KR101102002B1 - Nanocomposite for biomolecule binding and preparation method thereof - Google Patents

Abstract

The present invention relates to a nanocomposite for biomolecule binding and a preparation method thereof. The nanocomposite nanocomposite nanocomposite in which a metal oxide film and an ionic polymer film, which are nano-scale porous thin films, are sequentially formed on a solid support, can be stably bonded while the biological molecules maintain activity not only on the surface but also inside.

Nanocomposites, metal oxides, porous materials, polymer electrolytes, biomolecules, enzymes

Description

NANOHYBRID COMPOSITE FOR IMMOBILIZING BIOMOLECULE AND PREPARATION METHOD OF THE SAME

The present invention relates to a nanocomposite for biomolecule binding and a preparation method thereof. More particularly, the present invention relates to a biomolecule binding nanocomposite and a method for preparing the biomolecule, which can be stably bound to the inside of the nanocomposite surface as well as maintaining the activity.

Techniques for immobilizing biomolecules such as proteins and enzymes are widely used in the development of kits, protein chips, and biosensors for diagnosing diseases using indicator materials. However, it is difficult to produce a uniform and stable molecular film of biomolecules due to a small amount of fixed biomolecules fixed itself or inactivation due to degeneration of immobilized biomolecules, and cannot confirm interactions and recognition reactions with reactive substances. It has a disadvantage. Therefore, there is a need for the development of a new method of fixing a sufficient amount while maintaining the biomolecule.

To this end, a covalent bond method by amide bond (M. Zayats, E. Katz, I. Willner, JACS ., 2002, 124, 14724) and surface sol-gel is a method of immobilizing a biomolecule on a solid surface for the aforementioned purpose. The composite laminates of metal oxides and enzymes (I. Ichinose, R. Takaki, K. Kuroiwa, T. Kunitake, Langmuir , 2003, 19, 3883) were mainly used. However, the covalent bond method by amide bond has a disadvantage in that the biomolecule may lose activity during the reaction because the biomolecule must be reacted through the covalent bond. In addition, the fabrication of the laminated thin film by the surface sol-gel method can be uniformly fixed to the surface by a simple method using the electrostatic interaction of the metal oxide and the biomolecule, The biomolecules can be fixed inside, but there is a disadvantage that the denaturation of the biomolecules is very high due to the influence of the organic solvent used in the adsorption process of the metal oxide during the multilayer deposition process.

In order to stably fix a large amount of biomolecules on a solid support, a method of alternately stacking a polymer and a biomolecule using an ionic polymer having an electrostatic interaction is used. However, this method has a problem in that the reaction between the biomolecule and the reactor is prevented by the polymer layer covering the biomolecule, so that it does not work effectively on the reactor.

The present invention is to provide a nanocomposite for biomolecule binding that can be stably fixed while maintaining the activity of the biomolecule not only on the surface of the nanocomposite but also inside.

The present invention is also to provide a method for producing the nanocomposite for biomolecule binding.

The present invention provides a nanocomposite for biomolecule binding comprising a solid support, a composite thin film including a first metal oxide film and an ionic polymer film sequentially formed on the solid support, and a second metal oxide film formed on the composite thin film.

In addition, the present invention comprises the steps of forming a first metal oxide film on a solid support; Forming an ionic polymer film on the first metal oxide film; And providing a second metal oxide film on the ionic polymer membrane.

Hereinafter, a nanocomposite for biomolecule binding and a method for preparing the same according to embodiments of the present invention will be described in detail.

According to an embodiment of the present invention, a nanocomposite for biomolecule binding comprising a solid support, a composite thin film including a first metal oxide film and an ionic polymer film sequentially formed on the solid support, and a second metal oxide film formed on the composite thin film Is provided.

The nanocomposite for biomolecule binding according to the embodiment of the present invention includes a structure in which a first metal oxide film is formed on a solid support, and an ionic polymer film and a second metal oxide film are sequentially formed. In this case, the composite thin film including the first oxide film and the ionic polymer film may be formed as a single layer, but may be formed as two or more multilayered composite films. In this case, as will be described later, two or more layers of the first metal oxide film and the ionic polymer film are alternately formed, and the second metal oxide film is formed on the multilayer composite film.

In such a nanocomposite, the first or second metal oxide film serves as a nanoscale porous film to form a stable multilayer thin film and binds to biomolecules. In addition, the ionic polymer membrane can connect the layers of the multi-layered nano thin film by electrostatic attraction, hydrogen bonding, covalent bond or complex formation with a metal oxide, so that the nanocomposite can have a stable structure. In addition, the metal oxide film and the ionic polymer film are nano-scale porous membranes, which allow biomolecules to move and bind inside. In addition, since the biomolecules are bonded after the nanocomposite is prepared without using an organic solvent, there is no fear of reducing the activity of the biomolecules.

In one embodiment of the invention, the nanocomposite for biomolecule binding may have a structure as shown in FIG. 1 shows the structure of a nanocomposite conjugated with an enzyme using a quartz crystal microbalance (QCM) as a solid support. However, FIG. 1 is only one example of a biocombination nanocomposite according to one embodiment of the invention, and the nanocomposite is not limited thereto.

As a result of the experiments of the present inventors, the nanocomposite for biomolecule binding according to the embodiment forms a uniform and stable nano thin film layer due to the reaction between the metal oxide film and the ionic polymer, and the first and second metal oxide film and the ion Due to the large number of nanoscale pores present in the laminated thin film of the polymer membrane, the biomolecules have been shown to be able to bind large amounts of biomolecules stably, as well as to the inside of the nanocomposite surface.

On the other hand, in the biocomposite nanocomposite nanocomposite of an example of the invention, the solid support may include a surface active group, or may have an electrostatic interaction with the metal oxide. Such surface-active groups are introduced to the surface of the solid support through chemical / physical treatment, and mean active functional groups. The solid support may include a surface active group through plasma treatment, ozone treatment, ultrasonication in an alkali or acid solution, or a method of forming a self-assembled thin film using an alkane thiol molecule or a carboxyl group. And, the electrostatic interaction with the metal oxide can be used without limitation.

As a material of the solid support, a conventional non-metal substrate or a metal substrate may be used without particular limitation. For example, a substrate such as quartz, glass, silicon, or Teflon, which is a solid of nonmetals, may be used, and the solid of metals may be used. Substrates of gold, silver, copper, aluminum or platinum can be used, and further, quartz crystal microbalance (QCM) in which various metals are fixed, silicon substrates on which gold is deposited, or substrates coated with a conductive material Or a gold plate etc. can also be used. In addition, a polymer support such as polycarbonate (PC), PET, or acrylic, or a solid support on which gold, silver, platinum, etc. are deposited may be used, and natural cellulose, such as paper, cotton, or silk, may be used. It can be used as a solid support. In addition, various solid supports known to be usable in the nanocomposite for immobilization of biological molecules may be used without particular limitation.

In addition, the first metal oxide film may be bonded through a surface active group on the solid support. The surface active group may be introduced to the surface of the solid support through plasma treatment, ozone treatment, ultrasonication in an alkali or acid solution, or a method of forming a self-assembled thin film using an alkane thiol molecule or a carboxyl group. Examples of such surface active groups include active hydrogens, hydroxyl groups, carboxyl groups, sulfonic acid groups, phosphoric acid groups, amine groups, imine groups, ammonium groups, pyridine groups, and functional groups of other charged molecules. However, such surface-active groups are not limited to the above-mentioned types, and those which are introduced to the surface of the solid support and may be combined with the metal oxide may be used without particular limitation.

The first or second metal oxide film may include an oxide of a metal selected from the group consisting of titanium, zirconium, aluminum, boron, silicon, indium, tin, barium, and vanadium, or two or more composite metal oxides. The first or second metal oxide film may be electrostatically bonded to the solid support, the ionic polymer, or the biomolecule. In addition, the first or second metal oxide film may be formed by surface sol-gel reaction of a solid support or a product on which an ionic polymer is formed in a metal oxide precursor solution.

Precursors of the metal oxides for forming the first and second metal oxide layers include Ti (O- ⁿ Bu) ₄ , Zr (O- ⁿ Pr) ₄ , Al (O- ⁿ Bu) ₃ , and B (O- ⁿ Et) ₃ , Ti (acac) ₂ , Si (OMe) ₄ , In (OC ₂ H ₄ OMe) ₃ , Sn (O- ⁱ Pr) ₄ , InSn (OR) ₄ , BaTi (OR) ₄ or VO (O -metal alkoxides such as ⁱ Pr) ₄ can be used. However, in addition to the above examples, it is possible to form a bond by interaction with an activator of a biomolecule or a solid support, and various materials enabling the formation of a metal oxide may be used as the metal oxide precursor.

As shown in Experimental Examples 1-3, Experimental Examples 2, and Experimental Examples 3 and 6 to 10, the first and second metal oxide films and the ionic polymer membrane may be nano-scale porous membranes. Each of the membranes included in the nanocomposite for biomolecule binding contains a large number of pores on the nanoscale, so that even after the nanocomposite is manufactured, the biomolecules can move and bind to the inside of the nanocomposite through the porous membrane. have. Therefore, it is not necessary to alternately form the metal oxide film and the biomolecule film by using an organic solvent, and thus a nanocomposite having a stable and uniform composition can be manufactured while maintaining the activity of the biomolecule.

On the other hand, in one example of the invention, the ionic polymer membrane may be coupled by electrostatic interaction with the metal oxide membrane, or may be bonded by forming a complex via an active functional group. Since the metal oxide film partially has a negative charge, the metal oxide film may be coupled to the positively charged polymer electrolyte having cationic polyelectrolytes by electrostatic interaction. In addition to the bonds caused by electrostatic interactions, natural groups having active functional groups such as active hydrogens, hydroxyl groups, carboxyl groups, sulfonic acid groups, phosphoric acid groups, amine groups, imine groups, ammonium groups, pyridine groups, or functional groups of other charged molecules Alternatively, the polymer may be bonded through the formation of a complex between the synthetic polymer and the metal oxide.

The ionic polymer membrane is polyacrylic acid and its derivatives, cationic and anionic polysaccharides and their derivatives, nucleic acids, polymethacrylic acid and its derivatives, maleic anhydride copolymers, cationic acrylic esters and their copolymers, polyethylene imines, polyamines, poly It may include any one or more selected from the group consisting of amideamine and polydiallyldimethylammonium chloride. In addition, the ionic polymer membrane may include various polymers capable of interacting with the metal oxide.

Meanwhile, the nanocomposite for biomolecule binding according to an embodiment of the present invention may include a composite thin film including the first metal oxide film and the ionic polymer film as a single layer, or may include two or more composite layers. After forming the first metal oxide film and the ionic polymer film and before forming the second metal oxide film, the first metal oxide film and the ionic polymer film are sequentially added to include the composite thin film as two or more composite layers. Can be. Through this, the thickness of the nanocomposite for biomolecule binding can be controlled at the nanoscale level.

Each of the first and second metal oxide films may be substantially composed of a single molecule film of a metal oxide. The monomolecular film maintains intrinsic binding properties and activity of molecules, and can be appropriately controlled for the spacing and orientation between molecules in a molecular thin film, which is useful for the manufacture of molecular devices, biosensors, and the like.

In addition, the ionic polymer membrane may be substantially composed of a monomolecular membrane of the ionic polymer. The monomolecular film maintains intrinsic binding properties and activity of molecules, and can be appropriately controlled for the spacing and orientation between molecules in a molecular thin film, which is useful for the manufacture of molecular devices, biosensors, and the like.

Meanwhile, the nanocomposite for biomolecule binding according to an embodiment of the present invention may be a biomolecule coupled to the first or second metal oxide film. Since the nanocomposite includes a plurality of nanoscale porous membranes, the biomolecules may be bonded not only to the second metal oxide film but also to the first metal oxide film located therein when immersed in a solution in which the biomolecule is dissolved. In addition, in one embodiment of the invention, the nanocomposite for biomolecule binding does not need to go through a biomolecule binding step containing an organic solvent each time to form a metal oxide film can be stably bonded while maintaining the activity of the biomolecule To be.

Biomolecules bound to such nanocomposites include proteins, enzymes, antigens, antibodies, receptors and ligands, and more specifically, typical biomolecules such as myoglobin, lysozyme, peroxidase, and writings. Glucoamylase, glucosose oxidase, catalase, or cytochrome c , Cyt. C ). In addition, various biomolecules may be immobilized on the nanocomposite. Such biomolecules can be bound to the second metal oxide film through electrostatic interaction and can be immobilized in the cage of the porous space (holes, pores).

In addition, the biomolecule bound to the first or second metal oxide film may be an enzyme. In one embodiment of the invention, since the nanocomposite is made, the enzyme is bound so that there is no fear of reacting with the organic solvent, it can be stably maintained while maintaining the activity. Such enzymes include oxidoreductases, transferases, hydrolases, lyases, isomerases or ligases. In addition, various enzymes may be bound to the nanocomposite. The enzyme may be bound to the second metal oxide film through electrostatic interaction and may be immobilized in a cage of a porous space (hole, pore) inside the nanocomposite.

On the other hand, according to another embodiment of the invention, there is provided a method of manufacturing the above-mentioned biomolecule binding nanocomposite. Such a manufacturing method includes forming a first metal oxide film on a solid support; Forming an ionic polymer film on the first metal oxide film; And forming a second metal oxide film on the ionic polymer film.

According to an embodiment of the present invention, after forming the metal oxide film in the metal oxide precursor solution through a surface sol-gel method or the like in the metal oxide precursor solution, physically excess metal oxide adsorbed with a solvent and distilled water When hydrolyzed by use, an active first metal oxide film can be formed on the solid support. Thereafter, the prepared first metal oxide film may be reacted in a polymer electrolyte solution to form a composite thin film including an ionic polymer film and a metal oxide film. The formed thin film is reacted in a metal oxide precursor solution through a surface sol-gel method or the like to form a metal oxide film, and then the excess physically adsorbed metal oxide is removed with a solvent and hydrolyzed using distilled water. A metal oxide layer is formed.

Nanocomposite nanocomposite manufacturing method according to an embodiment of the present invention, before the step of forming the first metal oxide film, the ultrasonic treatment or self-assembled thin film formed in a plasma treatment, ozone treatment, alkali or acid solution on the surface of the solid support It may include the step. Through this step, the 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 charged molecule 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 an ultrasonic cleaning in a plasma, ozone treatment or ultrasonication in an alkali or acid solution. In the case where the solid support is a metal, after washing the surface, impurities are removed from the surface by an acidic solution, and then immersed in a solution containing an alkanehiol molecule or a carboxyl group whose one end is modified and self-assembled. Surface activity can be introduced by obtaining a self-assembled monolayer (SAM).

In addition, the forming of the first or second metal oxide may include performing a surface sol-gel reaction on the resultant in which the solid support or the ionic polymer film is formed in the metal oxide precursor solution. In the surface sol-gel reaction, the solid support or the ionic polymer membrane formed product is reacted in a solution in which a metal oxide precursor is dissolved, and then excess physical adsorption is removed with an appropriate solution, such as ethanol, in distilled water. And hydrolyzing and drying with nitrogen gas. Precursors for forming the first and second metal oxide films include Ti (O- ⁿ Bu) ₄ , Zr (O- ⁿ Pr) ₄ , Al (O- ⁿ Bu) ₃ , and B (O- ⁿ Et) ₃ , Ti (acac) ₂ , Si (OMe) ₄ , In (OC ₂ H ₄ OMe) ₃ , Sn (O- ⁱ Pr) ₄ , InSn (OR) ₄ , BaTi (OR) ₄ or VO (O- ⁱ Pr And metal alkoxides such as ₄ ). However, in addition to the above examples, it is possible to form an electrostatic bond with the activator of the biomolecule or the solid support, and various materials enabling the formation of the metal oxide may be used as the metal oxide precursor.

In addition, the forming of the ionic 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 of the first metal oxide film in a polymer electrolyte solution in which the ionic 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. The ionic polymer film may be formed on the metal oxide.

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

On the other hand, in the method for manufacturing a biomolecule binding nanocomposite according to an embodiment of the invention, before the forming step of the second metal oxide film, the step of forming the first metal oxide film and the step of forming the ionic polymer film alternately two or more times. Can be repeated The thickness of the nanocomposite can be controlled at the nanoscale level by forming two or more composite thin films including the first metal oxide film and the ionic polymer film.

In addition, the method of manufacturing a nanocomposite for biomolecule binding according to an embodiment of the present invention may further include coupling the biomolecule to the nanocomposite on which the second metal oxide film is formed. When the nanocomposite is reacted with a biomolecule, biomolecules may be coupled to the inside of the nanocomposite as well as the outermost second metal oxide film because a plurality of nanoscale porous membranes exist inside the nanocomposite. Therefore, in one embodiment of the invention, the step of preparing a nanocomposite for biomolecule binding does not include the activity of biomolecules without including a biomolecule binding step containing an organic solvent every time a metal oxide film is formed to bind the biomolecules therein. It is possible to bond stably while maintaining.

Such biomolecules include proteins, enzymes, antigens, antibodies, receptors and ligands, and more specifically, typical biomolecules such as myoglobin, lysozyme, peroxidase, and glucoamylase , Gluecose oxidase, catalase or cytochrome c (Cyt. C ). In addition, various biomolecules may be bound to the nanocomposite. Such biomolecules may be coupled to the second metal oxide film through electrostatic interaction and may be fixed in a cage of porous spaces (pores and pores) inside the nanocomposite.

In addition, the step of binding the biomolecule is the nanocomposite in a buffer solution in which the biomolecule is dissolved at 5 to 50 ℃, preferably 20 to 45 ℃, for 1 to 60 minutes, preferably 20 to 40 Can be reacted for minutes. Most biomolecules exhibit the greatest activity at 35-45 ° C., and become too active when too hot or cold. In addition, the buffer solution may be in the range of pH 2 to 10. In general, since biological molecules are greatly affected by pH, the use of a buffer solution may maintain activity at the step of binding the biological molecules, but may not serve as a buffer solution in too acidic or alkaline conditions.

In addition, in one embodiment of the invention, the biological molecule may be an enzyme. After the nanocomposite is formed, the enzyme is bound, so there is no fear of reacting with the organic solvent, so that it can be stably maintained while maintaining activity. Such enzymes include oxidoreductases, transferases, hydrolases, lyases, isomerases or ligases, and various enzymes may be coupled to the nanocomposite. These enzymes may be bound to the second metal oxide film through electrostatic interactions and may be fixed in cages of porous spaces (pores, pores) inside the nanocomposite.

Figure 2 shows an example of a method for manufacturing a nanocomposite for biomolecule binding according to an embodiment of the invention. In FIG. 2, a 9 MHz quartz crystal microbalance (QCM) was used as a solid support, a metal oxide film was formed in a metal alkoxide solution, and an enzyme was bound as a biomolecule. In addition, FIG. 2 shows that the process of forming a composite thin film including the first metal oxide film and the ionic polymer film may be repeated one or more times. However, Figure 2 shows only one example of a method for manufacturing a nanocomposite for biomolecule binding, the method for manufacturing a biocombination nanocomposite according to an embodiment of the invention is not limited thereto.

According to the present invention, a biomolecule binding nanocomposite and a method for preparing the biomolecule which can be stably bonded to the inside of the nanocomposite surface as well as maintaining the activity are provided.

Hereinafter, various embodiments of the present invention will be described. However, this is presented as an example of the invention, whereby the scope of the invention is not limited.

[ Example   : Preparation of Biomolecule Binding Nanocomposite]

< Example  One : QCM of As a solid support  One Nanocomposite>

Gold-deposited quartz crystal microbalance (QCM) was washed with piraha solution (96% sulfuric acid / 30-35.5% hydrogen peroxide, 3/1, v / v), followed by 10 mM 2-mertoethanol (2 A self-assembled thin film was prepared by reacting for 12 hours in a mercaptoethanol) / ethanol solution, which was used as a solid support. The solid support was ethanol / toluene (1/1) in which titanium (IV) -n-butoxide; (Ti (O- ⁿ Bu) ₄ ) / Acros Chem (USA) was dissolved at a concentration of 100 mM. , v / v) in solution for 3 minutes. Thereafter, the resultant metal oxide physically adsorbed in excess was removed using an ethanol solution and hydrolyzed for 1 minute in distilled water. After drying with nitrogen gas, a first metal oxide film was introduced onto the solid support.

The resultant on which the first metal oxide film is formed is reacted with a distilled water solution in which poly (acrylic acid, PAA) is dissolved at a concentration of 1wt% for 30 minutes, and the physical adsorption on the surface is washed twice with distilled water every 30 seconds. To remove. The resultant was dried using nitrogen gas to introduce an ionic polymer membrane (PAA membrane).

By forming the first metal oxide film and the ionic polymer film (PAA film) as one cycle, when the cycle was repeated, the thickness of the biomolecule binding nanocomposite thin film of the present invention was controlled at the nano level. After the process of forming the first metal oxide film and the ionic polymer film was repeated one or more times, a second metal oxide film was formed on the resultant produced by the same method as the method of preparing the first metal oxide film.

In this case, the (TiO ₂ / PAA) _N.5 nanocomposite means that N composite thin films including the first metal oxide film and the PAA are formed and a second metal oxide film is formed on the surface.

< Example  2: enzyme Combined  Nanocomposites>

Nanocomposite nanocomposite prepared by the method of Example 1 is an enzyme cytochrome seed (Cyt. C ) It was reacted for 30 minutes at room temperature in dissolved phosphate buffer solution (pH 7). 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: Quartz plate As a solid support  One Nanocomposite>

The quartz plate was washed with sulfuric acid (H ₂ SO ₄ 96.0%), and then ultrasonicated with KOH solution to introduce an active group to the surface of the quartz plate to use as a solid support. The solid support was prepared in the same manner as in Example 1 to prepare a metal oxide film and a PAA film, to prepare a nanocomposite including a TiO ₂ / PAA composite thin film. An enzyme was bound to the prepared nanocomposite by the method of Example 2.

[ Comparative example  1: Preparation of Metal Oxide Multilayer Thin Film]

Except for forming a PAA film, a metal oxide film was formed in the same manner as in Example 1 to prepare a metal oxide multilayer thin film including only the metal oxide film of TiO ₂ . Cyt.c was bonded to the metal oxide multilayer thin film prepared as described in Example 2.

[ Experimental Example  ]

< Experimental Example  One : QCM Frequency Change Measurement of>

By measuring the frequency of the QCM it was confirmed that each film is formed in the manufacturing process of the biomolecule binding nanocomposites.

One. Experimental Example  1-1

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 ionic polymer film, thereby obtaining the average frequency change when each film was formed.

In Figure 3 shows the frequency change of the QCM by the sequential formation of Ti (O- ⁿ Bu) ₄ and PAA. The average frequency change ΔF of 28 cycles by Ti (O- ⁿ Bu) ₄ binding was 28 ± 8 Hz, and the average frequency change ΔF of 26 cycles by PAA binding was 26 ± 16 Hz. The results were obtained by using a crystallization microbalance (QCM) manufactured by USI system (Japan) and a frequency measuring device of Hewlett Packard (USA), and the average frequency of 10 cycles using TiO ₂ / PAA composite thin film manufacturing process as one cycle. Change.

The crystallite microbalance (QCM) changes the resonance frequency in proportion to its mass even when a very small amount of nanogram material is adsorbed. Thus, the result shows that the composite thin film is uniformly formed at a molecular level on the solid support. It can be confirmed.

2. Experimental Example  1-2

Produced in Example 2 The formation of enzyme-bound nanocomposites was confirmed using QCM frequency changes. The frequency of the QCM was measured by the same method as in Experimental Example 1-1.

4 shows Cyt. The frequency change of c -bonded (TiO ₂ / PAA) _3.5 nanocomposite is shown. (TiO ₂ / PAA) _{3.5 The} average frequency change by Ti (O- ⁿ Bu) ₄ bonding of composite thin film was ΔF = 26 ± 11 Hz, and the average frequency change by PAA coupling was ΔF = 41 ± 20 Hz, Cyt. The frequency change of c was ΔF = 535 ± 34 Hz.

5 shows Cyt. The frequency change of the c -bonded (TiO ₂ / PAA) _10.5 nanocomposite was shown. (TiO ₂ / PAA) The average frequency change by Ti (O- ⁿ Bu) ₄ bond of _10.5 composite thin film was ΔF = 28 ± 8 Hz, and the average frequency change by PAA coupling was ΔF = 26 ± 16 Hz, Cyt. The change in frequency of c was ΔF = 1841 ± 526 Hz.

As shown in Figures 4 and 5, it was confirmed that the metal oxide film and the PAA film are uniformly and stably formed through the frequency change of the quartz crystal microbalance (QCM). In addition, Cyt. It was confirmed that the biomolecules were stably bound to the nanocomposite due to the frequency change due to the binding of c .

3. Experimental Example  1-3

The frequency change of the metal oxide multilayer thin film prepared in Comparative Example 1 and the Cyt.c was bonded to the nanocomposite prepared in Example 1 was compared. The frequency change was measured by using a crystallization microbalance (QCM) manufactured by USI system (Japan) and a frequency measuring instrument of Hewlett Packard (USA), and averaged the results of five replicates.

6 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. In the metal oxide multilayer thin film of Comparative Example 1, it was confirmed that the amount of Cyt.c bonded was constant regardless of the increase in the number of cycles. On the contrary, the nanocomposite of Example 1 was confirmed that the amount of the enzyme is also regularly increased as the cycle increases. These results show that the nanocomposite of Example 1, unlike the metal oxide multilayer thin film of Comparative Example 1, the enzyme moves to the inside and is bonded.

< Experimental Example  2: measurement of absorbance change>

Formation of the enzyme-bound nanocomposite prepared in Example 3 was confirmed using a change in the absorbance of the UV-vis spectrometer (the change in absorbance (λ _max = 409 nm) due to porphyrin molecules in the enzyme). The change in absorbance was confirmed using a UV-vis spectrometer (Lambda 35, Perkin Elmer) and showed an average peak after three replicates.

7 is Cyt. The UV-vis spectrometer changes of c -bonded (TiO ₂ / PAA) _3.5 nanocomposites. As it is seen in Figure _{7, (TiO 2 / PAA)} 3.5 nm For complexes in the vicinity of 250nm (TiO ₂ / PAA) present only characteristic peak by _3.5 nanocomposites but, Cyt. When c is combined, Cyt. at 409 nm. The characteristic peak by the porphyrin molecule in c appeared. The absorbance changes of the UV-vis spectrometer showed that the biomolecules were stably bound to the (TiO ₂ / PAA) nanocomposite.

8 is a nanocomposite of 1.5, 3.5, 5.5, 10.5 cycles by the method of Example 1 and Cyt. It shows the change of UV-vis spectrometer measured after binding c . In Figure 8 it can be seen that as the results of Experimental Example 1-3, the amount of enzyme bound is also increased regularly as the thickness of the nanocomposite increases by the number of cycles. This shows that the nanocomposite of Example 1, unlike the metal oxide multilayer thin film of Comparative Example 1, is enzymatically bound to the inside.

< Experimental Example  3: Cyclic Voltametry ( Cyclic voltammetry ; CV ) Measurement>

Cyclic voltammetry was used to confirm the electrochemical properties of the enzyme-bound nanocomposites.

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

9 shows (TiO ₂ ) ₃ , (TiO ₂ ) _{3.5 /} Cyt. In phosphate buffer solution (pH 7). c, (TiO ₂ / PAA) _{3.5 /} Cyt. c Changes in the electrochemical properties of the nanocomposite. For the third cycle the metal oxide multi-layer thin film ((TiO _{2) 3)} of the, 200 and though the current value changes in nearly 300mV vicinity or not appear, the enzyme-linked metal oxide multi-layer thin film _{((TiO 2) 3 / Cyt} . C) and nanocomposite _{((TiO 2 / PAA) 3.5} / Cyt. c), the iron's oxidation and reduction peaks in the enzyme were observed for.

In addition, the metal oxide multilayer films the enzymes are combined ((TiO _{2) 3)} nanocomposite of the more enzymes is combined _{TiO 2 / PAA ((TiO 2} / PAA) 3.5 / Cyt. C) three or more times the oxidation and the reduction in peak It was confirmed that there was an increase in the (current value). This is because the metal oxide multilayer thin film simply binds the enzyme to the outermost metal oxide surface, whereas in the nanocomposite of TiO ₂ / PAA, the enzyme moves and binds to the inside. Current value) is increased relatively.

Figure 10 shows the results of observing the electrochemical properties according to the thickness of the nanocomposite conjugated enzyme. Oxidation / reduction peak (current value) increased proportionally as the thickness of the enzyme-conjugated nanocomposite increased. These results were as in the results of Experimental Examples 1-3 and Experimental Example 2, and the thickness of the composite thin film. As is increased, the amount of enzyme bound is increased.

From the above results, it was confirmed that the biomolecule binding nanocomposite according to the embodiment of the present invention can bind the biomolecule to the inside of the nanocomposite while maintaining the activity.

< Experimental Example  4: Photoelectron spectroscopy experiment>

9.5 cycles of the nanocomposite (TiO ₂ / PAA) _9.5 and 9.5 cycles of the nanocomposite (TiO ₂ / PAA) _9.5 /Cyt.c prepared by the method of Example 1 or Example 2 The composition of each nanocomposite was confirmed using 210 products, VG Science Co., Ltd.).

In Fig. 11, (TiO ₂ / PAA) _9.5 overlaps the characteristic peaks due to Ti of the metal oxide near 465 eV, the carbon peak of PAA at 295 eV, and the oxygen peak of oxygen and PAA at 536 eV. Is showing. _{However, (9.5 / Cyt (TiO 2} / PAA). C) the enzyme is combined nanocomposite In got a new peak in the vicinity of 405 eV displayed by the nitrogen atom of the peptide molecules present in the enzyme as a whole peak of the metal oxide is an enzyme It can be seen that it does not appear superimposed on the peak of the molecule.

From these results, it can be seen that the enzyme is bound to the nanocomposite.

< Experimental Example  5: Atomic force  microscope( Atomic Force Microscope , AFM Surface observation with)

A nanocomposite was prepared in the same manner as in Example 1 except that the surface of the surface was washed with ethanol (Mica) as a solid support, and Cyt.c was bound by the method of Example 2. After that, (TiO ₂ / PAA) _3.5 And (TiO ₂ / PAA) _{3.5 /} Cyt. c The surface of the nanocomposite was observed using an atomic force microscope (JSPM-5200, JEOL). As a result, (TiO ₂ / PAA) _3.5 The root-mean-sequare (RMS) roughness of the nanocomposite is 0.524 nm, (TiO ₂ / PAA) _{3.5 /} Cyt. c The root-mean-sequare (RMS) roughness of the nanocomposite was 1.02 nm and the RMS roughness of the nanocomposite with the biomolecules was increased. These results indicate that Cyt. Pinned to the surface of the nanocomposite. c increases the RMS roughness. In addition, Cyt. It was confirmed that c penetrated to the inside and stably fixed, and as shown in the atomic force microscope measurement results, each of the membranes of the nanocomposite was also found to be porous from 2 nm to 100 nm.

12 and 13 are respectively (TiO ₂ / PAA) _3.5 And (TiO ₂ / PAA) _{3.5 /} Cyt. c AFM images of nanocomposites and root-mean-sequare (RMS) roughness.

< Experimental Example  6: electron microscope Scanning electron microscope , SEM Observation of Nanocomposites Using

Cyt.c was bound to the hex complex prepared by the method of Example 1 by the method of Example 2. The cross section of the (TiO ₂ / PAA) _10.5 / Cyt.c thus prepared was observed using an electron microscope (Carl LEO-1530_, Zeiss). As shown in FIG. 14, it was confirmed that the nanocomposite having a thickness of 45 ± 5 nm was uniformly formed on the QCM substrate.

Figure 1 shows an example of the structure of the nanocomposite for biomolecule binding.

Figure 2 shows an example of a method for producing a nanocomposite for biomolecule binding.

Figure 3 shows the frequency change of the QCM according to the sequential coupling of Ti (O- ⁿ Bu) ₄ and PAA.

Figure 4 shows the change in the frequency according to the (TiO ₂ / PAA) _3.5 nanocomposite forming step combined with Cyt.c.

Figure 5 shows the change in the frequency according to the (TiO ₂ / PAA) _10.5 nanocomposite forming step combined with Cyt.c.

FIG. 6 shows a comparison of Cyt.c coupling frequency changes according to thickness variation of a nanocomposite including a metal oxide multilayer thin film and a TiO ₂ / PAA composite thin film.

Figure 7 shows the UV-vis spectrometer changes of (TiO ₂ / PAA) _{3. 5} nanocomposites according to the binding of Cyt.c.

Figure 8 shows the UV-vis spectrometer changes according to the thickness change of the nanocomposite combined Cyt.c.

9 shows the difference in electrochemical properties of nanocomposites including a metal oxide multilayer thin film and a TiO ₂ / PAA composite thin film.

Figure 10 shows the electrochemical change according to the thickness of the nanocomposite including TiO ₂ / PAA composite thin film.

11 shows the composition of the nanocomposite and the Cyt. It shows the change of the nanocomposite composition when combining c .

Figure 12 (TiO ₂ / PAA) _3.5 AFM images of the nanocomposites and root-mean-sequare (RMS) roughness are shown.

Figure 13 (TiO ₂ / PAA) _{3.5 /} Cyt. c AFM images of nanocomposites and root-mean-sequare (RMS) roughness.

14 shows TiO ₂ / PAA) _{10.5 /} Cyt. c SEM photograph of the nanocomposite and the thickness of the nanocomposite cross section on the solid support.

Claims (21)

Solid supports comprising surface-active groups or electrostatically interacting with metal oxides; A composite thin film including a first metal oxide film and an ionic polymer film sequentially formed on the solid support; And A second metal oxide film formed on the composite thin film, The first or second metal oxide film includes an oxide of a metal selected from the group consisting of titanium, zirconium, aluminum, boron, silicon, indium, tin, barium and vanadium or two or more composite metal oxides, The ionic polymer membrane is polyacrylic acid and its derivatives, cationic and anionic polysaccharides and polysaccharide derivatives, nucleic acids, polymethacrylic acid and its derivatives, maleic anhydride copolymers, cationic acrylic esters and their copolymers, polyethylene imines, polyamines , At least one selected from the group consisting of polyamide amine and polydiallyldimethylammonium chloride, Nanocomposite for biomolecule binding. delete The method of claim 1, Wherein said surface active group is at least one functional group selected from the group consisting of active hydrogen, hydroxyl group, carboxyl group, sulfonic acid group, phosphoric acid group, amine group, ammonium group and pyridine group. delete The method of claim 1, The first and the second metal oxide film and the ionic polymer membrane is a nano-scale porous membrane nanocomposite binding for nanocomposite. The method of claim 1, The ionic polymer membrane is bonded to the metal oxide film by the electrostatic interaction, or a nanocomposite binding nanocomposite that is bonded by forming a complex via an active functional group. delete The method of claim 1, The nanocomposite for biomolecule binding further comprising at least one composite thin film comprising the first metal oxide film and the ionic polymer film. The method of claim 1, The first metal oxide film or the second metal oxide film is a nanocomposite for biomolecule binding comprising a monomolecular film of a metal oxide. The method of claim 1, The ionic polymer membrane is a nanocomposite for biomolecule binding comprising a monomolecular membrane of ionic polymer. The method of claim 1, The nanocomposite for biomolecule binding further comprising a biomolecule bonded to the first or second metal oxide film. The method of claim 11, The biomolecule is a nanocomposite for biomolecule binding comprising an enzyme. Forming a first metal oxide film on the solid support including the surface active group or in electrostatic interaction with the metal oxide; Forming an ionic polymer film on the first metal oxide film; And Forming a second metal oxide film on the ionic polymer film, The first or second metal oxide film includes an oxide of a metal selected from the group consisting of titanium, zirconium, aluminum, boron, silicon, indium, tin, barium and vanadium or two or more composite metal oxides, The ionic polymer membrane is polyacrylic acid and its derivatives, cationic and anionic polysaccharides and polysaccharide derivatives, nucleic acids, polymethacrylic acid and its derivatives, maleic anhydride copolymers, cationic acrylic esters and their copolymers, polyethylene imines, polyamines , At least one selected from the group consisting of polyamide amine and polydiallyldimethylammonium chloride, The method for producing a nanocomposite for biomolecule binding. The method of claim 13, Before the forming of the first metal oxide film, the surface of the solid support further comprising the step of ultrasonic treatment or self-assembled thin film forming treatment in plasma treatment, ozone treatment, alkali or acid solution. The method of claim 13, The forming of the first or second metal oxide film may include surface sol-gel reaction of a result of forming a solid support or an ionic polymer film in a metal oxide precursor solution (or metal oxide in a gas phase). Nanocomposite manufacturing method. The method of claim 13, Forming an ionic polymer film on the first metal oxide film comprises the step of reacting the product formed with the metal oxide film in a polymer electrolyte solution. The method of claim 16, The polymer electrolyte may include polyacrylic acid and derivatives thereof, cationic and anionic polysaccharides and polysaccharide derivatives, nucleic acids, polymethacrylic acid and derivatives thereof, maleic anhydride copolymers, cationic acrylic esters and copolymers thereof, polyethylene imines, polyamines, Polyamide amine and polydiallyldimethylammonium chloride. A method for producing a nanocomposite binding nanocomposite comprising any one or more selected from the group consisting of chloride. The method of claim 13, Before forming the second metal oxide film, repeating the forming of the first metal oxide film and the forming of the ionic polymer film two or more times. The method of claim 13, The method of manufacturing a nanocomposite for biomolecule binding further comprising the step of bonding a biomolecule to the nanocomposite on which the second metal oxide film is formed. The method of claim 19, The step of binding the biomolecules comprises the step of reacting the nanocomposite in a solution in which the biomolecules are dissolved for 1 to 60 minutes at 20 to 40 ℃. The method of claim 19, The biomolecule is a nanocomposite manufacturing method for binding a biomolecule comprising an enzyme.

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