KR20120130731A - Enzyme-immunoassay complex and manufacturing method thereof - Google Patents

Abstract

PURPOSE: A complex for an enzyme-immunoassay is provided to integrate a large amount of enzymes and to improve enzyme stability. CONSTITUTION: A complex for an enzyme-immunoassay comprises: an enzyme agglomerate which is supported in a pore between fibers of a fiber matrix containing three-dimensional network fibers; a fiber matrix complex with an enzyme-three dimentional network structure containing a shell; and an antibody which is conjugated on the complex. The three-dimensional network fiber is a microfiber or nanofiber. The shell has multiple layers. The enzyme is horse radish peroxidase.

Description

Enzyme-immunoassay complex and manufacturing method

The present invention relates to a complex for measuring enzyme immunity and a method for manufacturing the same, and more particularly, to maximize the amount of immobilized enzyme, while minimizing the generation of signals due to non-specific binding enzymes that can be optimally used in immunoassay systems The present invention relates to a measuring complex and a method of manufacturing the same.

Immunoassay is a generic term for techniques for detecting biomolecules. In general, it can be said to be a technology that tells the presence of microorganisms such as DNA and proteins or bacteria using specific binding such as hybridization of DNA and antibody-antigen binding. One of these immunoassays is the Enzyme-Linked ImmunoSorbent Assay (ELISA), which enables the presence and quantification of various antigens, such as viruses and pathogens, by using the specificity of the antibody antigen, substrate selectivity and catalytic reaction. It is a kind of immunoassay used to do. In order to detect the antigen to be analyzed, enzyme conjugated antibody is used in enzyme-linked immunosorbent assay. Compared to radioactive labels, which have been used for immunoassays since the 1960s, enzyme labels can be expected to have high efficiency due to substrate selectivity and catalytic reaction of enzyme itself, Safety is also excellent. Based on these advantages, two groups, Perrlmann and Schuurs, independently announced the enzyme-linked immunosorbent assay system to replace the radioimmunoassay (RIA) in 1971. Oraganon Teknika succeeded in commercializing the world's first commercially available enzyme-linked immunosorbent assay system for detecting hepatitis B surface antigen (HBsAg) (Haas, H. and Hotz, G., J.). Virol.Methods, 2, 1980). Enzyme-linked immunosorbent assays are still one of the most widely used techniques in diagnostic kits.

Conventional enzyme-linked immunosorbent assays are based on a mechanism that indicates the amount of antigen detected by an enzyme-labeled antibody as a signal of an enzyme reaction. However, in the existing enzyme-linked immunosorbent assay, there was no limit to improving sensitivity by binding one enzyme to one antibody. This is difficult to proceed with the immunoassay because some of the antigens that require a small amount of detection, there was a lot of errors in determining whether or not to be harmful enough or not. Incidentally, due to instability of the enzyme activity, even though the antigen was correctly detected through the antibody, it was difficult to obtain a consistent signal through the enzyme bound to the antibody according to the surrounding environment. Therefore, high stability and high sensitivity immunoassay is urgently needed for consistent results and improved signal detection.

In this context, various studies have suggested efforts to achieve high sensitivity immunoassays. As the most basic method, studies have been conducted to increase the number of enzymes per unit antibody. However, when maximizing the amount of enzymes, there is no problem that signals due to nonspecific binding are generated even when the size of the complex increases and there is no target antigen. In addition, most of these complexes have never considered stability. There is an urgent need for alternatives to solve these two problems.

The present invention has been made to solve the above problems, the problem to be solved of the present invention is to maximize the amount of the enzyme fixed, while minimizing the generation of signals due to non-specific binding enzymes that can be optimally utilized in immunoassay systems The present invention provides a complex for immunoassay, a method for preparing the same, and an immunoassay system.

In order to solve the above problems, the present invention, the cross-linked enzyme aggregate and the surface of the three-dimensional network fibers carried in the inter-fiber pores of the fiber matrix containing the three-dimensional network fibers and the diameter is larger than the inlet size of the pores A fibrous matrix complex of an enzyme-three-dimensional network structure comprising a shell comprising cross-linked enzymes surrounding; It provides a complex for measuring enzyme immunity; and an antibody conjugated to the complex.

According to a preferred embodiment of the present invention, the three-dimensional network fibers may be microfibers or nanofibers.

According to another preferred embodiment of the present invention, the three-dimensional network fibers are polyvinyl alcohol, polyacrylonitrile, nylon, polyester, polyurethane, polyvinyl chloride, polystyrene, cellulose, chitosan, polylactic acid, Polylactic-co-glycolic acid, polyglycolic acid polycaprolactone, collagen, polypyrrole, polyaniline and poly (styrene-co-maleic anhydride).

According to another preferred embodiment of the present invention, the shell may be a multilayer.

According to another preferred embodiment of the present invention, the enzyme is trypsin, trypsin, subtilisin, papain, lipase, holceradish peroxidase, soybean peroxidase, chloro peroxidase, manga Naese peroxidase, tyrosinase, laccase, cellulase, silanase, lactase, sucrase, organophosphohydrolase, chlorine terase, glucose oxidase, alcohol dehydrogenase, glucose dehydrate At least one selected from the group consisting of rosenase, hydrogenase and glucose isomerase.

According to another preferred embodiment of the present invention, the enzyme may be a holceradish peroxidase.

According to another preferred embodiment of the present invention, the method comprises the steps of: (1) adding an enzyme to a matrix comprising three-dimensional network fibers; (2) adding a precipitation agent to the matrix to prevent leakage of enzymes added into the pores of the matrix; (3) preparing an enzyme-fiber matrix complex by adding a crosslinking agent to form an enzyme aggregate to form crosslinks between the enzymes; And (4) conjugating the antibody to the complex.

According to another preferred embodiment of the present invention, the precipitation agent is methanol, ethanol, 1-propanol, 2-propanol, butyl alcohol, acetone, polyethylene glycol, ammonium sulfate, sodium chloride, sodium sulfate, sodium phosphate, potassium chloride , Potassium sulfate, potassium phosphate and aqueous solutions thereof may be single or mixed.

According to another preferred embodiment of the present invention, the crosslinking agent is diisocyanate, dianhydride, diepoxide, dialdehyde, diimide, 1-ethyl-3-dimethyl aminopropylcarbodiimide, glutar dialdehyde It may comprise any one or more compounds selected from the group consisting of, bis (imido ester), bis (succinimidyl ester) and diacid chloride.

According to another preferred embodiment of the present invention, the step of removing the precipitation agent and the crosslinking agent between the step (3) and (4).

According to another preferred embodiment of the present invention, step (1) may be an enzyme is adsorbed or covalently bonded to the matrix surface.

The enzyme immunoassay complex of the present invention can accumulate a large amount of enzyme as compared to other conventional complexes. In addition, the three-dimensional network structure of the nanofiber-based polymer can effectively protect the enzyme from external shock, thereby improving the stability of the enzyme.

In addition, by synthesizing the nanofiber-based polymer in the process of synthesizing the enzyme immunoassay complex and the conditions in the enzyme immobilization process to provide a fiber matrix complex of the enzyme three-dimensional network structure of various sizes. This provides the universality to amplify the signal while minimizing the nonspecific binding compared to other complexes that have to be fixed in size during the immunodetection process, which causes nonspecific binding to the substrate depending on the physical properties of the complex.

Therefore, when using the enzyme immunoassay complex of the present invention in the field of immunodetection, it is possible to improve the signal than when using a conventional single enzyme, by minimizing non-specific binding and improve the signal by synthesizing complexes of various sizes It can provide optimized results. Various platforms can also provide optimized results that minimize nonspecific binding and enhance signal.

Figure 1 is a schematic diagram showing the difference between the ELISA method using a conventional single enzyme and the ELISA method according to the present invention.
Figure 2a is a graph showing the synthesis of polyaniline nanofibers of various sizes depending on the amount of aniline added, Figure 2b shows the activity of the fiber matrix complex of the antibody-enzyme-three-dimensional network structure synthesized in polyaniline nanofibers of various sizes It is a graph.
Figure 3a is a graph comparing the enzyme activity of the fiber matrix complex of the three-dimensional enzyme-3D network structure synthesized as above, Figure 3b is a graph confirming the stability of the enzyme activity.
Figure 4a is a result of confirming the synergistic effect of the signal according to the detection of the target antigen hIgG at a specific concentration using the EAC and EAPC as a fiber matrix complex of the enzyme three-dimensional network structure, Figure 4b is magnetic This is confirmed by the nanoparticle-based immunoassay method.
Figure 5a is a result of confirming the signal according to the concentration of the target antigen hIgG using the EAC and EAPC as a fiber matrix complex of the enzyme three-dimensional network structure by a well plate-based immunoassay method, Figure 5b is a magnetic nanoparticle-based immunity The result confirmed by the measuring method.
Figure 6a is a result of confirming the selective binding ability in the immune response using the EAC and EAPC as a fiber matrix complex of the enzyme three-dimensional network structure by a well plate-based immunoassay method, Figure 6b is a magnetic nanoparticle-based immunoassay This is the result of checking the selective binding capacity by the method.
Figure 7 is a perspective view and a cross-sectional view of the polyaniline nanofiber matrix composite of the enzyme three-dimensional network structure according to an embodiment of the present invention.
8 is a schematic diagram showing a step of immobilizing an enzyme on a fiber matrix.

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

As described above, in the conventional enzyme-linked immunosorbent assay, the amount of the enzyme bound to the antibody was limited, so that the signal was weak even after reacting with the substrate, so it was difficult to measure the antigen at low concentration. The reliability of one signal was bound to decline. In addition, although various studies have been conducted to increase the number of enzymes per unit antibody, as the size increases, there is no problem that signals generated by nonspecific binding occur even in the absence of the target antigen.

Thus, in the present invention, the crosslinked enzyme aggregate carried in the interfiber pores of the fiber matrix including the three-dimensional network fibers and having a diameter larger than the inlet size of the pores and the crosslinked enzyme surrounding the surface of the three-dimensional network fibers. Fiber matrix complex of enzyme-three-dimensional network structure comprising a shell comprising a; And an antibody conjugated to the complex to provide a complex for measuring enzyme immunity. This allows the accumulation of a large amount of enzyme compared to other conventional complexes. In addition, the three-dimensional network structure of the nanofiber-based polymer can effectively protect the enzyme from external shock, thereby improving the stability of the enzyme.

First, the enzyme immunoassay complex of the present invention will be described. The enzyme immunoassay complex of the present invention comprises the steps of: (1) adding an enzyme to a matrix comprising three-dimensional network fibers; (2) adding a precipitation agent to the matrix to prevent leakage of enzymes added into the pores of the matrix; (3) preparing an enzyme-fiber matrix complex by adding a crosslinking agent to form an enzyme aggregate to form crosslinks between the enzymes; And (4) conjugating the antibody to the complex.

First, as step (1), the enzyme is adsorbed and / or covalently bonded to the porous matrix including the three-dimensional network fibers. If the surface of the three-dimensional network fiber contains a functional group capable of covalently bonding with the enzyme (modified for this), a covalent bond occurs between the enzyme and the fiber and is adsorbed if it does not contain it.

Fibers used in the present invention can be used without limitation as long as the three-dimensional network is formed during the spinning can form voids between the fibers and fibers, polyvinyl alcohol, polyacrylonitrile, nylon, polyester, Polyurethane, polyvinyl chloride, polystyrene, cellulose, chitosan, polylactic acid, polylactic-co-glycolic acid, polyglycolic acid polycaprolactone, collagen, polypyrrole, polyaniline and poly (styrene-co-maleic anhydride) It may be any one or more selected from the group consisting of, more preferably in consideration of cost and efficiency it is advantageous to use polyaniline nanofibers, but is not limited thereto.

If the type of spinning for producing the fiber also has a three-dimensional network structure between the fiber and the fiber can utilize a conventional polymerization and / or spinning process, it can be used both electrospinning, melt spinning and the like. The diameter of the fiber may also be applied to both nanofibers and microfibers, but it may be more advantageous to use nanofibers in consideration of the size of the enzyme assembly described below and the size of the pores formed between the fiber and the fiber. However, carbon nanotubes do not fall within the scope of the present invention because they cannot form a three-dimensional network even if they are woven into fibers.

The porous matrix according to the embodiment of the present invention may be configured to include some or all of the three-dimensional network fibers, wherein the porosity means the space (pore) between the fibers and the fibers forming the three-dimensional network.

The fiber of the present invention forms a three-dimensional network and can have a fiber matrix structure, and the matrix structure may be an amorphous structure in which fibers are intricately intertwined.

Enzymes included in the present invention can be used without limitation as long as it can be used in the enzyme immunoassay, preferably, trypsin, trypsin, subtilisin, papain, lipase, holceradish peroxidase, soybean peri Oxidase, chloro peroxidase, manganese peroxidase, tyrosinase, laccase, cellulase, silanase, lactase, sucrase, organophosphohydrolase, chlorinease, At least one selected from the group consisting of glucose oxidase, alcohol dehydrogenase, glucose dehydrogenase, hydrogenase and glucose isomerase. On the other hand, more preferably, the activity of the horseradish peroxidase is very excellent.

On the other hand, since the fiber matrix of the present invention can be applied to a fiber that substantially contains or does not contain any functional group (eg, an amino group) that can be covalently bonded to the enzyme on its surface, the fiber matrix is covalently bonded between the surface of the fiber and the enzyme. It may be bound or adsorbed between the surface of the fiber or the voids (space) formed between the fiber and the fiber. Therefore, when an external shock such as water washing is applied simply while the enzyme is adsorbed on the surface of the matrix, most of the adsorbed enzymes are separated from the matrix, and as a result, the fixation rate of the enzyme is significantly reduced.

As a next step (2), an enzymatic precipiatation is added to the matrix to prevent leakage of enzymes adsorbed and / or covalently bound into the pores of the matrix. Adsorbed enzymes are so small in size that they are hardly observed with the naked eye. Accordingly, when a precipitation agent is added to the matrix to precipitate the adsorbed enzymes, the adsorbed enzymes agglomerate with each other to increase their size, thereby eventually depositing between pores (spaces) formed between the surface of the fiber or between the fiber and the fiber. Done. This allows the formation of cross-linked enzyme aggregates with diameters larger than the inlet size of the inter-fiber pores, and if no precipitation agent is added, the aggregate size is smaller than the pore inlet size and the aggregates are then easily The gap between the gaps will be.

Precipitating agents that can be used at this time can be used without limitation as long as it can precipitate the enzyme with little effect on the activity of the enzyme, but preferably methanol, ethanol, 1-propanol, 2-propanol, butyl alcohol, Acetone, polyethylene glycol, ammonium sulfate, sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate, potassium phosphate and aqueous solutions thereof may be used alone or in combination, but is not limited thereto.

Next, in step (3), a crosslinking agent is added to form a crosslink between the enzymes to form an enzyme aggregate. Specifically, since the size of the precipitated enzyme is relatively small compared to the size of the pores (spaces) formed between the fibers, the precipitated enzyme may be leaked to the outside of the pores by external impact such as water washing. However, when the crosslinking agent is added to form a crosslink between the precipitated enzyme and the enzyme, the crosslinked enzymes form an aggregate, and the formed enzyme aggregate almost fills the interior of the pores. As a result, the size of the enzyme aggregate formed is larger than the inlet of the pores, so that the cross-linked enzyme aggregate does not easily flow out of the pores even by an external impact such as washing with water. As a result, even if time passes, the enzyme aggregate is located in the pores, so that even if a direct binding relationship such as covalent bond between the fiber and the enzyme is not formed, the enzyme aggregate can be provided in the fiber matrix for a long time. In addition, the complex formed between the enzyme three-dimensional network fiber matrix has a much higher amount of fixed enzyme than the conventional complex, so even when the complex is used in a biosensor or a biofuel cell, the performance of the complex is increased. It can be significantly improved compared to.

On the other hand, adding a crosslinking agent after treating the precipitation agent significantly improves the effect as compared with the case where only the crosslinking agent is added. When the crosslinking agent is added after the adsorption of the enzyme, the concentration of the enzyme becomes equal to the ambient concentration even if the inside of the pores formed between the fibers and the fibers is not filled or the filling of the pores. Enzymes of the same concentration crosslink and do not form a larger mass in the pores in the nanofiber than the inlet of the pores, so the crosslinked enzymes are likely to leak out during the washing process. Covalent bonds, except for covalent enzymes, do not form large masses and are more likely to be washed later.

However, in the case of the present invention, the enzymes are forced to fill the pores in the nanofibers more densely by precipitation after the adsorption of the enzymes, and the enzymes filled in the cooperatives form a large mass through crosslinking, and thus, as a bottleneck or ship in a bottle phenomenon. It is expected that losses will be less than EAC in the wash process.

Furthermore, in the case of polyaniline nanofibers having little functional group capable of covalently bonding with enzymes on the surface of the fiber, it is very difficult for the enzyme to be immobilized on the surface of the fiber. However, in the present invention, since the enzymes precipitated on the surface of the fiber are crosslinked to form a shell surrounding the surface of the fiber, even if a covalent bond is not substantially formed between the fiber and the enzyme like a hot dog, a large amount on the surface of the fiber Can be immobilized by forming a shell.

The crosslinking agent that can be used in the present invention can be used without limitation as long as it can form crosslinking between enzymes without inhibiting the activity of the enzyme, but is preferably diisocyanate, dianhydride, diepoxide, dialdehyde, At least one compound selected from the group consisting of diimide, 1-ethyl-3-dimethyl aminopropylcarbodiimide, glutar dialdehyde, bis (imido ester), bis (succinimidyl ester) and diacid chloride It may be used, including, but more preferably, glutar dialdehyde may be used, but is not limited thereto, it will be apparent to those skilled in the art to use without limitation cross-linking agents known in the art.

On the other hand, after the step (3), preferably, the process of removing the added cross-linking agent and precipitation agent by washing the enzyme three-dimensional network fiber matrix complex may be further performed. In the case of the matrix complex prepared by the conventional manufacturing method during the washing step, the enzyme immobilized between the pores flows out of the pores, but the enzyme-3D network fiber matrix complex prepared by the present invention is formed inside the pores. Since the size of the enzyme aggregate is larger than the inlet of the pores, the enzyme aggregate can be fixed inside the pores without flowing out despite the washing process.

Next, as step (4), the antibody is conjugated to the matrix containing the enzyme. In this case, the antibody to be used can be used in various ways according to the target antigen, and it is also possible to conjugate one antibody or a mixture of several antibodies.

Antibodies are conjugated to the matrix by the functional groups of the remaining crosslinker used in the preparation of the enzyme-3D network fiber matrix complex.

The composite of the present invention thus prepared is carried in the interfiber pores of the fiber matrix including the three-dimensional network fibers and surrounds the surface of the cross-linked enzyme aggregate and the surface of the three-dimensional network fibers having a diameter larger than the inlet size of the pores. Fiber matrix complexes of an enzymatic three-dimensional network structure comprising a shell comprising crosslinked enzymes; And antibodies conjugated to the complex.

Figure 7 is a perspective view and a cross-sectional view of the polyaniline nanofiber matrix composite of the enzyme three-dimensional network structure according to an embodiment of the present invention. Looking at this, a crosslinked enzyme aggregate having a diameter larger than the inlet size of the pores is carried in the pores of the fiber matrix of the three-dimensional network structure. In addition, a shell containing an enzyme aggregate surrounds the surface of the three-dimensional network fiber. 7 is a cross-sectional view of a portion of the shell of the three-dimensional network fibers wrapped around the shell of the three-dimensional network fibers as if the covalent bond is not substantially formed between the fibers and the enzyme aggregate forming the shell. Shells containing enzyme aggregates, such as hot dogs, surround the surface of three-dimensional network fibers.

As a result, the enzyme three-dimensional network fiber matrix complex of the present invention is significantly larger than the conventional enzyme complex because the size of the enzyme complex formed inside the pores is large enough so that the pores formed between the fibers and the fibers do not flow out. Positive enzymes can be immobilized on the matrix to form complexes. In other words, the size of the enzyme complex formed inside the pores is larger than the size of the inlet of the pores, which are the outflow passages from the pores formed between the fibers and the fiber, so that even if there is an external stimulus such as water washing, The complex can be maintained for a long time without forming a direct binding relationship between the enzyme and the matrix.

Furthermore, in the case of polyaniline nanofibers having little functional group capable of covalently bonding with enzymes on the surface of the fiber, it is very difficult to fix the enzyme on the surface of the fiber. However, in the present invention, since the enzymes precipitated on the surface of the fiber are crosslinked to form a shell surrounding the surface of the fiber, even if a covalent bond is not substantially formed between the fiber and the enzyme like a hot dog, a large amount on the surface of the fiber The enzyme of may form a shell and may be immobilized, and such shell may form a multilayer.

Figure 1 is a schematic diagram showing the ELISA method using the complex of the present invention, can have a significantly improved effect compared to the use of a conventional single enzyme.

Therefore, the enzyme immunoassay complex of the present invention can accumulate a large amount of enzyme as compared to other conventional complexes. In addition, the three-dimensional network structure of the nanofiber-based polymer can effectively protect the enzyme from external shock, thereby improving the stability of the enzyme.

In addition, by synthesizing the nanofiber-based polymer in the process of synthesizing the enzyme immunoassay complex and the conditions in the enzyme immobilization process to provide a fiber matrix complex of the enzyme three-dimensional network structure of various sizes. This provides the universality to amplify the signal while minimizing the nonspecific binding compared to other complexes that have to be fixed in size during the immunodetection process, which causes nonspecific binding to the substrate depending on the physical properties of the complex.

Therefore, when using the enzyme immunoassay complex of the present invention in the field of immunodetection, it is possible to improve the signal than when using a conventional single enzyme, by minimizing non-specific binding and improve the signal by synthesizing complexes of various sizes It can provide optimized results. Various platforms can also provide optimized results that minimize nonspecific binding and enhance signal.

Preparation Example 1 Size Change According to Synthesis and Conditions of 3D Polymer Nanofibers

Specifically, the polyaniline-based three-dimensional polymer nanofibers were prepared by oxidative polymerization using ammonium persulfate as an initiator. At this time, the growth of polyaniline may be controlled according to the amount of ammonium persulfate and the concentration of initial aniline, and when the initial aniline concentration is prepared as shown in FIG. 2 while the amount of ammonium peroxide is fixed, polyaniline nanofibers of various sizes are synthesized. Can be. 10 ml of the prepared ammonium persulfate solution was added to a 10 ml solution of aniline dissolved in HCl solution and mixed well. After this, the mixed solution was stirred for 24 hours at room temperature at 200 rpm. After the polymerization process was repeated several times the process of washing the solution with distilled water to neutralize the acidic solution to increase the pH to neutral, and then stored at 4 ℃.

Dynamic light scattering was used to confirm the size of the 3D polymer nanofibers synthesized through the above process. As a result, the size of the three-dimensional polymer nanofibers synthesized at the lowest initial aniline concentration (0.5% by weight) increases up to 700 nm, and as the initial aniline concentration increases, the size continues to decrease. When the concentration is 7.5% by weight, the size of the polymer nanofibers was confirmed to be reduced to 160 nm. In addition, solidification occurred when the initial concentration of aniline exceeded 10% by weight. These results indicate that by controlling the size of the fiber matrix complex of the enzyme three-dimensional network structure while changing simple conditions, it is possible to provide a fiber matrix complex of the enzyme three-dimensional network structure selectively according to its use. In order to proceed with the enzyme-linked immunosorbent assay as the use of the present invention, a concentration of 7.5% by weight of the initial aniline concentration, which is the smallest condition among these results, was used.

Preparation Example 2 Preparation of Polyaniline Nanofiber Matrix of 3D Network Structure

Specifically, the polyaniline-based three-dimensional polymer nanofibers were prepared by dissolving ammonium persulfate, an oxidizing agent, to 0.1 M in 1 M HCl solution, and aniline solution and 1 M hydrochloric acid, respectively 3: Put it in the ratio of 17 and mix well. 10 ml of the prepared ammonium persulfate solution was added to a 10 ml solution containing aniline and HCl and mixed well. After this, the well mixed solution was stirred for 24 hours at room temperature at 200 rpm. After the polymerization process was repeated several times the process of washing the solution with distilled water to neutralize the acidic solution to increase the pH to neutral, and then stored at 4 ℃. The polyaniline nanofibers thus synthesized are intricately connected to each other like corals to form a three-dimensional network structure, and depending on the concentration of aniline, as well as the pores of the outer branches of the nanofibers, there are many pores inside.

Comparative Example 1 Preparation of Fiber Matrix Complex of Enzyme-3D Network Structure According to EA

The polyaniline nanofiber matrix composite of the enzyme three-dimensional network structure was prepared according to enzyme adsorption (EA) for comparison with the case where the precipitation agent of FIG. 8 was added. Specifically, the mixture was mixed with a 1 mg solution of HRP (Horseradish peroxidase) at 10 mg / ml in the polyaniline nanofiber solution (initial aniline concentration of 7.5 wt%) prepared in Preparation Example 2, and then stirred at 150 rpm for 1 hour to be adsorbed. EA was prepared as a fiber matrix composite of -3D network structure. The prepared EA solution was centrifuged and washed with PB buffer to remove enzymes remaining without reaction.

Comparative Example 2 Preparation of Fiber Matrix Complex of Enzyme-3D Network Structure According to EAC

The polyaniline nanofiber matrix composite of the enzyme three-dimensional network structure was prepared according to enzyme adsorption and crosslinking (EAC) in order to compare the case with the precipitation agent of FIG. 8. Specifically, the mixture was mixed with a 1 mg solution of HRP (Horseradish peroxidase) of 10 mg / ml in the polyaniline nanofiber solution prepared in Preparation Example 2 (initial aniline concentration of 7.5 wt%), and the mixture was stirred at 150 rpm for 1 hour to be adsorbed well. Thereafter, the concentration of glutar dialdehyde as a crosslinking agent was added to 0.5%, followed by reaction for 1 hour in a refrigerator at 4 ° C. for sufficient reaction of the crosslinking agent to prepare an EAC as a fiber matrix complex having an enzyme three-dimensional network structure. It was. The prepared EAC solution was centrifuged and washed with PB buffer to remove the remaining enzymes without reacting.

Example 1 Preparation of Fiber Matrix Complex of Enzyme-3D Network Structure According to EAPC

The polyaniline nanofiber matrix composite of the enzyme three-dimensional network structure was prepared according to Enzyme adsorption and crosslinking (EAPC) of FIG. 8. Specifically, in the polyaniline nanofiber solution prepared in Preparation Example 2 (initial aniline concentration of 7.5% by weight) and mixed with a solution of 1 ml volume of HRP (Horseradish peroxidase) and GOx (Glucose oxidase) of 10mg / ml so that the adsorption is good Stir at 150 rpm for 1 hour. Thereafter, the concentration of ammonium sulfate as a precipitation agent was added to 50% (w / v), followed by stirring at 200 rpm for 30 minutes at room temperature so that precipitation of the enzyme proceeded sufficiently. Then, to crosslink the precipitated enzyme, the concentration of glutar dialdehyde as a crosslinking agent was added to 0.5% by weight, followed by reaction for 1 hour in a refrigerator at 4 ° C. for sufficient reaction of the crosslinking enzyme. EAPC was prepared as a fiber matrix composite of network structure. Centrifugation and washing of the prepared EAPC solution using PB buffer were repeated to remove enzymes that did not react.

Example 2 Antibody Immobilization of Fiber Matrix Complex of Enzyme-3D Network Structure

The fiber matrix complex of enzyme-3D network structure prepared in Example 1 and Comparative Examples 1 and 2 was mixed with hIgG antibody solution as a model protein for use in enzyme-linked immunosorbent assay and stirred at 200 rpm for 2 hours. . At this time, the antibody reacts with the functional group activated by the crosslinking agent remaining around the enzyme, so that the concentration of the antibody solution and the fiber matrix complex solution of the enzyme three-dimensional network structure is 1: 1 in mass ratio for more efficient reaction. It was made. The prepared antibody-enzyme-three-dimensional network structure of the fiber matrix complex solution using Tris buffer inactivates the remaining functional groups, and centrifugation and rinsing are repeated using the PBS buffer, which is generally used. Antibodies were removed.

Example 3 Determination of Enzyme Activity and Stability of Fiber Matrix Complex of Enzyme-3D Network Structure

The enzyme activity of the fiber matrix complex of each enzyme-three-dimensional network structure prepared through Comparative Example 1 and Example 1 was measured using a UV / Vis spectrometer. Specifically, the concentration of the fiber matrix complex of the enzyme three-dimensional network structure was prepared at a concentration of 10 μg / ml based on the amount of polyaniline nanofibers. To determine the enzyme reactivity of HRP, TMB (3,3,5,5-Tetramethylbenzidine) was used as a substrate, and DMSO (dimethyl sulfoxide) was used as an organic solvent for dissolving it. TMB was first dissolved in DMSO at high concentration and then diluted with phosphate buffer (PB buffer). This is because the enzyme reacts more stable on normal PB buffer than on organic solvent. Thereafter, the fiber matrix complex solution of the enzyme-3D network structure and the TMB reaction solution were mixed at a ratio of 5:95, and then the absorbance was measured through a spectrometer in real time, and the activity of the enzyme was measured through this data.

Figure 2b shows the activity of the fiber matrix complex of the antibody-enzyme-3D network structure synthesized using polyaniline nanofibers of various sizes using a UV / Vis spectrometer. Ab-HRP / EAPC activity (A655 / min) is 0.430, 0.570, and 1.105 when the initial aniline concentrations are 2.5, 5 and 7.5 wt%, respectively. In addition, Figure 3a is a graph measuring the activity of free enzymes, EA, EAC, and EAPC, wherein the activity of EA, EAC, and EAPC (A655 / min) is 1 μg / ml of polyaniline nanofibers, respectively. 0.005, 0.04, and 5.63. The activity of EAPC (Example 1) was more than 1000 times greater than EA and 140 times greater than EAC. FIG. 3b is a graph of continuous measurement and recording of enzyme activity at room temperature and strict stirring at 200 rpm. EA and EAC activity was 2% and 18% of initial activity, respectively, after 60 days, whereas EAPC was 69% of initial activity. You can see it keeps%. It can be said that EAPC is much more stable in activity as a fiber matrix complex of enzyme-three-dimensional network structure using precipitation method compared to simple free enzyme or simple crosslinked EAC approach.

Example 4 Antibody Immobilization of Magnetic Nanoparticles

In order to use magnetic nanoparticles for enzyme-linked immunosorbent assay, hIgG antibody was immobilized as a model protein. First, the magnetic nanoparticles were repeatedly separated and washed twice using sodium hydroxide solution, and then the magnetic separation and washing were repeated twice using distilled water to remove impurities from the surface of the magnetic nanoparticles. In order to replace the carboxyl group present in the magnetic nanoparticles with an amine group, a 16.4 mg / mL EDC (ethyl (dimethylaminopropyl) carbodiimide) solution was mixed and stirred at 200 rpm at 4 ° C. for 30 minutes. After washing with pH 6.0 and 25 mM MES buffer, the model protein hIgG antibody solution was mixed and stirred at 200 rpm for 1 hour. At this time, the antibody was immobilized by reacting with the functional group produced by the EDC solution. The prepared antibody-magnetic nanoparticle solution was inactivated with the remaining functional groups using 0.1% BSA solution (0.1% BSA in 10 mM PBS (pH 7.4)), and magnetic separation and washing using a commonly used PBS buffer. Was repeated to remove the remaining antibodies without reaction.

Example 5 Analysis of Target Antigen Using Well Plate

Enzyme-linked immunosorbent assay using a well plate was performed to confirm what characteristics the fiber matrix complex of the antibody-enzyme-3D network structure prepared in Example 2 had in analyzing the target antigen. More specifically, dilute the antibody (binding antibody) that binds the target antigen to a well plate in a concentration of 10 μg / ml in 100 mM sodium bicarbonate buffer (pH 9.4) and add 50 μl to each well. Binding antibodies were adsorbed onto well plates at 4 ° C. for 12 hours. Thereafter, in order to prevent nonspecific binding of the well plate and the complex, 3 w / v% BSA solution (100 μl) was added to each well and reacted for 1 hour. Remove all the reaction BSA solution and wash each well five times with PBS-T [0.05% Tween-20 in 10 mM PBS (pH 7.4)] solution. To confirm the detection limit, 50 μl of various target antigen solutions (5-0.0001 μg / ml) diluted with PBS solution were added and kept warm at 37 ° C. for 1 hour to induce antigen-antibody binding. Wash 5 times with PBS-T solution to remove unbound target antigen. Subsequently, 50 μl of the fiber matrix complex solution of the antibody-enzyme-three-dimensional network structure that specifically binds the target antigen was diluted in 1 μg / ml in PBS-T solution, and it was put at 37 ° C. for 1 hour 30 Insulate for minutes to finally induce an antibody-antigen binding based bridge between the fiber matrix complex of the enzyme-3D network structure and the well plate. After washing 10 times with PBS-T solution, the remaining fiber matrix complex of antibody-enzyme-3D network structure is removed. After this, 50 μl of the same TMB solution as in Example 3 was added to quantify the target antigen, followed by stirring for 30 minutes. The absorbance was measured at a wavelength of 655 nm through a plate reader.

In order to compare the diagnostic performance of the fiber matrix complex of the enzyme three-dimensional network structure, the experiment was conducted using the EAPC and EAC as described above. Figure 4a is a result of confirming the performance of the target antigen hIgG by using a well plate-based immunoassay using EAPC (Example 1) and EAC (Comparative Example 2) as the fiber matrix complex of the enzyme three-dimensional network structure. As a result of confirming the signal according to the concentration of EAPC rather than EAC based on the concentration of the same polyaniline nanofibers, when the concentration of hIgG as the target antigen was 500 ng / ml, more than six times improved signal was observed. In addition, based on these results, as shown in FIG. 5A, the target antigens of various concentrations were reacted to confirm the detection limit and the detection range of the fiber matrix complex of the enzyme-3D network structure. As a result, it was confirmed that the detection limit of EAPC was 1 ng / mL hIgG (6.7 pM), and the detection range was 1 to 500 ng / ml.

Example 6 Analysis of Antigen Using Magnetic Nanoparticles

Enzyme-linked immunity using the antibody-magnetic nanoparticles prepared in Example 4 to determine what characteristics the fiber matrix complex of the antibody-enzyme-3D network structure prepared in Example 2 has in analyzing the target antigen Adsorption test was performed. First, the prepared antibody-magnetic nanoparticles are washed three times with PBS-T [0.05% Tween-20 in 10 mM PBS (pH 7.4)] solution. In order to confirm the detection limit, 1 ml of the target antigen solution (5-0.0001 μg / ml) diluted with PBS solution was added in 1 ml and stirred at 200 rpm for 1 hour to induce antigen-antibody binding. Wash three times with PBS-T solution to remove unbound target antigen. Subsequently, a solution of the fiber matrix complex of the antibody-enzyme-three-dimensional network structure that specifically binds to the target antigen was diluted in a concentration of 1.5 μg / ml in PBS-T solution, and added in 1 ml each, at 200 rpm for 30 minutes. Stirring finally leads to the formation of bridges based on antibody-antigen binding between the fiber matrix complex of the enzyme-3D network structure and the antibody-magnetic nanoparticles. After washing 4 times with PBS-T solution, the remaining fiber matrix complex of antibody-enzyme-3D network structure is removed. After this, 1 ml of TMB, glucose, and HRP solution were added to quantify the target antigen, followed by stirring at 200 rpm for 30 minutes. The absorbance was measured at 655 nm using a UV / vis spectrometer.

Figure 4b is a result of confirming the diagnostic performance of the target antigen hIgG using the magnetic nanoparticles based immunoassay using EAPC as a fiber matrix complex of the enzyme three-dimensional network structure. In addition, based on the results, as shown in FIG. 5B, the target antigens of various concentrations were reacted to confirm the detection limit and the detection range of the fiber matrix complex of the enzyme-three-dimensional network structure. As a result, it was confirmed that EAPC had a detection limit of 0.1 ng / mL hIgG (0.67 pM) and a detection range of 1 to 500 ng / ml.

Example 7 Confirmation of Selective Binding Ability of Fiber Matrix Complex of Enzyme-3D Network Structure

For the EAPC of Example 2, Figure 6a shows the target antigen as a selective binding ability to three antigens, such as hIgG, rIgG (rabbit immunoglobulin G), mIgG (rat immunoglobulin G) confirmed by well plate-based immunoassay Results 6b is a result confirmed through the magnetic nanoparticle-based far station inspection. As shown in the graph it was confirmed that only selectively reacts with human immunoglobulin G.

The complex for measuring enzyme immunity of the present invention and a method of preparing the same may be usefully used in the field of immunoassay.

Claims (12)

A shell containing a crosslinked enzyme aggregate contained within the interfiber pores of the fiber matrix comprising three-dimensional network fibers and having a diameter larger than the inlet size of the pores and the crosslinked enzymes surrounding the surface of the three-dimensional network fibers. Fiber matrix complex of enzyme-three-dimensional network structure comprising; And
Enzyme immunoassay complex comprising an antibody conjugated to the complex. The complex of claim 1, wherein the three-dimensional network fiber is a microfiber or a nanofiber. The method of claim 2,
The three-dimensional network fibers are polyvinyl alcohol, polyacrylonitrile, nylon, polyester, polyurethane, polyvinyl chloride, polystyrene, cellulose, chitosan, polylactic acid, polylactic-co-glycolic acid, polyglycolic acid Polycaprolactone, collagen, polypyrrole, polyaniline and poly (styrene-co-maleic anhydride) is any one or more selected from the group consisting of enzyme complex immunoassay complex. The complex of claim 1, wherein the shell is a multilayer. The method of claim 1,
The enzyme is mitripsin, trypsin, subtilisin, papain, lipase, holceridish peroxidase, soybean peroxidase, chloro peroxidase, manganese peroxidase, tyrosinase, lacca Aases, Cellulase, Silanese, Lactase, Sucrase, Organophosphohydrolase, Chlorinesterase, Glucose oxidase, Alcohol dehydrogenase, Glucose dehydrogenase, Hydrogenase and Glucose isomerase Enzyme immunoassay complex, characterized in that any one or more selected from the group consisting of. The method of claim 5,
The enzyme is an enzyme immunoassay complex, characterized in that the Holceradi peroxidase. (1) adding an enzyme to a matrix comprising three-dimensional network fibers;
(2) adding a precipitation agent to the matrix to prevent leakage of enzymes added into the pores of the matrix; And
(3) preparing an enzyme-fiber matrix complex by adding a crosslinking agent to form an enzyme aggregate to form crosslinks between the enzymes; And
(4) conjugating the antibody to the complex; a method for preparing an enzyme immunoassay complex comprising a. The method of claim 7, wherein
The precipitation agent is methanol, ethanol, 1-propanol, 2-propanol, butyl alcohol, acetone, polyethylene glycol, ammonium sulfate, sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate, potassium phosphate and aqueous solutions thereof alone Or a method for producing an enzyme immunoassay complex, characterized in that the mixture. The method of claim 7, wherein
The crosslinking agent is diisocyanate, dianhydride, diepoxide, dialdehyde, diimide, 1-ethyl-3-dimethyl aminopropylcarbodiimide, glutar dialdehyde, bis (imido ester), bis (succinimi Dill ester) and diacid chloride, any one or more compounds selected from the group consisting of a method for producing an enzyme immunoassay complex characterized in that it comprises. The method of claim 7, wherein
Method for producing an enzyme immunoassay complex, characterized in that further comprising the step of removing the precipitation agent and cross-linking agent between step (3) and (4). The method of claim 7, wherein
Step (1) is a method for producing an enzyme immunoassay complex, characterized in that the enzyme is adsorbed or covalently bonded to the matrix surface.  Enzyme immunoassay method using the complex of any one of claims 1 to 6.

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