WO2016099096A1 - Organic/inorganic nanocomposite for biochemical material immobilization, method for preparing same, and biosensor or adsorption apparatus comprising same - Google Patents

Abstract

The present invention relates to an organic/inorganic nanocomposite for biochemical material immobilization, a method for preparing the same, and a biosensor or adsorption apparatus comprising the same and, specifically, to an organic/inorganic nanocomposite for biological material immobilization, capable of maintaining activity of the biochemical materials, stably immobilizing a large quantity of biochemical materials, shortening the preparing time, and reducing the preparing costs, to a method for preparing the same, and to a biosensor or adsorption apparatus comprising the same.

Description

 【Specification】

 [Name of invention]

 Organic / inorganic nanocomposite for fixing biochemicals, preparation method thereof and biosensor or adsorption device comprising the same

 Technical Field

 The present invention relates to an organic / inorganic nanocomposite for fixing biochemicals, a method for preparing the same, and a biosensor or adsorption device including the same. Specifically, the present invention can not only stably hold a large amount of biochemicals while maintaining the activity of the biochemicals, but also shorten the manufacturing time and reduce the manufacturing cost of the organic / inorganic nanocomposite for fixing biochemicals, the preparation thereof A method and a biosensor or adsorption device comprising the same.

 [Technique to become background of invention]

 Techniques for fixing biochemicals such as proteins and enzymes are widely used in the development of kits, protein chips and biosensors for diagnosing diseases using indicators. It occupies an important position in the pharmaceutical industry.

 In particular, since enzymes in biochemicals have high catalytic activity and selectivity as biocatalysts, efforts have been made to easily separate them from the solid supports by collecting them after fixing them on a suitable solid support.

 However, when the biochemical is fixed only on the surface of the solid support, the amount of biomolecules fixed by itself is small, or when the biochemical is immobilized through a multilayer fixing thin film formed on the surface of the solid support. There is a problem of denaturation and inactivation, and when the biochemical is used as a fixed solid support self-dispersing biosensor, adsorption device, etc., it is possible to confirm the interaction and recognition reaction of the reaction with the fixed biochemical. It has a downside.

Conventionally, for the above-mentioned purposes, a covalent bond method using an amide bond by immobilizing a biochemical enzyme or protein on a solid surface (M. Zayats, E. Katz, I. Wier, JACS. , 2002, 124, 14724), complex thin film of metal oxide and enzyme using surface sol-gel method (I. Ichinose, R. Takaki, K. Kuroiwa, T. Kunitake, Langmuir, 2003, 19, 3883), 3 The method using the mesoporous molecular sieve of (three) dimensional pore structure (J.M.Kisler, GWStevens, AJ.O 'Connor, Mater. Phys.Mech. 2001, 4, 89), etc. was mainly used.

 However, the covalent bond method by the amide bond has a disadvantage that the biochemical material may lose activity during the covalent bond reaction, since the biochemical must be reacted through the covalent bond.

 In addition, the formation of the laminated thin film by the surface sol-gel method is a simple method using the electrostatic interaction of the metal oxide and the biochemical material, it is possible to uniformly fix the biochemical material on the surface of the metal oxide, metal oxide and biochemical material Biochemicals can be fixed to a plurality of metal oxide thin films laminated through alternating lamination, but the modification of the fixed biochemicals due to the effect of organic solvents used in the adsorption of biochemicals to metal oxides during the multi-layer lamination process The disadvantage is that it is very likely.

 In addition, the method using the mesoporous molecular sieve having a three-dimensional pore structure does not reversibly adsorb / desorb the biochemical to the meso material, and thus has a problem of recovering and recycling the adsorbed biochemical.

Furthermore, as a method of stably fixing a large amount of biochemicals on a solid support, a method of alternately stacking electrolyte polymers and biochemicals using an electrolyte polymer that has an electrostatic interaction with the biochemicals is used (Yuri Lvov _: atsuhiko Ariga , Izumi Ichinose, Toy ok i Kuni take, JACS. 1995, 117, 6117), this method is blocked by the reaction of biochemicals and reactions because the biochemicals are blocked by the polymer layer covering the biochemicals. There is a problem that can not be directly applied to biosensors, adsorption devices.

Meanwhile, Korean Patent Laid-Open Publication No. 10-2010-0128110 discloses a biochemical-fixing nanocomposite formed by alternating stacking of a metal oxide film and an ionic polymer film on a solid support to stably fix a large amount of biochemicals. These nanocomposites are used to fix abundant amounts of biochemicals. In order to stack the multilayer film excessively, there is a problem that the manufacturing time is delayed and the manufacturing cost increases.

 Therefore, there is an urgent need to develop a new biochemical fixing complex that can not only fix a sufficient amount while maintaining the activity of the biochemical, but also shorten the manufacturing time and reduce the manufacturing cost.

 [Content of invention]

 Problem to be solved

 An object of the present invention is to provide a biochemical fixed organic / inorganic nanocomposite, a method for manufacturing the same and a biosensor or adhesion device comprising the same, which can fix a sufficient amount while maintaining the activity of the biochemical.

 In addition, an object of the present invention is to provide an organic / inorganic nanocomposite for fixing a biochemical material, a manufacturing method thereof, and a biosensor or adsorption device including the same, which can reduce manufacturing time and reduce manufacturing cost.

 [Measures to solve the problem]

 In order to solve the above problems, the present invention,

 Solid support; At least one composite thin film including a first metal oxide film formed on the solid support and an electrolyte polymer film formed on the first metal oxide film and being a nanoscale porous film; And a second metal oxide film formed on the composite thin film and being a nanoscale porous film, wherein the polymer forming the electrolyte polymer film has a molecular weight of 500 or more.

 Here, there is provided a nanocomposite for fixing a biochemical, characterized in that the molecular weight of the polymer is 2,000 or more.

 In addition, the solid support provides a nano-composite for fixing a biochemical, characterized in that the first metal oxide film can be formed by the surface sol-gel reaction.

In addition, the surface of the solid support is a plasma treatment, ozone treatment, ultrasonic treatment in an alkali or acid solution and using alkanediol molecules or carboxyl groups Provided is a nanocomposite for fixing a biochemical, characterized in that at least one treatment selected from the group consisting of self-assembled thin film formation treatments has been performed.

 Furthermore, the solid support has at least one surface active group selected from the group consisting of active hydrogen, hydroxyl group, carboxyl group, sulfonic acid group, phosphoric acid group, amine group, imine group, ammonium group, pyridine group and functional groups of charged molecules on its surface It provides a nanocomposite for fixing a biochemical, characterized in that is introduced.

 In addition, the metal oxide forming the first metal oxide film, the second metal oxide film, or both thereof may include at least one metal selected from the group consisting of titanium, zirconium, aluminum, boron, silicon, indium, tin, barium, and vanadium. Provided is a nanocomposite for fixing a biochemical, comprising an oxide.

 On the other hand, the polymer forming the electrolyte polymer membrane is polyacrylic acid, cationic or negative polysaccharide, nucleic acid, polymethacrylic acid, maleic anhydride copolymer, cationic acrylic acid ester, polyethylene imine, polyamine, polyamideamine, polydiallyldi Methylammonium chloride and derivatives thereof, characterized in that it comprises one or more selected from the group consisting of, biochemicals for fixing nanocomposites.

 In addition, the first metal oxide film, the second metal oxide film, the electrolyte polymer film or all of them are made of a monomolecular film, provides a nano-composite for fixing a biochemical.

 In addition, the combination of the first metal oxide film and the electrolyte polymer film, the combination of the electrolyte polymer film and the second metal oxide film, or both of these are bonds by electrostatic interaction, nano-fixed biochemicals fixed To provide a complex.

On the other hand, by forming a first metal oxide film on a solid support and then repeating the process of forming an electrolyte polymer film on the first metal oxide film one or more times to form one or more composite thin film is a nano-scale porous film; And forming a second metal oxide film, which is a nanoscale porous film, on the top of the composite thin film. Here, the formation of the first and the second metal oxide film is provided by a surface sol-gel method, provides a method for producing a nano-composite for fixing a biochemical.

 Myoglobin, Lysozyme, Peroxidase, Glucoamylase, Gluecose oxidase, Catalase and Cytochrome c, Cyt c. It provides a biosensor formed by fixing at least one biochemical material selected from the group consisting of)) to the biochemical-fixed nanocomposite according to claim 1 or 2.

 In addition, myoglobin, Lysozyme, peroxidase

1 or more biochemicals selected from the group consisting of (Peroxidase), Glucoamylase, Gluecose oxidase, Catalase and Cytochrome c, (Cyt. C) Or it provides an adsorption device formed by being fixed to the biochemicals fixing nanocomposite according to claim 2.

 【Effects of the Invention]

 The organic / inorganic nanocomposite for immobilizing biochemicals according to the present invention has an excellent effect of stably fixing a large amount of biochemicals by alternately stacking a metal oxide film and an electrolyte polymer film on a solid support.

 In addition, the organic / inorganic nanocomposite for fixing the biochemical material according to the present invention is larger than the thin film of the conventional nanocomposite fixing nanocomposite by fixing the biochemical material inside the electrolyte polymer membrane by controlling the molecular weight of the polymer forming the electrolyte polymer membrane. Even a thin film of reduced thickness can fix a large amount of biochemicals, thus reducing the manufacturing time and reducing the manufacturing cost.

 [Brief Description of Drawings]

1 schematically illustrates a method for preparing an organic / inorganic nanocomposite for fixing a biochemical according to the present invention. Figure 2 shows the frequency change of the QCM according to the sequential combination of TK0-¾u) ₄ and PAA _X.

Figure 3 shows the change in the frequency according to the (Ti0 ₂ / PAA ₂ ) _3.5 nanocomposite forming step combined with Cyt.c.

4 is a combination of Cyt.c (Ti0 ₂ / PM ₂₅ ) ₃ . ₅ shows the change in frequency according to the nanocomposite formation step.

Figure 5 (Ti0 ₂ / PAA45o) ₃ Cyt.c combined. ₅ shows the change in frequency according to the nanocomposite formation step.

FIG. 6 shows the change of the frequency according to the (Ti0 ₂ ) ₄ metal oxide multilayer thin film formation process combining Cyt.c.

FIG. 7 shows the change of the frequency according to the (Ti0 ₂ ) ₇ metal oxide multilayer thin film formation process combining Cyt.c.

8 is a metal oxide multilayer thin film (Ti0 ₂ / PAA _x ) ₃ . ₅ shows the comparison of Cyt.c binding frequency change according to the PM molecular weight of the nanocomposite including the composite thin film. FIG. 9 illustrates the variation of the frequency of QCM according to the sequential combination of Zr (0- ⁿ Pr) ₄ and PAA ₂₅

Combined Cyt.c (Zr0 ₂ / PM ₂₅ ) ₃ . ₅ shows the change in frequency according to the nanocomposite formation step.

Figure 10 shows the change of the frequency according to the sequential coupling of PDDA and PAA ₂₅ and the frequency according to the (PDDA / PAA ₂₅ ) 3.5 nanocomposite formation step combined with Cyt.c.

11 is a combination of the frequency change of the QCM and Cyt.c according to the sequential coupling of PEI and PAA ₂₅ (PEI / PM ₂₅ ) 3. ₅ shows the change in frequency according to the nanocomposite formation step.

12 is a combination of the frequency change of the QCM and Cyt.c according to the sequential coupling of PAH and PAA ₂₅ (ΡΑΗ / ΡΜ ₂₅ ). ₅ shows the change in frequency according to the nanocomposite formation step.

FIG. 13 shows the sequential coupling of Ti (0- ⁿ Bu) ₄ and PAA ₂ and the coupling of Cyt.c (Ti0 ₂ / PM ₂ ) ₃ . ₅ shows the UV-vis spectrometer changes of nanocomposites. 14 shows the sequential coupling of Ti (0— ⁿ Bu) ₄ and PAA ₂₅ and the coupling of Cyt.c (Ti () ₂ / PAA ₂₅ ) ₃ . ₅ shows the UV-vis spectrometer changes of nanocomposites.

15 shows the sequential binding of TK0- ⁿ Bu) ₄ and PM450 and the binding of Cyt.c (ΤΚνΡΑΑ4 ₅₀ ) ₃ . ₅ shows the UV-vis spectrometer changes of nanocomposites.

FIG. 16 illustrates changes of UV-vis spectrometer according to the (Ti0 ₂ ) ₃ metal oxide multilayer thin film formation step combining Cyt.c.

FIG. 17 illustrates changes of UV-vis spectrometer according to the (Ti0 ₂ ) ₆ metal oxide multilayer thin film formation step combining Cyt.c.

18 is a metal oxide multilayer thin film (Ti0 ₂ / PAA _X ) ₃ . ₅ shows the change in absorbance at 409 nm due to Cyt.c binding according to the PAA molecular weight of the nanocomposite including the composite thin film.

19 is Cyt.c conjugated (Ti0 ₂ / PM ₂₅ ) ₃ . _The UV-vis spectrometer changes due to the desorption of Cyt.c from the ₅ nanocomposite thin film.

20 is Cyt.c conjugated (Ti0 ₂ / PAA _X ) ₃ . _The change in absorbance at 409 nm due to the desorption of Cyt.c from the ₅ nanocomposite thin film is shown.

21 is Cyt.c bound (Ti0 ₂ / PM _x ) ₃ . _The reproducibility of the absorbance change at 409 nm due to the desorption of Cyt.c from the ₅ nanocomposite thin film.

22 is a Cyt.c-coupled metal oxide multilayer thin film and (Ti0 ₂ / PM _x ) ₃ . ₅ shows the difference in electrochemical properties of the nanocomposite including the composite thin film.

23 is Cyt.c conjugated (Ti0 ₂ / PM _x ) ₃ . ₅ shows the difference in electrochemical properties of composite thin films.

FIG. 24 shows the difference by Amperometric titration of Cyt.c-bonded (Ti0 ₂ / PM _x ) _3.5 composite thin film.

FIG. 25 shows AFM images and root-mean-sequare (RMS) roughness of (Ti0 ₂ ) ₃ metal oxide multilayers.

FIG. 26 shows AFM images and root-mean-sequare (RMS) roughness of Cyt.c-bonded (Ti0 ₂ ) ₃ metal oxide multilayers.

27 (Ti0 ₂ / PM ₄₅₀ ) ₃ . ₅ Show the AFM image and root—mean-sequare (RMS) roughness of the composite film. 28 is Cyt. c is bound (Ti0 ₂ / PAA4 ₅₀ ) ₃ . It shows an AFM picture and root- mean-sequare (RMS) roughness of the thin film composite _5.

 [Specific contents to carry out invention]

 Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided to enable the disclosed contents to be thorough and complete, and to fully convey the spirit of the present invention to those skilled in the art.

 The organic / inorganic nanocomposite for fixing a biochemical according to the present invention is a solid support L One or more composite thin films formed on the solid support and made of a first metal oxide film and an electrolyte polymer film stacked on the first metal oxide film, and It may include a second metal oxide film formed on the at least one composite thin film.

In the present invention, the solid support can be used without any particular limitation, such as a conventional non-metal substrate or a metal substrate, for example, a non-metal substrate such as quartz, glass, silicon, Teflon, etc. can be used, the metal gold, Substrates such as silver, copper, aluminum, and metal can be used, and further, quartz crystal microcroba lance (QCM) on which various metals are deposited, and silicon substrates on which gold is deposited _: a substrate coated with a conductive material or a gold plate Etc. can also be used. _:

 In addition, a polymer support such as polycarbonate (PC), polyethylene terephthalate (PET) or acrylic, or a solid support deposited with gold, silver, platinum, or the like can be used, and paper, cotton, silk, etc., which are natural cellulose can be used as a solid support. Can be used In addition, various solid supports known to be usable in the nanocomposite for fixing biochemicals may be used without particular limitation.

The solid support may more stably bond with the first metal oxide through electrostatic interaction and / or chemical bonding with the metal oxide of the first metal oxide film. The means for causing the solid support to have electrostatic interaction and / or chemical bonding with the metal oxide of the first metal oxide film is not particularly limited, and for example, a surface active group may be introduced onto the solid support. The surface-active group is not particularly limited as long as it can be combined with the metal oxide of the first metal oxide film as a functional group having an activity, and for example, active hydrogen, hydroxyl group, carboxyl group, sulfonic acid group, phosphoric acid group, amine group, imine group, It may be selected from an ammonium group, a pyridine group or other functional groups of the charged molecule and the like. In addition, the surface active group may be introduced to the surface of the solid support through chemical / physical treatment, for example, the solid support may be a full plasma treatment, ozone treatment, sonication in an alkali or acid solution, or an alkanthi molecule. It may be introduced through a method of forming a self-assembled thin film using a carboxyl group or the like.

 In the present invention, the first metal oxide film and / or the second metal oxide film constituting the at least one composite thin film is combined with a biochemical to perform the function of fixing the biochemical. On the other hand, the electrolyte polymer film constituting the at least one composite thin film and laminated on the first metal oxide film is laminated on the upper and lower portions thereof through electrostatic attraction, hydrogen bonding, covalent bond or complex formation with metal oxide, respectively. The first metal oxide film and the first metal oxide film deposited thereon and the second metal oxide film stacked thereon may be connected to each other, thereby allowing the nanocomposite to have a stable structure.

 The electrolyte polymer film may be bonded to the metal oxide film by electrostatic interaction or may be bonded by forming a complex through an active functional group. Since the metal oxide film partially has a negative charge, the metal oxide film may be coupled with a positively charged polyelectrolytes having a positive charge by electrostatic interaction. And, in addition to the bonds caused by electrostatic interaction, there is also a natural group having active functional groups such as active hydrogen, hydroxyl group, carboxyl group, sulfonic acid group, phosphoric acid group, amine group, imine group, ammonium group, pyridine group or other functional group of charged molecule. Alternatively, the polymer may be bonded through the formation of a complex between the synthetic polymer and the metal oxide.

The first and second metal oxide films and the electrolyte polymer film may be formed as a stable multi-layered thin film as a nanoscale porous film, and biochemicals to be fixed may not only be used as the porous film but also the surface of the nanocomposite according to the present invention. The formed first and second metal oxide films and the electrolyte polymer film The nanocomposite according to the present invention exhibits an excellent effect of fixing a large amount of biochemicals by being moved and fixed to the inside of the entire lamination film, that is, the metal oxide film disposed below through the numerous micro-scale micropores present.

The first and second metal oxide films and the electrolyte polymer film may be formed of a monomolecular film of a metal oxide or an electrolyte polymer. The monomolecular film maintains the inherent binding properties and activity of the metal oxide molecule or the electrolyte polymer, and is useful in the manufacture of biosensors, molecular devices, and the like, because the monomolecular film can properly control the spacing and orientation between molecules.

 The first and second metal oxide films may include an oxide of a metal selected from the group consisting of back titanium, zirconium, aluminum, boron, silicon, indium, tin, barium, and vanadium or two or more kinds of composite metal oxides. The first and second metal oxide films may be electrostatically bonded to the solid support, the electrolyte polymer film, and the biochemical.

 The electrolyte polymer membrane includes polyacrylic acid and derivatives thereof, cation and anionic polysaccharides and derivatives thereof, nucleic acids, polymethacrylic acid and derivatives thereof, maleic anhydride copolymers, cationic acrylic esters and copolymers thereof, polyethylene imines, polyamines, polyamides It may include any one or more selected from the group consisting of amine and polydiallyldimethylammonium chloride. In addition, the electrolyte polymer membrane may include various polymers capable of interacting with the metal oxide.

 In particular, the inventors of the present invention, when the molecular weight of the polymer forming the electrolyte polymer film is about 500 or more, preferably about 2,000, or more, the electrolyte polymer film is the function of connecting the metal oxide film laminated on the upper and lower portions thereof, as well as the metal The present invention was completed by experimentally confirming that the biochemical can be fixed in the same as the oxide film, while the molecular weight of the polymer is about 450,000, and the content of the fixed biochemical is maximized.

The inventors of the present invention, wherein the electrolyte polymer membrane is capable of fixing a biochemical material therein, the electrolyte polymer membrane also includes a (-) charge that can bind to and fix the biochemicals such as cationic enzymes therein, As the molecular weight of the polymer forming the electrolyte polymer membrane increases, more space for accommodating biochemicals is formed between the polymer chains, and the space formed is maximized when the molecular weight of the polymer is about 450, 000. I guess. Therefore, the organic / inorganic nanocomposite for fixing a biochemical according to the present invention may fix the biochemical within the metal oxide film as well as the electrolyte polymer film by controlling the molecular weight of the polymer forming the electrolyte polymer film. Under the premise of fixing the same amount of biochemicals, the number of laminated thin films can be significantly reduced compared to conventional biochemicals fixing composites, thereby reducing the manufacturing time and reducing the manufacturing cost of the nanocomposites. .

 In addition, the organic / inorganic nanocomposite for fixing a biochemical according to the present invention does not need to stack biochemicals between the metal oxide films using an organic solvent to stack the first and second metal oxide films. By suppressing the decrease in the activity of the biochemicals by the exhibits an excellent effect that can be stably fixed the biochemicals.

 The present invention relates to a method for preparing an organic / inorganic nanocomposite for fixing a biochemical. 1 schematically illustrates a method for preparing an organic / inorganic nanocomposite for fixing a biochemical according to the present invention.

 As shown in FIG. 1, according to the present invention, a method of manufacturing a nanocomposite is repeated one or more times after forming a first metal oxide film on a solid support and then forming an electrolyte polymer film on the first metal oxide film. Thereby forming one or more composite thin films, and forming a second metal oxide film on top of the composite thin film.

Specifically, after the solid support is formed in the metal oxide precursor solution through a surface sol-gel method, the first metal oxide film is removed, and then physically excessively adsorbed metal oxide is removed with a solvent and hydrolyzed using distilled water. The first metal oxide film having an activity on the solid support may be formed. Thereafter, the prepared first metal oxide film may be reacted in a polymer electrolyte solution to form a composite thin film including an electrolyte polymer film and a metal oxide film. The formed composite After the thin film is repelled in a metal oxide precursor solution through a surface sol-gel method or the like to form a metal oxide film, an excess of physically bound metal oxide is removed with a solvent and hydrolyzed using distilled water to form a second metal oxide layer. Is formed. According to an embodiment of the present invention, a method for manufacturing a nanocomposite for immobilizing a biochemical may include ultrasonic treatment or self-assembling thin film in a plasma treatment, ozone treatment, alkali or acid solution on the surface of the solid support before forming the first metal oxide film. It may further comprise the step of forming. Through this step, surface active groups such as active hydrogen, hydroxyl group, carboxyl group, sulfonic acid group, phosphoric acid group, amine group, imine group, ammonium group, pyridine group or other functional groups of charged molecules can be introduced into the solid support.

 In the case where the solid support is a non-metal, it is possible to introduce a molecule having activity by performing ultrasonic treatment in a plasma treatment, ozone treatment, or ultrasonic treatment in an alkali or acid solution. In addition, when the solid support is a metal, after washing the surface, the impurities present on the surface are removed by an acidic solution, and then the self-assembled thin film is immersed in a solution containing an alkanethiol (alkanethiol) molecule or a carboxyl group at one end thereof. Surface activity can be introduced by forming a sel f-assembled monolayer (SAM).

 In addition, the forming of the first or second metal oxide may include performing a surface sol-gel reaction on the result of the solid support or the electrolyte polymer film formed in the metal oxide precursor solution. In the step of performing the surface sol-gel reaction, the resultant on which the solid support or the electrolyte polymer membrane is formed is reacted in a solution in which the metal oxide precursor is dissolved, and then the excess physical adsorption component is removed with an appropriate solvent such as ethanol, and then in distilled water. And hydrolyzing and drying with nitrogen gas.

Precursors for the formation of the first and second metal oxide films include Τί (0- ^ι1) ₄ ' ^{Zr (} °- ^{/ 2Pr)} 4'

AKO-flBu), B (O-nEt), Ti (acac), Si (OMe), In (0C Η OMe), Sn (O-iPr), InSn (OR), BaTi (OR) or VO (O- metal alkoxides such as iPr) 3 3 2 4 2 4 3 4 4 4 4 can be used. However, in addition to the above examples, a variety of materials capable of forming an electrostatic bond with an activator of a biochemical or a solid support and forming a metal oxide may be used as the metal oxide precursor. In addition, the forming of the electrolyte polymer film on the first metal oxide film may include reacting the resultant product on which the metal oxide film is formed in the polymer electrolyte solution. After reacting the resultant product formed with the first metal oxide film in a polymer electrolyte solution in which the electrolyte polymer is dissolved at room temperature, the polymer that is unstablely adsorbed on the surface is washed with a suitable solvent such as distilled water and dried using nitrogen gas. An electrolyte polymer film may be formed on the metal oxide.

 The polymer electrolyte may include polyacrylic acid and its derivatives, cationic and anionic polysaccharides and polysaccharide derivatives, nucleic acids, polymethacrylic acid and its derivatives, maleic anhydride copolymers, silver esters and copolymers thereof, polyethylene imines, and polyamines. It may include any one or more selected from the group consisting of polyamide amine and polydiallyldimethylammonium chloride. In addition, the electrolyte polymer may include various polymers capable of interacting with a metal oxide.

In addition, after fixing the biochemicals using the biocomposite-fixed nanocomposites, the fixed biochemicals can be easily desorbed again, and the nanocomposites in which the biochemicals are immobilized in ammonia solution 5 to 50 ^° C., preferably Preferably it can be desorbed at 20 to 45'C for 1 to 60 minutes, preferably for 10 to 20 minutes. In addition, the nanocomposite from which the biochemical material is desorbed may be reacted again in a complete layer solution in which the biochemical material is dissolved.

The present invention relates to a biosensor or adsorption device including the biocomposite-fixed nanocomposite described above. The biosensor or adsorption device may be prepared by reacting the biochemicals-fixed nanocomposite according to the present invention with a biochemical.

The biocomposite-fixed nanocomposite is composed of a plurality of nanoscale porous membranes, as described above, so that not only the outermost second metal oxide film but also the first metal oxide film and the electrolyte polymer film, which are inside the nanocomposite, are biochemicals. Can be combined and fixed. Bio Sensor or Adsorption The device is capable of detecting or adsorbing a substance which reacts with the biochemical.

The biochemicals include proteins, enzymes, antigens, antibodies, receptors and ligands, and more specifically, typical biochemicals such as myoglobin, lysozyme, peroxidase _: (Peroxi dase), and glycoamylase ( Glucoamyl ase, Gluecose oxi dase, Catalase or Cytochrome c, (Cyt. C). In addition, various biochemicals may be fixed to the nanocomposite.

Specifically, the step of fixing the biochemical with the nanocomposite is the nanocomposite in 5 ~ 50 ^° C, preferably 20 to 45 ^° C in a complete solution in which the biochemical is dissolved, 1 to 60 minutes During the reaction, preferably by reacting with each other for 20 to 40 minutes. Most biochemicals show the highest activity ^at 35 to 45 ^° C, and are too low or too low. In addition, the buffer solution may be in the range of pH 2 to 10. In general, because biochemicals are greatly affected by pH, the use of a complete solution may maintain activity at the step of binding the biochemical, but may not serve as a complete solution in too acidic or alkaline conditions.

 [Example: Preparation of Nanocomposite for Biochemical Fixation]

 Example 1 Nanocomposite with QCM as Solid Support

Gold-deposited quartz crystal microbalance (QCM) was washed with pi raha solut ion: 96% sulfuric acid /30-35.5% hydrogen peroxide, 3/1, v / v, and then 10 mM 2-mercaptoethanol. A self-assembled thin film was prepared by reacting for 12 hours in a (2—mercaptoethanol) / ethanol solution, which was used as a solid support. This solid support was prepared by ethanol / luene in which titanium butoxide (Ti tani um (IV) -n-but oxi de; (Ti (0-nBu) 4) / Acros Chem, USA) was dissolved at a concentration of 100 mM. It was reacted for 3 minutes in (1/1, v / v) solution. Thereafter, the metal oxide adsorbed excessively to the resultant was removed using an ethanol solution and hydrolyzed in distilled water for 1 minute. Then, it was dried with nitrogen gas to introduce a first metal oxide film on the solid support. Distilled water in which the polymetallic acid (Poly (acryl ic acid) PAA) having a molecular weight of 2000 (PAA2), 25000 (ΡΑΑ25), and 450,000 (ΡΑΑ450), respectively, was dissolved at a concentration of 1 wt%. The solution was reacted for 30 minutes, and the surface physical adsorption was washed by distilled water twice for 30 seconds. The resultant was dried using nitrogen gas to introduce an electrolyte polymer membrane (P membrane).

By forming the first metal oxide film and the electrolyte polymer film (PM film) as one cycle, the amount of enzyme immobilization of the nanocomposite-fixed nanocomposite thin film of the present invention was controlled by forming an electrolyte polymer film having a different molecular weight. My

After the process of forming the first metal oxide film and the electrolyte polymer film was repeated one or more times, a second metal oxide film was formed on the resultant obtained by the same method as the method of preparing the first metal oxide film.

 In this case, the (Ti02 / PMx) N.5 nanocomposite means that N composite thin films including the first metal oxide film and other PAAs having an X molecular weight are formed and a second metal oxide film is formed on the surface.

 Also, under the same conditions, Zr02 was deposited on a solid support using a zirconium hydroxyside (Zrconium (IV) -nZ propoxide; (Zr (0-nPr) 4) / Aldrich Co., USA) and PAA25 as a metal oxide precursor. /PAA25).3.5 nanocomposites were formed.

 Example 2 Nanocomposite with Enzyme Coupling

 The nanocomposite-fixed nanocomposite prepared by the method of Example 1 was reacted at room temperature for 30 minutes in a complete phosphate solution (pH 7) in which the enzyme cytochrome seed (Cyt.c) was dissolved. Thereafter, unstable immobilized excess enzyme adsorption was removed using distilled water and dried with nitrogen gas to prepare a nanocomposite conjugated with enzyme.

 <Example 3: Nanocomposite using quartz plate as solid support>

The quartz plate was washed with sulfuric acid (¾SO ₄ 96.0%), and then ultrasonicated with a K0H solution to introduce an active group onto the surface of the quartz plate to use as a solid support. The solid

The support was prepared in the same manner as in Example 1 to prepare a metal oxide film and a PAA film, A nanocomposite including a Ti0 ₂ / PAA composite thin film was prepared. An enzyme was bound to the prepared nanocomposite by the method of Example 2.

 Comparative Example: Fabrication of Multilayer Thin Film

 Comparative Example 1 Manufacture of Metal Oxide Multilayer Thin Film

 In the same manner as in Example 1 except for forming a PM film

By preparing a metal oxide film, a metal oxide multilayer thin film including only the metal oxide film of Ti0 ₂ was prepared. Thus prepared metal oxide multilayer thin film by the method of Example 2 Cyt. c was bound.

 Comparative Example 2: Fabrication of Electrolyte Polymer Multilayer Thin Film

 Solid support is polydiallyldimethylammonium as a polymer electrolyte

Chloride (poiydial lyldimethyl ammonium chloride, PDDA),

Polyethylenimine (PEI), or Polyarylamine Hydro

React in a distilled water solution in which polylylamine hydrochloride (PAH) is dissolved at a concentration of lwt% for 30 minutes, and the physical adsorption on the surface is repeated twice for 30 seconds.

It was removed by washing with distilled water. The resultant was dried using nitrogen gas to introduce an electrolyte polymer membrane of PDDA, PEI or PAH. A PM membrane was introduced in the same manner as in Example 1 to the resultant in which the first electrolyte polymer membrane was formed, thereby preparing an electrolyte polymer multi-though thin film. Thus prepared electrolyte polymer multi-pump thin film by the method of Example 2 Cyt. c was bound.

 Experimental Example

 Experimental Example 1 Measurement of Frequency Change of QCM

The frequency of QCM was measured to confirm the formation of each film during the preparation of the biocomposite-fixed nanocomposite.

 Experimental Example 1-1

 In the nanocomposite fabrication process of Example 1, the frequency of the QCM was measured immediately before and after the formation of the first metal oxide and immediately after the formation of the electrolyte polymer film, thereby obtaining the average frequency change when each film was formed.

In Figure 2 by the sequential formation of Ti (0- ⁿ Bu) ₄ and PAA _X of various molecular weight

The frequency change of the QCM is shown. For PAA ₂ nanocomposite thin films, Ti (0- ⁿ Bu) ₄ bonds The average frequency change AF of 1 cycle by was 21 ± 4 Hz, and the average frequency change AF of 1 cycle by combining PM ₂ was 20 Hz 3 Hz. In the case of the PAA ₂₅ nanocomposite thin film, the average frequency change of one cycle by Ti (0- ⁿ Bu) ₄ bond AF = 29 ± 8 Hz, and the average frequency change of one cycle by PM ₂₅ bond ΔΡ = 26 士 9 Hz. In case of P / so nanocomposite thin film, the average frequency change of 1 cycle by ¹¹ Bu) ₄ bonds with TK

AF = 34 土 10 Hz,? The average frequency change of 1 cycle by coupling of ₄₅₀ was ᅀ = 36 ± 10 Hz. The results were obtained by using a quartz crystal microcrystal (QCM) manufactured by USI system (Japan) and a frequency measuring device of Hewlett Packard (USA), and the average of 10 cycles using Ti0 ₂ / PAA _x composite thin film manufacturing process as one cycle. Frequency change was shown. The crystallite micro low (QCM) has a resonance frequency in proportion to its mass even when a very small amount of nanogram material is adsorbed. Thus, as a result, the composite thin film is uniformly formed at a molecular level on a solid support. It can be confirmed.

 2. Experimental Example 1-2

 The formation process of the nanocomposite conjugated with the enzyme prepared in Example 2 was confirmed using a QCM frequency change. The frequency of the QCM was measured by the same method as in Experimental Example 1-1.

In Figure 3 Cyt.c is combined (Ti0 ₂ / PM ₂ ) ₃ . The frequency change of the ₅ nanocomposite is shown. (Ti0 ₂ / PM ₂ ) ₃ . The frequency change of bound Cyt.c of the ₅ composite thin films was AF = 271 ± 22 Hz. In Figure 4 Cyt.c is combined (Ti0 ₂ / PM ₂₅ ) ₃ . The frequency change of the ₅ nanocomposite is shown. (Ti0 ₂ / PM ₂₅ ) ₃ . The frequency change of bound Cyt.c of the ₅ composite thin films was AF = 480 Hz 42 Hz. 5 is Cyt.c bound (Ti0 ₂ / PAA4 ₅₀ ) ₃ . The frequency change of the ₅ nanocomposite is shown. (Ti0 ₂ / PAA45o) ₃ .5 composite bakmakwa frequency change of the combined Cyt.c was AF = 735 ± 100 Hz. 3, 4 and 5, crystal decision

It was confirmed that the metal oxide film and the PAA _X film are uniformly and stably formed by changing the frequency of the micro-lower (QCM). In addition, it was confirmed that the enzyme molecules were stably bound to the nanocomposite due to the frequency change caused by the binding of Cyt.c.

3. Experimental Example 1-3 Formation process of the metal oxide multilayer thin film prepared in Comparative Example 1 and Cyt. The binding of c was confirmed using the QCM promotion number change. The frequency of the QCM was measured by the same method as in Experimental Example 1-1. 6 shows Cyt. The frequency change of the c-bonded (Ti0 ₂ ) ₄ metal oxide multilayer thin film was shown. The average frequency change due to Ti (0- ⁿ Bu) ₄ binding was AF = 13 cm 2 Hz and the combined Cyt. The frequency change of c was AF = 144 * 18Hz. 7 Cyt. The frequency change of the (Ti0 ₂ ) ₇ metal oxide multilayer thin film bonded with c was shown. The average frequency change due to Ti (0- ⁿ Bu) ₄ binding was AF = 13 sul 3 Hz and the combined Cyt. The frequency change of c was AF = 148 ± 13 Hz. As shown in FIGS. 6 and 7, it was confirmed that the metal oxide multilayer film was uniformly and stably formed through the frequency change of the quartz crystal microbalance (QCM). In addition, regardless of the number of cycles (thickness) of the metal oxide multilayer film, Cyt. The change in frequency due to the binding of c was almost similar, indicating that the enzyme molecules were bound only to the surface of the metal oxide multilayer.

 8 shows Cyt. In the metal oxide multilayer thin film of Comparative Example 1 and the nanocomposite of Example 1. The difference of the frequency change by combining c is shown. Nanocomposite of Example 1 was confirmed that the increase in the amount of the enzyme bound saturation curve as the molecular weight of PAA increases. These results show that the nanocomposite of Example 1 is different from the metal oxide multilayer thin film of Comparative Example 1, and the enzyme is bound to the inside of the polymer electrolyte membrane. As the molecular weight of PAA increases, the enzyme may bind to the nanocomposite thin film. It shows that more porous spaces (holes, pores) can be created.

 4. Experimental Example 1—4

 In the nanocomposite fabrication process of Example 1, the frequency of QCM was measured immediately before and after the formation of the first metal oxide and immediately after the formation of the electrolyte polymer film, thereby obtaining the average frequency change when each film was formed.

In Figure 9 shows the change in the frequency of the QCM by the sequential formation of Zr (0- ⁿ Pr) ₄ and PAA ₂₅ . Average frequency change in one cycle by Zr (0- ⁿ Pr) ₄ bond \ F = 39 士 9

Hz, and the average frequency change AF of 1 cycle by PAA ₂₅ binding was 42 cm 10 Hz. (Zr0 ₂ / PAA ₂₅ ) 3.5 Combined Cyt of Composite Thin Films. The frequency change of c is F = 594 ± 72 Hz. As shown in Figure 9, through the frequency change of the quartz crystal microbalance (QCM) through the frequency change of the quartz crystal microbalance (QCM)

It was confirmed that the zirconium oxide film and the PAA ₂₅ film were formed uniformly and stably, and due to the change of frequency caused by the bonding of Cyt.c,

It was confirmed that the enzyme molecules were stably bound not only on the surface but also on the inside.

 5. Experimental Example 1-5

Formation process of the electrolyte polymer multilayer thin film prepared in Comparative Example 2 and the binding of Cyt.c were confirmed using the QCM frequency change. The frequency of the QCM was measured by the same method as in Experimental Example 1-1. 10 shows Cyt.c bound to (PDDA / PM ₂₅ ) 3. ₅ shows the frequency change of the multilayer polymer electrolyte thin film. The average frequency change by PDDA coupling was ΔΙ = 39 Hz 12 Hz and the average frequency change AF = 28 ± 16 Hz by PM ₂₅ coupling. (PDDA / PAA ₂₅ ) ₃ . ₅ Electrolyte bound to polymer multilayer thin film

The frequency change of Cyt.c was Δί = 13 ± 8 Hz. Figure 11 is a combination of Cyt.c

(PEI / PAA _{25) 3} .5 shows the frequency changes of polyelectrolyte multilayers. The average frequency change by PEI binding was ΔΙ ^ 31 ± 10 Hz, and the average frequency change AF = 28 ± 27 Hz by PM ₂₅ binding. (PDDA / PM ₂₅ ) ₃ . The frequency change of Cyt.c bound to the ₅ electrolyte polymer multilayer thin film was ᅀ F-12 ± 7 Hz.

12 shows Cyt.c bound to (ΡΑΗ / ΡΜ ₂₅ ) 3. ₅ shows the frequency change of the multilayer polymer electrolyte thin film. The average frequency change by PAH coupling was AF = 25 Hz 6 Hz,

The average frequency change due to the binding of PM ₂₅ was士AF = 28 9 Hz. (PAH / PAA ₂₅ ) ₃ . The frequency change of Cyt.c bonded to the ₅ electrolyte polymer multilayer thin film was AF = 40 ± 18 Hz. As shown in FIGS. 10, 11, and 12, the crystal micro-crystals (QCM) through the change of the frequency of the quartz crystal microbalance (QCM) through the change of the electrolyte polymer and the

It was confirmed that the PM ₂₅ film was formed uniformly and stably. Due to the frequency change caused by the binding of Cyt.c of the electrolyte polymer multilayer thin film, it was confirmed that the enzyme molecules only adhered to the surface finely and could not penetrate and bond inside. Compared with the composite thin film using metal oxide, the bond amount of P is similar, but the frequency change due to the bonding of Cyt.c is more than 10 times. These results indicate that the enzyme inside the nanocomposite thin film of metal oxide Although there is a sufficient space for binding, it can be confirmed that there are almost no spaces or functional groups to which enzymes can bind in the case of an electrolyte polymer multilayer film.

 Experimental Example 2 Measurement of Absorbance Change

 6. Experimental Example 2-1

Formation of the enzyme-conjugated nanocomposite prepared in Example 3 was confirmed using a change in absorbance ((λ _∞χ = 409ηιιι) absorbance change by porphyrin molecules in the enzyme) of the UV- _vis spectrometer. Change of absorbance is UV-vis spectrometer

(Perk in Elmer, Lambda 35) was confirmed using the average peak after three replicates.

13 shows Cyt. c bonded to (Ti0 ₂ / PAA ₂ ) ₃ . The UV_vis spectrometer of ₅ nanocomposites is shown. As shown in FIG. 13, the characteristic peak due to Ti0 ₂ increased regularly as the number of cycles increased around 250 nra due to Ti (0- ⁿ Bu) ₄ binding, but the characteristic peak due to Ti¾ was slightly decreased when PAA was bonded. Decreased. This is due to the desorption of Ti0 ₂ which is unstable in bonding with PM. (Ti0 ₂ / PAA ₂ ) ₃ . ₅ Nanocomposites (Ti0 ₂ / PAA ₂ ) at around 250 nm ₃ . ₅ Only characteristic peaks due to nanocomposites exist, but Cyt. When c is combined, at 409 nm

Cyt. Characteristic peaks due to porphyrin molecules in c are shown. The absorbance change of the UV-vis spectrometer showed that the enzyme molecules were stably bound to the (Ti0 ₂ / PM ₂ ) nanocomposite. 14 is Cyt. c combined

(Ti0 ₂ / PAA ₂₅ ) 3.5 nanocomposite, Figure 15 Cyt. c-linked (Ti¾ / PAA4 ₅₀ ) ₃ . The UV-vis spectrometer of the ₅ nanocomposite was shown. Molecular weight of P can increase

Characteristic peak and Cyt by Ti0 _2. The characteristic peak by binding of c increased. These results are the same as the QCM frequency change, and it can be confirmed that the enzyme is stably bound to the nanocomposite thin film.

16 is Cyt. c-linked (Ti0 ₂ ) ₃ metal oxide multilayer thin film, Figure 17 is Cyt. The UV-vis spectrometer of c-bonded (Ti0 ₂ ) ₆ metal oxide multilayer thin film was shown.

The characteristic peaks due to Ti0 ₂ in the vicinity of 250 nm by Ti (0- ⁿ Bu) ₄ bond showed a regular increase as the number of cycles increased. at 409 nra by combination of c The peak was almost constant regardless of the number of cycles. These results indicate that the inside of the nanocomposite thin film of the metal oxide has a space in which the enzyme can bind, but in the case of the metal oxide multilayer film, there is no space for the enzyme to bind, but only the surface thereof.

 18 shows Cyt. In the metal oxide multilayer thin film and nanocomposite of Example 3. FIG. The variation of UV-vi s spectrometer according to c binding was shown. As the molecular weight of the P nanocomposites increased, the amount of enzyme bound was increased by drawing a saturation curve. These results show that as the molecular weight of PAA increases, more porous spaces (pores, pores) are formed inside the nanocomposite thin film.

 6. Experimental Example 2-2

 Absorbance of the UV-vis spectrometer using the enzyme-linked nanocomposite prepared in Example 3 using 1 wt% ammonia water to remove enzyme and form recombination.

(( _Max = 409 nm) absorbance change by porphyrin molecule in enzyme) was confirmed using the change.

In FIG. 19, a process of separating the bound enzyme by reacting the enzyme-bound (Ti0 ₂ / PM ₂ ) _3.5 nanocomposite with 1 wt% ammonia water was confirmed using a UV_vis spectrometer change. As shown in FIG. 19, approximately 94% of the bound enzyme was reacted by reacting with ammonia water for about 5 minutes (Ti0 ₂ / PAA ₂₅ ) ₃ . ₅ nanocomposite thin films could be removed. In weak base ammonia water and reaction (Ti0 ₂ / PM ₂₅ ) ₃ . _By confirming that the characteristic peak due to Ti0 ₂ of the ₅ nanocomposite thin film was not changed, the bound enzyme could be successfully removed without damaging the nanocomposite. 20 is Cyt.c is combined

(Ti0 ₂ / PAA _X ) 3. ₅ Nanocomposites were reacted with 1 wt% ammonia water and bound to Cyt. The process of separating c was confirmed from the change in absorbance at 409 nm. It was confirmed that the saturation was reached in about 5 minutes. After 20 minutes of ammonia and reaction, Cyt .c was combined (Ti0 ₂ / PM _x ) ₃ . Isolation of Cyt .c from ₅ nanocomposites _{(Ti0 2 / PAA 2) 3} .5 for nanocomposites, about _{99.7¾, (Ti0 2 / PAA 25} ) 3. _{About 5} % for ₅ nanocomposites,

(Ti0 ₂ / PAA4 ₅₀ ) ₃ . _{For 5} nanocomposites, the separation capacity was about 86%. As the molecular weight of PAA increases, the removal rate of enzyme from nanocomposite thin film decreases. Indicated. These results indicate that as the molecular weight of P increases, the enzyme bound to the nanocomposite thin film is farther from the surface and more stable. 21 is Cyt.c bound (Ti0 ₂ / PM _x ) ₃ . _The results of reproducibility using the change of absorbance at 409 nm by the separation of Cyt.c from ₅ nanocomposites. As for the binding of Cyt.c, the separation of Cyt.c was repeated for 20 minutes in 1 wt% ammonia water in the same manner as in Example 3, and it was confirmed that the binding and separation proceeded repeatedly repeatedly. From these results, it was confirmed that the nanocomposite thin film can be reused very stably.

 Experimental Example 3 Cyclic Voltammetry (CV) Measurement>

 In order to confirm the electrochemical properties of enzyme-bound nanocomposites

Cyclic voltammetry was used.

 The change in CV was measured by using 'IvinumStat' (Ivium Technologies, The Netherlands) on a QCM electrode in which an enzyme is bound to the nanocomposite prepared by the method of Example 1 in the method of Example 2.

22 shows (Ti0 ₂ ) ₃ , (Ti0 ₂ ) ₃ .5 / Cyt.c, in a complete phosphate solution (pH 7).

Electrochemical properties of (Ti0 ₂ / PM _x ) _{35 /} Cyt.c nanocomposite were shown.

In the case of the 3-cycle metal oxide multilayer thin film ((Ti0 ₂ ) ₃ ), the change of the current value was hardly observed in the vicinity of 200 to 300 mV, but the metal oxide multilayer thin film containing the enzyme was bound.

_{((Ti0 2) 3 / Cyt.c} ) and nanocomposite _{((Ti0 2 / PAA x)} 3. 5 / Cyt.c) For, the oxidation of iron in the enzyme's? A reduction peak was observed. Metal Oxides with Enzymes

A multi-layer thin film ((Ti0 _{2) 3)} than the enzyme-linked nanocomposites _{((Ti¾ / PM X) 3} . 5 / Cyt.c) oxidation and increase of the reduction in peak was confirmed. In addition, as the molecular weight of PAA increased, the oxidation / reduction peak increased. This is because the metal oxide multilayer thin film simply binds the enzyme to the outermost metal oxide surface, whereas in the nanocomposite of Ti0 ₂ / PAA, the enzyme moves and binds to the inside, so the oxidation / reduction peak from the enzyme is increased due to the increase of immobilized enzyme. (Current value) is relatively increased. From the above results, it can be seen that the biocomposite-fixed nanocomposite according to the embodiment of the present invention can be bound to the inside of the nanocomposite while maintaining the activity of the biochemical. Experimental Example 4 Amperometric Titration Measurement

 Biosensor Characterization of Enzyme-bound Nanocomposites

Amperometric titration was used.

The concentration of hydrogen peroxide (¾0 ₂ ) in the complete phosphate solution (pH 7) was obtained by using the 'IvinumStat' Gvium Technologies, Netherlands, which employs a QCM electrode in which the enzyme is conjugated to the nanocomposite prepared by the method of Example 1. The change in current value due to the increase was measured.

23 is added, and at 30-second intervals from the hydrogen peroxide solution applied voltage of 500mV yut _{ΙμΜ, (Ti0 2) 3,} (Ti0 2) 3 .5 / Cyt.c, (Ti0 2 / PAA x) 3.5 / Cyt.c nano The change in the current value of the composite was measured. In the case of the 3-cycle metal oxide multilayer thin film ((Ti0 ₂ ) ₃ ), no change in current value was observed, but the enzyme-bonded metal oxide multilayer thin film ((Ti0 ₂ ) _{3 /} Cyt.c) and the nanocomposite ((Ti0 ₂ / for _{PAA x) 3. 5 / Cyt.c} ), the current value increased due to increased concentrations of hydrogen peroxide was observed. In particular, the greater the amount of enzyme bound, the greater the current value.

 Experimental Example 5: Surface Observation Using Atomic Force Microscope (AFM)

 The surface of the mica (Mica) washed with ethanol as a solid support, except for the self-particulate thin film process, a metal oxide multilayer thin film prepared in the same manner as in Comparative Example 1 and a nanocomposite prepared in the same manner as in Example 1 Cyt.c

Combined. Then, (Ti0 ₂ ) ₃ , (Ti0 ₂ ) _{3 /} Cyt.c, (Ti0 ₂ / PAA ₄₅₀ ) ₃ . ₅ watts

(TiO ₂ / PM ₄₅₀ ) ₃ . _{5 /} Cyt.c thin films were observed on the surface using an atomic force microscope (JSPM-5200, JE0L). As a result, the root-mean-sequare (RMS) roughness of the (Ti0 ₂ ) ₃ metal oxide multilayer thin film is 0.148 nm, and the root-mean-sequare (RMS) roughness of the ((Ti0 ₂ ) _{3 /} Cyt.c nanocomposite is 1.048. nm RMS roughness was gajeungga of nanocomposite enzyme is coupled in. in _{addition, (TiO 2 / PM 450)} 3. ₅ nanocomposite root- mean- sequare (RMS) roughness is 1.148nm, (Ti () 2 / PAA4 50 The root-mean-sequare (RMS) roughness of the 3.5 / Cyt.c nanocomposite was 1.848 nm, which increased the RMS roughness of the enzyme-coupled nanocomposite 7), which resulted in Cyt.c immobilized on the surface of the nanocomposite. RMS roughness has increased, and Cyt.c of about 3nm size From the point of penetration and stable fixation and the results of atomic force microscopy, each membrane of the nanocomposite was confirmed to be porous.

25 and 26 are (Ti0 ₂ ) ₃ metal oxide multilayer thin films and (Ti0 ₂ ) _{3 /} Cyt. AFM image and root-mean-sequare (RMS) roughness of c nanocomposite are shown, and FIGS. 27 and 28 are respectively (Ti0 ₂ / PAA4 ₅₀ ) ₃ . ₅ nanocomposite thin films and (Ti0 ₂ / PAA ₄₅ o) 3.5 / Cyt. AFM photographs of c nanocomposites and root- mean- sequare (RMS) roughness

 Although the present specification has been described with reference to preferred embodiments of the invention, those skilled in the art may variously modify and change the invention without departing from the spirit and scope of the invention as set forth in the claims set forth below. Could be done. Therefore, a modified implementation is basically of the present invention

If included in the scope of the claims, all of the technical scope of the present invention

It should be considered to be included.

Claims

[Patent Claims]

 [Claim 11

 Solid support;

At least one composite thin film including a first metal oxide film formed on the solid support and an electrolyte polymer film formed on the first metal oxide film and being a nanoscale porous film; And

 A second metal oxide film formed on the composite thin film and being a nanoscale porous film;

 The molecular weight of the polymer forming the electrolyte polymer membrane is 500 or more, biochemical material fixing nanocomposite.

 [Claim 2]

 The method of claim 1,

 Characterized in that the molecular weight of the polymer is 2,000 or more, biochemical material fixing nanocomposite.

 [Claim 3]

 The method of claim 1 or 2,

 The solid support is characterized in that the first metal oxide film can be formed by a surface sol-gel reaction thereon, biochemicals fixed nanocomposite.

 [Claim 4]

 The method according to claim 1 or 2,

 The solid support is characterized in that the surface thereof is subjected to at least one treatment selected from the group consisting of plasma treatment, ozone treatment, ultrasonic treatment in an alkali or acid solution, and self-assembled thin film formation treatment using alkanedi molecules or carboxyl groups, Nanocomposite for Biochemical Fixation.

 [Claim 5]

 The method of claim 4,

The solid support is formed of an active hydrogen, a hydroxyl group, a carboxyl group, a sulfonic acid group, a phosphate group, an amine group, an imine group, an ammonium group, a pyridine group and a charged molecule on its surface. At least one surface active group selected from the group consisting of functional groups, characterized in that the biochemicals fixed nanocomposite.

 [Claim 6]

 The method according to claim 1 or 2,

 The metal oxide forming the first metal oxide film, the second metal oxide film, or both thereof includes an oxide of at least one metal selected from the group consisting of titanium, zirconium, aluminum, boron, silicon, indium, tin, barium and vanadium. Biocomposites-fixed nanocomposites, characterized by comprising.

 [Claim 7]

 The method according to claim 1 or 2,

 The polymer forming the electrolyte polymer membrane may be polyacrylic acid, cationic or anionic polysaccharide, nucleic acid, polymethacrylic acid, maleic anhydride copolymer, cationic acrylic acid ester, polyethyleneimine, polyamine, polyamideamine, polydiallyldimethylammonium It characterized in that it comprises one or more selected from the group consisting of chlorides and derivatives thereof, biochemicals for fixing nanocomposites.

 [Claim 8]

 The method according to claim 1 or 2,

 The first metal oxide film, the second metal oxide film, the electrolyte polymer film, or both, characterized in that made of a monomolecular film, a biochemical fixed nanocomposite.

 [Claim 9]

 The method according to claim 1 or 2,

 The combination of the first metal oxide film and the electrolyte polymer film, the combination of the electrolyte polymer film and the second metal oxide film, or both of them characterized in that the bond by the electrostatic interaction, biochemical material fixing nanocomposite.

[Claim 10] Forming at least one composite thin film which is a nanoscale porous film by repeating a process of forming an electrolyte polymer film on the first metal oxide film one or more times after forming the first metal oxide film on the solid support; And

 And forming a second metal oxide film, which is a nanoscale porous membrane, on the top of the composite thin film.

 [Claim 11]

 The method of claim 10

 Forming the first and the second metal oxide film is characterized in that it is carried out by a surface sol-gel method, a method for producing a nanocomposite for fixing a biochemical.

 [Claim 12]

 Myglobin, Lysozyme, Peroxidase, Glucoamylase, Gluecose oxidase, Catalase and Cytochrome c, (Cyt.c) A biosensor formed by immobilizing at least one biochemical selected from the group consisting of the nanocomposite for fixing a biochemical according to claim 1.

 [Claim 13]

 Myglobin, Lysozyme, Peroxidase, Glucoamylase, Gluecose oxidase, Catalase and Cytochrome c, (Cyt.c) At least one biochemical material selected from the group consisting of the adsorption device formed by being fixed to the biochemical-fixing nanocomposite according to claim 1 or 2.