hydrogel functional layer.
    According to one embodiment, a hydrogel functional layer can control the sensitivity to multiple bonds by adjusting the ratio between the components and is thus not limited to the examples described herein. According to one aspect, with increasing specific amount of acrylic acid and with decreasing specific amount of BIS, the reactivity (degree of deswelling) of the hydrogel functional layer with respect to multiple bonds between the target protein and the binding mediator can increase.
    According to one aspect, the transducer surface of the biochip can be modified in order to increase the adhesive strength to the hydrogel functional layer.
    According to one aspect, the portion of the hydrogel functional layer in which the physical properties change can be the entire hydrogel functional layer or a part of the surface of the hydrogel functional layer.
    That portion of the hydrogel functional layer in which the refractive index changes due to multiple bonds in the target protein is referred to as an “activated layer”. The activated layer can be equal to the thickness of the entire hydrogel functional layer or smaller than the thickness of the hydrogel functional layer. The activated layer can be formed over a certain depth from the surface of the hydrogel functional layer.
    According to one aspect the surface of the hydrogel functional layer can be modified by forming a binding mediator, according to another aspect the surface of the hydrogel functional layer can be modified using at least one of nanoparticles or protein as a coupling fragment.
    As described above, the binding mediator can be at least one of a receptor, a ligand, DNA, or RNA. Depending on the embodiment, the binding mediator can be a mixture of two or more of the receptor, ligand, DNA and RNA. The binding mediator formed on the hydrogel functional layer 110 can be specifically designed to detect multiple bindings with a specific target protein.
    The binding mediator can be formed on the hydrogel functional layer by carbodiimide crosslinkers, crosslinkers in the form of a Schiff base, azlactone crosslinkers, carbonyldiimidazole (CDI) crosslinkers, iodoacetyl crosslinkers, hydrazide crosslinkers, Mannich crosslinkers or maleimide crosslinkers be. According to one embodiment, a functional carboxylic acid group (COOH), which is present on the surface of the hydrogel functional layer, and an NH2 <+> group, which is present in the protein, can be used to bind the binding mediator with the surface of the hydrogel. Link functional layer.
    FIG. 4 illustrates an example of the above-described carbodiimide crosslinker or maleimide crosslinker.
    According to one aspect, at least one crosslinking agent selected from the group consisting of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) hydrochloride, dicyclohexylcarbodiimide (DCC), sodium cyanoborohydride ( NaCNBH3), azlactone, carbonyldiimidazole (CDI), iodoacetyl, hydrazide, diaminodipropylamine (DADPA) and N-hydroxysuccinimide (NHS) esters. The crosslinking agent can be adjusted in such a way that the binding properties between the binding mediator and the hydrogel functional layer are controlled.
    <Examples of a biochip>
    (1) Biochip containing the hydrogel functional layer + waveguide
    In a conventional light-based biochip, an optical signal applied from a light source (not shown in the drawings) is transmitted and transmitted into the waveguide, and a part is transmitted to the surface of the optical waveguide in the form of an evanescent field . Thus, when chemical or biological phenomena occur on the surface of the waveguide, the change in the index of refraction of the surface material of the waveguide may occur on the waveguide based on its presence or extent. As a result, the change in the effective refractive index of the waveguide changes the optical signal transmission properties inside the waveguide. A device designed to detect a change in a chemical / biological phenomenon occurring on the surface of an SPR chip via a change in the characteristic of the optical transmission inside a waveguide due to a change in the refractive index of the surface material, is referred to per se as an optical detector or a refractive index detector.
    Optical detectors or refractive index detectors that measure a biomolecular binding relationship have a higher throughput and better sensitivity compared to other biochemical chips, do not require fluorescent labeling and can detect the biomolecular binding relationship in real time. They represent a significant advantage. In several approaches, the refractive index, which was caused by a change in a concentration of a biochemical molecule, was also quantified. However, no light detector technology has yet been implemented that is capable of detecting target proteins with multiple bonds in addition to the usual single bonds. Technology for accurately quantifying the quantitative amount of proteins with multiple bonds that are distinguishable from single bonds can be the basis for thrombus / immunity / cancer related therapies and for new drug discovery tests. There is thus a need for a technology that is capable of effectively detecting and measuring these.
    FIG. 5 illustrates an example of a biochip in accordance with one aspect. Referring to FIG. 5, the biochip 500 contains a hydrogel functional layer 510 and a transducer 520, which is designed to supply a displacement signal, which corresponds to a change in a refractive index of the hydrogel functional layer 510 due to multiple bonds between the target protein and the binding mediator, to the analysis instrument.
    The transducer 520 can be a waveguide. The waveguide may be one of a surface plasmon resonance (SPR) waveguide, a ring resonator waveguide, a long period fiber grating waveguide, a grating coupler, and a grating waveguide.
    The transducer 520 outputs a displacement signal, such as an optical signal or an electrical signal, indicative of a change in at least one of transmission / diffraction / scattering / refraction / reflection / resonance characteristics of the optical signal due to the change in the refractive index corresponds to the hydrogel functional layer 510. The displacement signal is provided to the analytical instrument, which can analyze the multiple bindings between the target protein and the binding mediator corresponding to the displacement signal.
    According to one aspect, the hydrogel functional layer can have a thickness of 10 nm to 1000 nm. For example, if a light source of approximately 500 nm to 1700 nm is used, the thickness of the hydrogel functional layer can be from 10 nm to 1000 nm and can be calculated using an optical waveguide simulation (e.g. a finite difference time domain ( FDTD) method or a finite difference method (FDM)). At the same time, the hydrogel functional layer can show a change in the effective refractive index at a thickness of 10 nm to 1000 nm. If the thickness of the hydrogel functional layer is less than 10 nm or exceeds 1000 nm, it may be difficult to detect the multiple bonds between the target protein and the binding mediator because it is difficult to determine the extent of the change in the refractive index.
    (2) Biochip containing hydrogel functional layer + gold thin film
    FIG. 6 illustrates a biochip in accordance with another aspect. Referring to FIG. 6, a biochip 600 contains a hydrogel functional layer 610 and a transducer 620, which is designed to transmit a displacement signal, which corresponds to a change in a refractive index of the hydrogel functional layer 610 due to multiple bonds between the target protein and the binding mediator, to an analysis instrument .
    The transducer 620 may be a layered structure composed of a gold thin film 621 and a glass 622. Surface plasmon resonance (SPR) occurs in the gold thin film 621 due to a change in the refractive index in at least a part of the hydrogel functional layer 610. More specifically, the input signal introduced into the biochip 600 by the analysis instrument (not shown) is changed in propagation angle due to surface plasmon resonance generated in the gold thin film 621, or a leak signal may be generated on the gold thin film 621. In this way, displacement signals (light output signals or corresponding electrical signals), such as signals that have changed angles of propagation or leak signals, are transmitted to the analysis instrument. The analytical instrument can analyze the displacement signal to analyze multiple bindings between the target protein and the binding mediator.
    (3) Biochip containing hydrogel functional layer + piezoelectric element
    The biochip according to one aspect includes a transducer that supplies a displacement signal, which corresponds to a change in a volume of a hydrogel functional layer due to multiple bonds between the target protein and the binding mediator, to an analytical instrument. As with reference to FIG. 2, swelling occurs in at least one section of the hydrogel functional layer due to the multiple bonds between the target protein and the binding mediator, and this changes the volume of the hydrogel functional layer. A piezoelectric element contained in the transducer outputs a displacement signal based on the change in the volume of the hydrogel functional layer. The displacement signal is transmitted to the analysis instrument. The analytical instrument can analyze the multiple bonds between the target protein and the binding mediator that correspond to the change in volume.
    (4) Multi-channel biochip
    FIG. 7 illustrates a biochip in accordance with another aspect.
    The biochip 700 according to one aspect may include two or more physically distinct hydrogel functional layers 711, 712 and 713 to detect multiple bonds between one or more target proteins and a binding mediator.
    In another aspect, the application region of the hydrogel functional layer formed on the surface of the transducer 720 is physically divided two or more times, and different hydrogel functional layers 711, 712 and 713 are formed in each region. According to another aspect, only an application region is formed on the surface of the transducer 720 to form various binding media on the hydrogel functional layers 711, 712, 713, and in this way multi-channel biochips can be implemented.
    Multiple bonds 741 take place between the binding mediator 721 formed in the region A711 of the hydrogel functional layer and the target protein A, which results in a de-swelling region 731 in the region A 711.
    Multiple bonds occur between the binding mediator formed in region B712 of the hydrogel functional layer and the target protein B, which results in a de-swelling region in region B.
    Multiple bonds 743 occur between the binding mediator formed in region C 713 of the hydrogel functional layer and the target protein C, which results in a de-swelling region in region C.
    The transducer 720 supplies a displacement signal corresponding to each of the regions A 711 to C 713 to an analysis instrument based on a change in physical properties of each of the regions A 711 to C 713 due to the de-swelling regions in each of the regions A. to C of the hydrogel functional layer.
    Shift signals can be analyzed using analysis equipment, and they can analyze multiple bindings of target proteins for each region corresponding to a change in the physical properties of each region.
    <Method of making the biochip>
    (1) Method for producing the biochip using hydrogel in the form of nanoparticles: FIG. 8 illustrates a hydrogel synthesis method for forming a hydrogel functional layer of a biochip in accordance with one aspect.
    According to one aspect, a hydrogel for forming the hydrogel functional layer of the biochip can be produced as follows: Mixing 801 of the total monomers, from 55 to 98% of a main monomer, 2 to 40% of comonomers and 0.1 to 5% of one Include crosslinking agent so that the sum of the monomers is 100%; Heating 802 an aqueous solution containing the monomers; Initiating a reaction by adding an initiator 803; and step 804 of obtaining an aqueous hydrogel solution generated by the reaction.
    In step 801, in one aspect, the major monomer and comonomer are polymerized with a crosslinking agent to form the hydrogel. Monomers capable of forming a hydrogel functional layer that is preferably sensitive to heat or pH can be used.
    In one aspect, the major monomer can be selected from the group consisting of N-isopropyl acrylamide, poly (N-acryloylglycine amide), hydroxypropyl cellulose, poly (vinyl caprolactam), and polyvinyl methyl ether. The comonomer can be selected from the group consisting of allylamine (AA), dimethylaminoethyl methacrylate (DMAEMA), dimethylaminoethyl acrylate (DMAEA), acrylic acid (AAc), polyethylene glycol (PEG), and methacrylic acid (MAAc).
    In one aspect, the hydrogel can include 55 to 98% of the major monomer, 2 to 40% of the comonomer, and 0.1 to 5% of the crosslinking agent.
    In one aspect, when the content of the main monomer is less than 55%, the reactivity may decrease, when it exceeds 98%, the ability to detect the dimerization of the protein may decrease.
    In another aspect, when the content of the comonomer is less than 2% or exceeds 40%, the ability to detect the dimerization of the protein may decrease. If the amount of the crosslinking agent is less than 0.1%, it may be difficult to form the hydrogel functional layer, and if the content of the crosslinking agent exceeds 5%, the dissolving ability, protein dimerization may decrease.
    For example, the major monomer can be N-isopropyl acrylamide (a temperature sensitive hydrogel) and the comonomer can be an acrylic acid (i.e., a pH sensitive hydrogel). In this example, a crosslinking agent can be N, N'-methylene-bis-acrylamide (BIS).
    In one aspect, the hydrogel is at least one selected from the group consisting of poly (N-isopropylacrylamide-co-allylamine) (poly (NIPAM-co-AA)), poly (N-isopropylacrylamide-co- 2- (dimethylamino) ethyl methacrylate) (poly (NIPAM-co-DMAEMA)), poly (N-isopropylacrylamide-co-2- (dimethylamino) ethyl acrylate) (poly (NIPAM-co-DMAEA)), poly (N-isopropylacrylamide- co-acrylic acid) (poly (NIPAM-co-AAc)), poly (N-isopropylacrylamide-co-polyethylene glycol-acrylic acid) (poly (NIPAM-co-PEG-AAc)), [oly (N-isopropylacrylamide-co-methacrylic acid ) (Poly (NIPAM-co-MAAc)), N-isopropyl acrylamide, poly (N-acryloylglycine amide), hydroxypropyl cellulose, poly (vinyl caprolactam) and polyvinyl methyl ether.
    In one aspect, the hydrogel can include 55 to 98% N-isopropyl acrylamide, 2 to 40% acrylic acid, and 0.1 to 5% of a BIS crosslinking agent. In particular, when acrylic acid is used as the comonomer, it is preferably included in at least 2% of the materials forming the hydrogel functional layer.
    The aqueous solution containing the main monomer and comonomers is heated in step 802.
    The reaction is initiated by adding the initiator to the heated aqueous solution in step 803. In one aspect, ammonium persulfate (APS) can be used as the initiator.
    The hydrogel aqueous solution generated by the reaction is obtained in 804. In step 804, a step of maintaining an oxygen-free environment while the aqueous solution is being heated can also be carried out. If an oxygen-free environment is maintained, the size uniformity of the hydrogel obtained in step 804 may increase.
    In another aspect, step 804 may include dialysis to purify unreacted monomer.
    The hydrogel described above can control the sensitivity to multiple bonds between the target protein and the binding mediator by adjusting the ratio between the components. The composition and the manufacturing method of the hydrogel are not limited to the examples described herein. According to one aspect, with increasing specific proportion of acrylic acid and with decreasing specific proportion of the BIS crosslinking agent, the reactivity (degree of deswelling) of the hydrogel functional layer with respect to the multiple bonds can increase.
    On the basis of the hydrogel synthesis method shown in FIG. 8, a hydrogel can be synthesized in the form of nanoparticles.
    FIG. 9 illustrates a method of making a biochip by forming a hydrogel obtained by the hydrogel synthesis process of FIG. 8 was synthesized on a transducer.
    The method for producing a biochip according to an embodiment comprises the steps of synthesizing a hydrogel in a form of nanoparticles 901; activating 902 a surface of the transducer with at least one of positive charge, negative charge, an epoxy, or a mercapto; and applying 903 the hydrogel to the surface of the transducer to form a hydrogel functional layer.
    In step 901, the hydrogel can be in the form of nanoparticles based on the hydrogel synthesis method of FIG. 8 can be synthesized.
    In step 902, the surface of the transducer is activated with at least one of a positive charge, a negative charge, epoxy, or mercapto.
    The step 902 of activating the surface of the transducer with at least one of the positive charges of the epoxy and the mercapto can be performed by an aqueous phase reaction using at least one of aminosilane, epoxysilane and mercaposilane.
    The aminosilane is (3-aminopropyl) -triethoxysilane, bis [(3-triethoxysilyl) propyl] amine, (3-aminopropyl) -trimethoxysilane, bis [(3-trimethoxysilyl) propyl] amine, (3-aminopropyl) - methyldiethoxysilane, (3-aminopropyl) -dimethylethoxysilan, (3-aminopropyl) -diethoxymethylsilan, aminoethylaminopropyltrimethoxysilane, aminoethylaminopropylmethyldimethoxysilane, Diethylentriaminopropylmethyldimethoxysilan, piperazinylpropylmethyldimethoxysilane, (N-phenylamino) methyltrimethoxysilane, (N-phenylamino) methyl triethoxysilane, (N-phenylamino) propyltrimethoxysilane, Dimethylaminoethyltriethoxysilan, Diethylaminomethylmethyldiethoxysilan and diethylaminopropyltrimethoxysilane.
    The epoxysilane is 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropyltriethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane and 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane, and 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane and 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane and 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane, and 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane.
    The mercaptosilane can be any of (3-mercaptopropyl) trimethoxysilane (MPTMS), (3-mercaptopropyl) triethoxysilane, and (3-mercaptopropyl) methyldimethoxysilane (MPDMS), where the mercaptosilane has a thiol group.
    In step 903, the hydrogel is applied to the surface of the transducer in order to form a functional hydrogel layer.
    In order to apply the hydrogel functional layer evenly to the surface of the transducer according to one aspect, the surface of the transducer is positively charged (+) and the hydrogel functional layer can be activated beforehand with a negative charge (-).
    In one aspect, when a hydrogel functional layer is formed in a form of nanoparticles, an aqueous free radical precipitation polymerization method can be used.
    According to one aspect, a surface of the hydrogel functional layer can be modified by forming a binding mediator, which can be at least one of a ligand, a receptor, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
    According to another aspect, the surface of the hydrogel functional layer can include at least one of a nanoparticle or a protein as a linking compound to form a binding mediator, the at least one of a ligand, a receptor, a deoxyribonucleic acid (DNA) or Ribonucleic acid (RNA) is.
    The binding mediator can be linked to the hydrogel functional layer by at least one of carbodiimide crosslinkers, crosslinkers in the form of a Schiff base, azlactone crosslinkers, carbonyldiimidazole (CDI) crosslinkers, iodoacetyl crosslinkers, hydrazide crosslinkers, Mannich crosslinkers and maleimide -Linker to be linked. A carboxylic acid functional group (COOH) present on the surface of the hydrogel functional layer and an NH2 + group present on a protein can be used to form the binding mediator on the surface of the hydrogel functional layer.
    For the crosslinking, at least one crosslinking agent can be used which is selected from the group consisting of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) hydrochloride, dicyclohexylcarbodiimide (DCC), sodium cyanoborohydride (NaCNBH3), Azlactone, carbonyldiimidazole (CDI), iodoacetyl, hydrazide, diaminodipropylamine (DADPA) and N-hydroxysuccinimide (NHS) esters. The crosslinking agent can be adjusted to control the binding properties between the binding mediator and the hydrogel functional layer.
    According to one aspect, the hydrogel functional layer can be formed by a method for forming a binding mediator on the surface of the hydrogel functional layer using at least one crosslinking agent selected from the group consisting of 1-ethyl-3- (3- dimethylaminopropyl) carbodiimide (EDC) hydrochloride, dicyclohexylcarbodiimide (DCC), sodium cyanoborohydride (NaCNBH3), azlactone, carbonyldiimidazole (CDI), iodoacetyl, hydrazide, diaminodipropyluccamine (DADPA) and N-hydroxymide are modified. In this case, the crosslinking agent can be adjusted in order to control the binding properties between the binding mediator and the hydrogel functional layer.
    (2) Method for producing the biochip using hydrogel in the form of gel mass:
    The biochip according to one aspect can be formed with a hydrogel functional layer in the form of a gel mass.
    FIG. 10 illustrates a method of making a biochip using a hydrogel in a form of a gel mass. In this case, a method of initiating a polymerization reaction after filling the reactant (liquid phase) on the surface of the transducer can be used.
    The method of manufacturing a biochip using a hydrogel in a form of gel mass according to one aspect includes activating 1001 the surface of the transducer with at least one of positive charge, negative charge, epoxy, or mercapto; Applying 1002 the aqueous hydrogel solution to the surface of the transducer; and forming 1003 a hydrogel functional layer in the form of a gel mass by adding an initiator to the aqueous hydrogel solution.
    In one aspect, the aqueous hydrogel solution can include 55 to 98% of the major monomer, 2 to 40% of the comonomer, and 0.1 to 5% of the crosslinking agent.
    In one aspect, the major monomer can be selected from the group consisting of N-isopropyl acrylamide, poly (N-acryloylglycine amide), hydroxypropyl cellulose, poly (vinyl caprolactam), and polyvinyl methyl ether. The comonomer can be selected from the group consisting of allylamine (AA), dimethylaminoethyl methacrylate (DMAEMA), dimethylaminoethyl acrylate (DMAEA), acrylic acid (AAc), polyethylene glycol (PEG), and methacrylic acid (MAAc).
    In one aspect, the aqueous hydrogel solution may include at least one selected from the group consisting of poly (N-isopropylacrylamide-co-allylamine) (poly (NIPAM-co-AA)), poly (N-isopropylacrylamide- co-2- (dimethylamino) ethyl methacrylate) (poly (NIPAM-co-DMAEMA)), poly (N-isopropylacrylamide-eo-2- (dimethylamino) ethyl acrylate) (poly (NIPAM-co-DMAEA)), poly (N- isopropylacrylamide-co-acrylic acid) (poly (NIPAM-co-AAc)), poly (N-isopropylacrylamide-co-polyethylene glycol acrylic acid) (poly (NIPAM-co-PEG-AAc)), [oly (N-isopropylacrylamide-co -methacrylic acid) (poly (NIPAM-co-MAAc)), N-isopropyl acrylamide, poly (N-acryloylglycine amide), hydroxypropyl cellulose, poly (vinyl caprolactam) and polyvinyl methyl ether.
    The surface of the transducer is activated with at least one of a positive charge, a negative charge, an epoxy, or a mercapto in step 1001. Since step 1001 is the same as step 902 described above, detailed description is omitted.
    The aqueous hydrogel solution is applied to the surface of the transducer in step 1002. When the aqueous hydrogel solution is applied to the surface of the transducer, the thickness of the hydrogel functional layer can be adjusted by adjusting the water level.
    In step 1003, an initiator is added to the aqueous hydrogel solution in order to form a hydrogel functional layer in the form of a gel mass. Because the manner of adding the initiator is the same as that shown in step 1003 of FIG. 7, the detailed description is omitted.
    According to one aspect, it can further include modifying the surface of the hydrogel functional layer by forming a binding mediator on the surface of the hydrogel functional layer.
    According to another aspect, it can further include the step of modifying the surface of the hydrogel functional layer using at least one of nanoparticles and protein as a coupling fragment to form a binding medium on the surface of the hydrogel functional layer.
    As described above, the binding mediator may include at least one of a receptor, a ligand, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and two or more selected from a receptor, a ligand, deoxyribonucleic acid (DNA) or Ribonucleic acid (RNA) are selected to be mixed.
    Since the modification of the surface of the hydrogel functional layer has already been described above, a detailed description thereof will be omitted.
    <Experimental example of the biochip>
    Applicants used a “biochip that includes a gold thin film with a surface modified with PEG / COOH”, which is commonly used as a biochip for SPR sensors, and a “biochip that has one on a surface of gold - Contains a thin film formed hydrogel functional layer ", which is produced by a special technology. The two types of biochips were each mounted in Reichert® SPR sensors, which are analytical instruments. The biochip including the hydrogel functional layer formed on the surface of the gold thin film has been described in detail with reference to FIG. 6, and therefore its detailed description will be omitted.
    The “biochip containing the hydrogel functional layer formed on the surface of the gold thin film” was modified using a crystallized cystamine dihydrochloride to form a hydrogel functional layer.
    A binding mediator was formed on the “biochip including the gold thin film with the surface modified with PEG / COOH” and on the “biochip including the hydrogel functional layer formed on the surface of the gold thin film” . They are specially designed so that when the state of binding is measured after the introduction of the target protein, either a single bond or a multiple bond is generated.
    Thus, when the target protein is introduced into the two types of biochips, the following four cases may arise if both the biochip type and the protein binding state are taken into account: 1) Simple bindings of protein to the “biochip that contains the gold thin film with the surface modified with PEG / COOH ”, 2) simple bonds of protein to the“ biochip, which contains the hydrogel functional layer formed on the surface of the gold thin film ”; 3) multiple bonds of protein to the “biochip that contains the gold thin film with the surface modified with PEG / COOH”; and 4) multiple bonds of protein to the “biochip containing the functional hydrogel layer formed on the surface of the gold thin film”.
    The same concentration of target protein was delivered to each of the two types of biochips described above. Binding reactions between the target protein and the binding mediators were started in each biochip and after a certain time the results were measured using the SPR sensor.
    FIG. 11 is a diagram illustrating a performance test result of a biochip according to an embodiment.
    The left side of FIG. 11 (a) illustrates the response magnitude 317 of the SPR sensor in case 1). The right side of FIG. 11 (a) illustrates the response magnitude 226 of the SPR sensor in case 2).
    The left side of FIG. 11 (b) illustrates the response magnitude 287 of the SPR sensor in case 3). The right side of FIG. 11 (b) illustrates the response magnitude 504 of the SPR sensor in case 4).
    Referring to the graphs on the left of FIGS. 11 (a) and (b), in the case of using the commonly used “biochip which contains the gold thin film with the surface modified with PEG / COOH”, it is not possible, based on the observed magnitude of the SPR response, between single and multiple Distinguish ties. The magnitude of the SPR response for single bonds is rather higher than that for multiple bonds, and it can act as noise when attempting to measure multiple bonds.
    Referring to the graphs on the right of FIGS. 11 (a) and (b), in the case of using the “biochip which contains the hydrogel functional layer formed on the surface of the gold thin film”, a meaningful response of the SPR sensor is shown, which is able to distinguish between the simple and multiple ties. In other words, it was observed that the magnitude of the SPR response in the region in which the binding medium was formed in the hydrogel functional layer was greater in the case of multiple bonds than in the case of single bonds. In the experimental example above, the SPR response due to multiple bonds can be calculated by subtracting the magnitude of the SPR response observed in the PEG / COOH layer from the magnitude of the SPR response in multiple bonds of the region of the Hydrogel functional layer with formed receptor can be obtained. This result confirms that the amount of multiple bonds can be easily judged even when any protein of which the multiple bond and single bond states are not known is applied to the target protein.
    Although the embodiments have been described by the limited embodiments and drawings described above, various modifications and variations from the foregoing description are possible for those skilled in the art. For example, adequate results can be achieved if the preceding techniques are carried out in a different order than the method described and / or the components described are combined or combined in a different form than the method described or are substituted or exchanged for other components or equivalents.
    Therefore, with respect to the claims, various implementations, alternative embodiments thereto and their equivalents are contained within the scope of protection of the following claims.
    权利要求:
    Claims (10)
    [1]
    1. Biochip comprising:a hydrogel functional layer, on the surface of which a binding mediator is linked, and with physical properties which are changed by a reaction between a target protein to be introduced and the binding mediator; anda transducer which supplies a displacement signal, which corresponds to a change in one of the changeable physical properties of the hydrogel functional layer, to an analysis instrument,wherein the hydrogel functional layer comprises a copolymer formed from a main monomer and a comonomer and a crosslinking agent, and the main monomer 55 to 98% of the hydrogel functional layer, the comonomer 2 to 40% of the hydrogel functional layer and the crosslinking agent 0.1 to 5% make up the hydrogel functional layer, with the proviso that the sum of the monomers is 100%,wherein the reaction involves multiple bonds between the target protein and the binding mediator and swelling occurs in at least one part of the hydrogel functional layer due to the multiple bonds,wherein the changed physical property is the refractive index of at least part of the hydrogel functional layer and the refractive index is changed by the de-swelling, andwherein the binding mediator is a ligand, a receptor, a deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA), andthe binding mediator being linked to the surface, if desired, by means of a protein as a coupling fragment.
    [2]
    2. biochip according to claim 1, whereinthe transducer comprises a waveguide, andthe displacement signal is an output signal of the waveguide.
    [3]
    3. biochip according to claim 1, whereinthe transducer comprises a gold thin film, andthe displacement signal is a signal corresponding to a surface plasmon resonance (SPR) occurring in the gold thin film.
    [4]
    4. biochip according to claim 1, whereinthe main monomer is selected from the group consisting of N-isopropyl acrylamide, N-acryloylglycine amide, vinyl caprolactam and vinyl methyl ether, andthe comonomer are selected from the group consisting of allylamine (AA), dimethylaminoethyl methacrylate (DMAEMA), dimethylaminoethyl acrylate (DMAEA), acrylic acid (AAc) and methacrylic acid (MAAc).
    [5]
    5. Biochip according to claim 1, wherein the hydrogel functional layer is at least one copolymer selected from the group consisting of poly (N-isopropylacrylamide-co-allylamine), (poly (NIPAM-co-AA)), poly (N-isopropylacrylamide -co-2- (dimethylamino) ethyl methacrylate), (poly (NIPAM-co-DMAEMA)), poly (N-isopropyl acrylamide-co-2- (dimethylamino) ethyl acrylate), (poly (NIPAM-co-DMAEA)), poly (N-isopropylacrylamide-co-acrylic acid), (poly (NIPAM-co-AAc)), poly (N-isopropylacrylamide-co-polyethylene glycol-acrylic acid), (poly (NIPAM-co-PEG-AAc)), poly (N -isopropylacrylamide-co-methacrylic acid), (poly (NIPAM-co-MAAc)) and hydroxypropyl cellulose is selected.
    [6]
    6. biochip according to claim 1, whereinthe surface of the hydrogel functional layer is made of nanoparticles and / or is modified using protein as a coupling fragment.
    [7]
    7. Biochip according to claim 1, wherein the hydrogel functional layer is divided into at least two regions for reaction with the target protein.
    [8]
    8. The method of manufacturing a biochip of claim 1, wherein the method comprises:Synthesizing a hydrogel in the form of nanoparticles;Activating a surface of the transducer with positive charge, negative charge, epoxy and / or mercapto using aminosilane, carboxysilane, epoxysilane and / or mercaptosilane;Forming the hydrogel functional layer by applying the hydrogel to the surface of the transducer; andModifying a surface of the hydrogel functional layer by linking the binding mediator to the hydrogel functional layer.
    [9]
    9. The method according to claim 8, wherein the modification of the surface of the hydrogel functional layer is carried out using protein as a coupling fragment.
    [10]
    10. The method of manufacturing a biochip of claim 1, wherein the method comprises:Activating the surface of the transducer with positive charge, negative charge, epoxy and / or mercapto using aminosilane, carboxysilane, epoxysilane and / or mercaptosilane;Applying an aqueous hydrogel solution to the surface of the transducer; andForming the hydrogel functional layer in the form of a gel mass by adding an initiator to the aqueous hydrogel solution; andModifying the surface of the hydrogel functional layer by linking the binding mediator to the hydrogel functional layer.
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    同族专利:
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    US10359422B2|2019-07-23|
    GB2560293A|2018-09-05|
    DE112016006111T5|2018-09-06|
    US20190302106A1|2019-10-03|
    GB2560293B|2020-04-01|
    KR20170081152A|2017-07-11|
    KR101754774B1|2017-07-06|
    CN108463725A|2018-08-28|
    GB201811528D0|2018-08-29|
    US20180149641A1|2018-05-31|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    KR20150188729|2015-12-29|
    KR20160038665|2016-03-30|
    KR20160081008|2016-06-28|
    KR1020160149507A|KR101754774B1|2015-12-29|2016-11-10|Biochip and Method of manufacturing the Biochip|
    PCT/KR2016/015172|WO2017116078A1|2015-12-29|2016-12-23|Biochip and method for manufacturing biochip|
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