KR20150001375A - Platinum Nanoparticles-Magnetic Nanoparticles-Graphene Oxide Hybrid and Method for Preparing the Same - Google Patents

Abstract

The present invention relates to a platinum nanoparticle-magnetic nanoparticle-graphene oxide hybrid material and to a method for preparing the same. The platinum nanoparticle-magnetic nanoparticle-graphene oxide hybrid material having platinum nanoparticles fixed on the surface of the magnetic nanoparticles and a graphene oxide is manufactured by a method which comprises the steps of (a) fixing magnetic nanoparticles on a graphene oxide to manufacturing a magnetic nanoparticle-graphene oxide hybrid material; and (b) fixing platinum nanoparticles on the magnetic nanoparticle-graphene oxide hybrid material to manufacture platinum a nanoparticle-magnetic nanoparticle-graphene oxide hybrid material. The hybrid material has high activity as an extremely improved peroxidase compared with a case of using only existing magnetic nanoparticles to fix separately or with an organic enzyme to be used as a substitute for a peroxidase in the enzyme-linked immunosorbent assay (ELISA), biosensor, or immunodiagnosis chemistry using enzymes.

Description

TECHNICAL FIELD The present invention relates to platinum nanoparticles, magnetic nanoparticles, and graphene oxide grains,

The present invention relates to a platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid and a preparation method thereof, and more particularly, to an enzyme immunoassay (ELISA) using an enzyme, a biosensor, or a substitute for a peroxidase Usable platinum nanoparticles-magnetic nanoparticles-oxidized graphene hybrids and methods for their production.

Currently, enzyme-linked immunosorbent assay (ELISA) -based immunodiagnostic methods based on enzyme reactions are widely used for the detection of a wide range of diseases and environmentally harmful substances. The principle of ELISA is to detect the presence or absence of an antigen or an antibody with a high sensitivity by causing a reaction between a specific substrate and an enzyme using a primary antibody that binds to an antigenic substance and a secondary antibody-enzyme complex that binds thereto. Because this method has the advantage of being simple and inexpensive, to date, most commercialized immunoassay kits follow this approach. In such ELISA techniques, the enzyme most widely used for secondary antibody-enzyme conjugates is an organic enzyme such as peroxidase or phosphatase. However, since the organic enzyme used in the conventional ELISA method is not sufficiently sensitive due to limited enzyme activity, enzyme activity greatly changes depending on the surrounding environment, reaction conditions, and storage time, resulting in a negative problem in the reliability of diagnosis .

The organic peroxidase is also widely used as a biosensor in combination with various oxidases (Oxidase). When the target substance to be detected is present in the sample, hydrogen peroxide (H ₂ O ₂ ) is generated through the reaction of the oxidase using the target substance as a substrate, and the organic peroxidase oxidizes the specific substrate while reducing the organic peroxidase, A target substance is detected by inducing a signal. However, in the above-described biosensor, there is a problem that the activity of the enzyme decreases with time, and the enzyme activity greatly changes depending on the surrounding environment or the reaction condition, resulting in a problem in the reliability of the result.

Various types of enzyme analogues have been developed to solve the problems of organic enzymes as described above. Recently, it has been reported that oxidized graphene has activity of peroxidase (Song, YJ; Qu, KG; Zhao, C .; Ren, JS; Qu, XG Advanced Materials 2010 , 22 , 2206-2210). They have found new properties of oxidized graphene and, coupled with glucose oxidase reaction, have shown that glucose concentration in the sample can be quantitated by colorimetric analysis. In addition, graphene oxide can be mass-produced chemically inexpensively and is more stable to external conditions such as acidity and temperature than an organic peroxidase having inevitable instability according to reaction time. Therefore, .

In addition, by binding antibody to graphene graphene grains, it is possible to capture and isolate target cancer cells and proteins, and then to detect the complex activity of hemin with its role as a peroxidase enzyme or as a peroxidase enzyme in neutral acidity by obtaining a signal shown by the possibility of a new immunodiagnostic system development (Song, YJ; Chen, Y .; Feng, LY; Ren, JS;. Qu, XG Chemical Communications 2011, 47, 4436-4438 Qu, FL; Li, T. Yang, MH Biosensors & Bioelectronics 2011 , 26 , 3927-3931). They capture target cancer cells or proteins using antibodies and then isolate them using centrifugation or magnetic separation to diagnose color by using TMB (3,3 ', 5,5'-tetramethylbenzidine) as a substrate, It has been demonstrated that fins can be used as peroxidase substitutes.

However, the graphene oxide has insufficient activities and sensitivities per unit of the graphene, so that it is not easy to use in actual immunodiagnosis, and accessibility with other organic enzymes is insufficient.

The present inventors have made intensive efforts to solve the above problems. As a result, when the platinum nanoparticles are immobilized on the surface of the oxidized graphene together with the magnetic nanoparticles, the activity of the peroxidase is dramatically improved. As a result, And can be used in enzyme immunoassay, biosensor, or immunohistochemistry. The present invention has been completed based on this finding.

An object of the present invention is to provide a platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid which can be used as a substitute for peroxidase in enzyme immunoassay, biosensor or immunohistochemistry and has excellent stability and sensitivity, and a method for producing the same have.

In order to achieve the above-mentioned object, the present invention provides a method for manufacturing a magnetic recording medium, which comprises a magnetic nanoparticle having magnetic nanoparticles fixed on the surface of a graphene oxide and platinum nanoparticles fixed on the surfaces of the magnetic nanoparticles and the graphene oxide, Thereby providing an oxide graphene hybrid body.

The present invention also provides a method of manufacturing a magnetic nanoparticle-grafted oxide composite, comprising: (a) fixing magnetic nanoparticles to an oxidized graphene to produce a magnetic nanoparticle-oxidized graphene hybrid; And (b) fixing the platinum nanoparticles to the magnetic nanoparticle-oxidized graphene hybrid to produce a platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid, wherein the platinum nanoparticle-magnetic nanoparticle-oxidized Thereby producing a graphene hybrid body.

The present invention is also characterized in that the magnetic nanoparticles are fixed on the surface of the graphene oxide, the platinum nanoparticles are fixed on the surface of the magnetic nanoparticles and the graphene oxide, and the active ester is fixed on the surface of the graphene oxide Platinum < / RTI > nanoparticle-magnetic nanoparticle-oxidized graphene hybrid.

The present invention also relates to a method for producing a platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid comprising the steps of mixing N- (3-Dimethylaminopropyl) -N '(3-dimethylaminopropyl) N-Hydroxysulfosuccinimide sodium salt (sulfo-NHS) is reacted with N-hydrocarbodiimide hydrochloride (EDC) to form a platinum nanoparticle-magnetic nano- Platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid, characterized in that it comprises the steps of: preparing an active ester-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid; .

The present invention also provides an immunoassay method comprising reacting the active ester-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid with an antibody.

The present invention is also characterized in that the magnetic nanoparticles are fixed on the surface of the graphene oxide, the platinum nanoparticles are fixed on the surface of the magnetic nanoparticles and the graphene oxide, and the oxidizing enzyme is accumulated on the surface of the graphene oxide Platinum < / RTI > nanoparticles-magnetic nanoparticles-oxidized graphene hybrids.

The present invention also provides a method for producing an oxidase-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid comprising the step of accumulating an oxidase in the platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid do.

The present invention also provides a method for color development diagnosis comprising the step of reacting the oxidase-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid with a chromogenic substrate.

The platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid according to the present invention exhibits a high activity as a peroxidase, which is remarkably improved as compared with the case of using only conventional magnetic nanoparticles. Therefore, Can be used as a highly sensitive inorganic signal derivative of a diagnosis, has an effect of showing a high-sensitivity color development diagnosis of a substance to be a substrate of an enzyme, and is reusable and economical.

1 is a view showing a reaction mechanism of a platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid according to an embodiment of the present invention, and a color development diagnosis of a target cancer cell.
FIG. 2 is a graph showing the results of measurement of (a) graphene oxide (GO), (b) magnetic nanoparticle-oxide graphene hybrid (10 wt% Magnetite nanoparticles on graphene oxide (GO_MNP- c) platinum nanoparticles-oxidized graphene oxide (GO_Pt-10), 10% by weight platinum nanoparticles-magnetic nanoparticles-oxidized graphene hybrid [10 wt% Magnetite nanoparticles & 10% by weight platinum nanoparticles on graphene oxide (GO_MNP-10 / Pt-10)] by a transmission electron microscope (TEM).
FIG. 3 is a graph showing the results of measurement of (a) graphene oxide (GO), (b) magnetic nanoparticle-oxidized graphene hybrid (10 wt% magnetic nanoparticle-oxidized graphene: GO_MNP-10) (c) a magnetic nanoparticle-oxidized graphene hybrid (30 wt% magnetic nanoparticles-oxidized graphene: GO_MNP-30), (d) platinum nanoparticles-oxidized graphene hybrid (10 wt% (Graphene: GO_Pt-10), (e) platinum nanoparticles-magnetic nanoparticles-oxidized graphene hybrid (10 wt% magnetic nanoparticles-10 wt% platinum nanoparticles-oxidized graphene: GO_MNP-10 / ) and (f) platinum nanoparticles-magnetic nanoparticles-oxidized graphene hybrid (30 wt% magnetic nanoparticles-10 wt% platinum nanoparticles-oxidized graphene: GO_MNP-30 / Pt-10).
FIG. 4 is a graph showing the activity of the platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid according to an embodiment of the present invention as an activity of (a) peroxidase and (b) 10, (6): GO_MNP-30 / Pt-10, (7): GO_MNP-10, : Negative control].
FIG. 5 is a graph showing the state-of-the-art steady-state (TG) state of a platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid according to an embodiment of the present invention using TMB (3,3 ', 5,5'-tetramethylbenzidine) 10, (c, d), (10), (10), (10) : Platinum nanoparticles, (e, f) organic peroxidase (HRP)].
FIG. 6 is a graph showing a reaction formula for measuring and quantifying a target substrate with a color change using an oxidase-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid according to an embodiment of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

In the present invention, the platinum nanoparticle-magnetic nano-particle-oxide graphene hybrid prepared by integrating the platinum nanoparticles and the magnetic nanoparticles on the surface of the oxidized graphene has excellent peroxidase activity, so that it can be detected by enzyme immunoassay, And can be used in immunohistochemistry.

In the present invention, platinum nanoparticles are integrated with the magnetic nanoparticles on the surface of the oxidized graphene. As a result, it was confirmed that the activity of the peroxidase was remarkably improved in the platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid.

That is, in one embodiment of the present invention, it was confirmed that the platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid can be used instead of peroxidase in enzyme immunoassay, biosensor, and immunohistochemistry.

Therefore, in one aspect of the present invention, there is provided a magnetic recording medium comprising magnetic nano particles fixed on the surface of a graphene oxide, platinum nano particles fixed on the surface of the magnetic nano particles and oxidized graphene, Graphene hybrid.

In the present invention, the magnetic nanoparticles have a particle diameter of 4 nm to 6 nm, an average diameter of about 5 nm, platinum nanoparticles have a particle diameter of 3 nm to 5 nm, and an average diameter of about 4 nm . The activity as the highest enzyme is exhibited when the particle diameter is in the range of the particle diameter, and the activity is seriously inhibited when each nanoparticle has a diameter of 10 nm or more.

Also, in the present invention, the magnetic nanoparticles and the platinum nanoparticles are each prepared at a weight ratio of 10 wt% to 30 wt%, and the highest enzyme activity is measured at the above range.

In the present invention, the graphene oxide provides a large surface area of 332 m ² / g, resulting in the formation of inorganic nanoparticles of ~ 5 nm in size and inorganic nanoparticles of ~ 4 nm in size, And by immobilizing various organic enzymes on the remaining surface area, a simple organic / inorganic hybrid substance can be realized. In addition, by using the synergistic effect of magnetic nanoparticles and platinum nanoparticles, the activity as a peroxidase is increased, and the presence and concentration of the substrate of the enzyme is colorimetrically reacted through the linkage reaction of the immobilized enzyme, magnetic nanoparticles and platinum nanoparticles So that it can be quantified easily and quickly.

In the present invention, the graphene graphene is prepared by the Hummers method based on the oxidation of graphite powder (SP-II graphite) (WS Hummers, RE Offeman, J. Am. Chem. Soc. 1958 , 80 , 1339-1339 ). ≪ / RTI > The graphite powder was mixed with H ₂ SO ₄ , K ₂ S ₂ O ₈ and P ₂ O ₅ and oxidized at 80 ° C. for 4 hours to form a pre-oxidized graphite oxide. After filtration and separation and purification H ₂ SO ₄ and KMnO _4, and further oxidized at 35 ° C for 2 hours, reacted again at 35 ° C for 2 hours, and further reacted with 30% H ₂ O ₂ to obtain a yellowish brown powder. The resulting powder is washed 5 times with 1 M HCl, washed with distilled water, sonicated for 2 hours, and the oxidized graphite is removed to obtain the graphene oxide.

In the present invention, the magnetic nanoparticles are generally ferromagnetic particles having a size of about 10 nm. Sorters may include iron oxide _{(Fe 2 O 3, Fe 3} O 4) and Ferrite (Fe ₃ O ₄ Fe in one other magnetic related reactor changed _{form, CoFe 2 O 4, MnFe 2} O 4). When magnetic nanoparticles are practically used, magnetic nanoparticles are not used in powder form because of their ease of use, but they are used in various fields by making them dispersed in liquid. In this case, magnetic particles in ferrofluid Since it must be dispersed well without being known, appropriate surfactants or hydrocarbon rings are attached to the surface of magnetic particles to induce dispersion well, and co-precipitation method is mainly used.

According to another aspect of the present invention, there is provided a method of manufacturing a magnetic nanoparticle-grafted oxide composite, comprising: (a) preparing a magnetic nanoparticle-oxidized graphene hybrid by immobilizing magnetic nanoparticles on the graphene oxide; And (b) fixing the platinum nanoparticles to the magnetic nanoparticle-oxidized graphene hybrid to produce magnetic nanoparticle-platinum nanoparticle-oxidized graphene, wherein the platinum nanoparticle-magnetic nanoparticle-oxidized graphene And a method for producing a hybrid body.

In the present invention, the fixing of step (a) is performed by mixing FeCl ₃ / FeCl ₂ solution with oxidized graphene and precipitating under basic conditions of adjusting the acidity to pH 10 using an aqueous ammonia solution. In a preferred embodiment The preparation of the magnetic nanoparticle-graphene oxide grains in step (a) is performed by mixing FeCl ₃ : FeCl ₂ = 2: 1 molar ratio with oxidized graphene, and mixing the mixture at a pH of 10 with ammonia water of 30% Reaction, and uniform precipitation, whereby the magnetic nanoparticle-oxidized graphene hybrid body can be produced.

In the present invention, the magnetic nanoparticle-platinum nanoparticle-oxidized graphene in the step (b) is prepared by first mixing ethylene glycol solution with magnetic nanoparticles-oxidized graphene and uniformly dispersing the solution, ₂ PtCl ₆ 6H ₂ O at 125 to 150 ° C for 2 hours The platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid particles can be produced.

In the present invention, the magnetic nanoparticle-oxidized graphene hybrid body can be easily produced by uniformly depositing the magnetic nanoparticles on the surface of the oxidized graphene, and the platinum nanoparticles are also immobilized on the oxidized graphene, - oxidized graphene hybrid. The deposition is preferably performed using a high-temperature deposition method.

In the present invention, it was possible to develop magnetic nanoparticle-platinum nanoparticle-oxidized graphene, which is an inorganic hybrid material capable of quantitative analysis with high sensitivity using color without using an organic peroxidase by performing immobilization.

Meanwhile, it can be predicted that the active nanoparticle-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid material can be used for immunodiagnosis by producing an active ester through EDC + NHS chemistry and binding to an amine group of an antibody.

Therefore, the present invention provides a method for manufacturing a magnetic recording medium in which magnetic nanoparticles are fixed on the surface of a graphene oxide, the platinum nanoparticles are fixed on the surfaces of the magnetic nanoparticles and the graphene oxide, Platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid having the formula (1), wherein the ratio of the carboxyl group (-COOH) of the platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid to the active ester- -Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC) with sodium hydroxide (sulfo-NHS) in the presence of N-hydroxide to form platinum nanoparticles-magnetic nano- Comprising the steps of: preparing an active ester-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid with an ester on its surface Ester-platinum nanoparticles-magnetic nano-particles-oxide graphene hybrid.

The present invention also relates to an immunodiagnostic method comprising the step of reacting the above-mentioned active ester-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid with an antibody.

In the present invention, the magnetic nanoparticles, the platinum nanoparticles, and the oxidized graphene are the same as those used in the platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid.

Also, in the present invention, it is preferable to use a mixture of the N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC), the N-hydroxy pseudosuccinimide sodium salt (sulfo-NHS) The magnetic nanoparticles and the oxidized graphene hybrid are used at a concentration of 20 to 200 mM, 5 to 50 mM, and 1 to 2 mg / mL, respectively. When the concentration is within the above range, the active ester is most appropriately formed and a large amount of antibody is modified .

The platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid of the present invention can be applied as an effective signal derivative for high-sensitivity immunodiagnosis by obtaining activity as a peroxidase which is remarkably improved as compared with the case of using only magnetic nanoparticles. Although the peroxidase is inherently an organic enzyme, it has inevitable denaturation of protein structure and a decrease in activity over time. However, since the hybrid substance of the present invention uses an inorganic magnetic nanoparticle that can be recovered instead of a peroxidase enzyme, The use of nanoparticles results in synergistic effects for increased activity as peroxidase. In addition, it is possible to carry out high sensitivity quantitative analysis by increasing the activity per unit individual by integrating in a large surface area graphene oxide, and it is possible to separate and reuse more efficiently by magnetic force. On the other hand, quantitative analysis of important substances through linkage reaction using additional immobilization of organic enzymes is an excellent method in terms of diagnostic cost since it can be easily performed by color difference through the naked eye.

The magnetic nanoparticle-platinum nanoparticle-oxidized graphene developed by the present invention binds a specific antibody thereto, thereby providing a signal derivative of an immunodiagnosis of proteins, DNA, pathogens and the like, which causes diseases that provide specific information on the progress of disease .

According to another aspect of the present invention, there is provided a magnetic recording medium comprising magnetic nanoparticles fixed on the surface of a graphene oxide, platinum nanoparticles fixed on the surfaces of the magnetic nanoparticles and the graphene oxide, Platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid, wherein the oxidized enzyme-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid is integrated with the oxidized enzyme-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid - Platinum nanoparticles - Magnetic nanoparticles - Oxidation graphene hybrid bodies.

In the present invention, the oxidizing enzyme is selected from the group consisting of a glucose oxidase, a cholesterol oxidase, a galactose oxidase, a pyruvate oxidase and an alcohol oxidase, but is not limited thereto.

In the present invention, the magnetic nanoparticles, the platinum nanoparticles, and the oxidized graphene are the same as those used in the platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid.

(EDC), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), platinum nanoparticles-magnetic nanoparticles-oxidized grains (N-ethylcarbodiimide hydrochloride The pinholes are used in concentrations of 20 to 200 mM, 5 to 50 mM, and 1 to 2 mg / mL, respectively. When the concentration is within the above range, active ester is appropriately formed and a large amount of antibody is modified.

In the present invention, by immobilizing the magnetic nanoparticle-platinum nanoparticle-oxidized graphene together with the organic oxidizing enzyme, the magnetic nanoparticle-platinum nanoparticle-oxidized graphene is immobilized on the substrate of the enzyme for a small molecule such as glucose, galactose, cholesterol, liver function factor (GPT, GOT) And the concentration can be measured. In order to efficiently immobilize the organic oxidizing enzyme, the magnetic nanoparticle-platinum nanoparticle-active ester on the surface of the oxidized graphene can be cross-linked with the amine of the enzyme and can be accumulated at high density on the surface.

The present invention also relates to a method for color development diagnosis comprising the step of reacting the oxidase-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid with a chromogenic substrate.

In the present invention, the coloring substrate may be ABTS (3-ethylbenzothiazoline-6-sulphonic acid), TMB (3,3 ', 5,5'-tetramethylbenzidine) Diamine dihydrochloride (OPD), 3,3'-diaminobenzidine (DAB) and 10-acetyl-3,7-dihydroxyphenoxazine (10-acetyl- 3,7-dihydroxyphenoxazine, Amplex-Red). However, the present invention is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

[Example]

Example  One: Platinum  Nanoparticles - Magnetic nanoparticles - Oxidation Grapina  Manufacture of hybrid and application to immunodiagnosis

<Step 1> Preparation of oxidized graphene

Oxidative graphene is characterized by the synthesis of graphite powder (SP-II graphite) based on the Hummers method (WS Hummers, RE Offeman, J. Am. Chem. Soc., 1958, 80, 1339-1339) do.

The graphite powder was mixed with H ₂ SO ₄ , K ₂ S ₂ O ₈ and P ₂ O ₅ and oxidized at 80 ° C for 4 hours to form pre-oxidized graphite oxide. After filtration and separation, H ₂ SO ₄ and KMnO _4, and further oxidized at 35 ° C for 2 hours, followed by reaction at 35 ° C for 2 hours and further reaction with 30% H ₂ O ₂ to obtain a yellow powder. The powder was washed five times with 1M HCl, washed with distilled water, and sonicated for 2 hours to remove the oxidized graphite, thereby obtaining oxidized graphene.

<Step 2> Immobilization of magnetic nanoparticles on graphene oxide

The immobilization of magnetic nanoparticles on the graphene oxide was carried out by mixing the graphene oxide with FeCl ₃ : FeCl ₂ = 2: 1 (molar ratio) in the preparation of magnetic nanoparticles-graphene oxide grains, The reaction was conducted at 90 DEG C for 4 hours and uniformly precipitated to prepare a magnetic nanoparticle-graphene oxide hybrid. The presence of the inorganic magnetic nanoparticles on the surface of the oxidized graphene was confirmed by TEM (JEOL EM-2010 microscope) photograph. The result is shown in FIG. 2 and the XRD (Rigaku D / Max 2500) 3 (b), (c), (e) and (f).

<Step 3> Magnetic nanoparticles of platinum nanoparticles- immobilized on oxidized graphene

The immobilization of platinum nanoparticles on GO_MNP-10 and GO_MNP-30 was performed by first mixing ethylene glycol solution with magnetic nanoparticles-graphene oxide and uniformly dispersing the solution, and then hydrolyzing H ₂ PtCl ₆ 6H ₂ O at 125 ° C 2 (d), 3 (e), and 3 (f), XRD was used to prepare uniformly precipitated platinum nanoparticles-magnetic nanoparticles-oxidized graphene hybrid. The nanoparticle size was confirmed.

<Step 4> platinum nanoparticle-magnetic nanoparticle-oxide yes application of the immunodiagnostic of pin hybrid

EDC-sulfo NHS was reacted with the prepared platinum nanoparticle-magnetic nanoparticles-oxidized graphene hybrid to activate the ester using the carboxyl group on the surface. And then reacted with an antibody of the specific antibody to bind the antibody and the prepared hybrid. The platinum nanoparticle-magnetic nanoparticle-oxidized graphene-antibody hybrid prepared above was applied to the color development diagnosis of the target substance of the antibody. In order to observe the activity of the developed hybrid as a peroxidase, TMB was used as a chromogenic substrate and its activity as a peroxidase was examined. The activity was obtained from all the peroxidase analogues, and the highest activity was obtained especially when the platinum nanoparticles were immobilized with magnetic nanoparticles at an appropriate ratio (GO_MNP-10 / Pt-10). (A) and the absorbance scanning (Cary 100 Conc UV-Visible spectrophotometer (Varian, Inc.)) were used to measure the activity of various magnetic nanoparticle carriers including the platinum nanoparticles-magnetic nanoparticles- (B) of Palo Alto, CA) is shown in Figure 4. By observing the enzyme reaction parameters through steady state kinetics for more accurate analysis, we confirmed the activity of platinum as a high peroxidase The results are shown in FIG. 5, and the reaction parameters (catalytic parameters) as the peroxide enzyme calculated using the Lineweaver-Burk plot are summarized The values are shown in Table 1.

Comparison of catalytic parameters Concentration (μg mL ^-1 ) K _m (mM) V _max (nMs ^-1 ) Free GO 50 0.080 70.9 Free MNPs 20 0.173 172.3 GO_MNP-10 50 0.144 162.6 GO_MNP-30 50 0.237 225.2 Free Pt NPs 10 3.417 4002.2 GO_Pt-10 50 0.619 1267.3 GO_MNP-10 / Pt-10 50 0.442 2180.9 GO_MNP-30 / Pt-10 50 0.519 2126.3 HRP 0.0025 0.606 204.7

Example  2: Magnetic nanoparticles - Platinum  Nanoparticle - Oxidation Grapina - Preparation of oxidase enzyme hybrids and Color  Application to sensor

<Step 1> Immobilization of oxidase in platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid

Cholesterol, galactose, and liver function factors (GPT, GOT), which are major factors of health screening, such as glucose oxidase, cholesterol oxidase, galactosoxidase and pyruvate oxidase, (GO_MNP-10 / Pt-10, GO_MNP-30 / Pt-10) through the cross-linking using the carboxyl group on the surface of the hybrid body. To this end, EDC (90 mM) + sulfo-NHS (5 mM) was reacted with the developed hybrid in 30 minutes MES buffer (0.1 M, pH 6.0) Lt; / RTI &gt; The platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid can be produced which has the advantages of the magnetic nanoparticles of the present invention (easy separation and recovery by magnetic force) and can obtain activity as a higher peroxidase FIG. 6 shows the composition of the organic / inorganic hybrid material by further immobilization of various organic enzymes to the oxidized graphene, and the colorimetric sensor and the reaction mechanism of the target substance.

<Step 2> Oxidizing Enzymes - Magnetic Nanoparticles - Platinum Nanoparticles - Oxidative Graphene Application to color sensor

(ABTS, [3-ethylbenzo-thiazoline-6-sulfonic acid] diammonium salt, TMB, Amplex-Red) was dissolved in a sodium acetate buffer solution (sodium acetate buffer) at room temperature to measure the amount of H ₂ O _{2 in the} reaction solution. It was confirmed that the color developed when only a specific target substrate was present, and thus, the absorbance scanning was confirmed. In addition, the biosensor can be easily recovered in 20-30 seconds using magnetism, and it can be confirmed that the original activity is retained even during the reuse of the original activity about 20-30 times.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the invention will be defined by the claims and their equivalents.

Claims (25)

The magnetic nanoparticles are fixed on the surface of the graphene oxide,
Platinum nanoparticle-magnetic nano-particle-oxide graphene hybrid wherein platinum nanoparticles are fixed on the surfaces of the magnetic nanoparticles and the oxidized graphene.

The method according to claim 1,
Wherein the magnetic nanoparticles have a particle diameter of 4 nm to 6 nm, and the platinum nanoparticles have a particle diameter of 3 nm to 5 nm.
The method according to claim 1,
Wherein the magnetic nanoparticles are 10 wt% to 30 wt%, and the platinum nanoparticles are 10 wt% to 30 wt%.
The method according to claim 1,
Wherein the magnetic nanoparticles are iron oxide or ferrite. The platinum nanoparticle-magnetic nanoparticle-oxide graphene hybrid according to claim 1, wherein the magnetic nanoparticle is iron oxide or ferrite.
5. The method of claim 4,
The iron oxide is Fe ₂ O ₃ or Fe ₃ O ₄ , and the ferrite is CoFe ₂ O ₄ or MnFe ₂ O ₄ , wherein the platinum nanoparticle-magnetic nano-particle-oxide graphene hybrid.
A method for producing a platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid comprising the steps of:
(a) fixing the magnetic nanoparticles to the oxidized graphene to produce a magnetic nanoparticle-oxidized graphene hybrid; And
(b) fixing the platinum nanoparticles to the magnetic nanoparticle-oxidized graphene hybrid to prepare a platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid.
The method of claim 6, wherein the fixing of the step (a) is characterized in that the precipitate under basic conditions in combination with the oxidation Yes FeCl ₃ / FeCl ₂ solutions pin.
The method of claim 6, wherein the fixing of step (b) is performed by first mixing ethylene glycol solution with magnetic nanoparticles-graphene oxide and dispersing it, and then depositing H ₂ PtCl ₆ 6H ₂ O How to.
The magnetic nanoparticles are fixed on the surface of the graphene oxide,
Platinum nanoparticles are fixed on the surfaces of the graphene grains and the magnetic nanoparticles,
An active ester-platinum nanoparticle-magnetic nanoparticle-oxide graphene hybrid wherein the active ester is immobilized on the surface of the oxidized graphene.
10. The method of claim 9,
Platinum nanoparticle-magnetic nanoparticle-oxide graphene hybrid characterized in that the magnetic nanoparticle has a particle diameter of 4 nm to 6 nm and the platinum nanoparticle has a particle diameter of 3 nm to 5 nm.
10. The method of claim 9,
Wherein the magnetic nanoparticles are iron oxide or ferrite. The active ester-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid according to claim 1, wherein the magnetic nanoparticle is iron oxide or ferrite.
11. The method of claim 10,
The iron oxide is Fe ₂ O ₃ or Fe ₃ O ₄ , and the ferrite is CoFe ₂ O ₄ Or MnFe ₂ O ₄ is an active ester-platinum nanoparticle-magnetic nanoparticle-oxide graphene hybrid.
Reacting the platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid of claim 1 with N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride and N-hydroxy pseudosuccinimide sodium salt Platinum nanoparticle-magnetic nanoparticle-oxide graphene hybrid on a surface of a platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid material, wherein the active ester- Platinum nanoparticle - Magnetic nanoparticle - Method for producing oxidized graphene hybrid.
14. The method of claim 13,
The concentrations of the N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride, the N-hydroxy pseudocinnamide sodium salt and the platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid were 20 Platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid, characterized in that the active ester-platinum nanoparticle is in the range of 1 to 200 mM, 5 to 50 mM, and 1 to 2 mg / mL.
A method for immunological diagnosis comprising the step of reacting the active ester-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid of claim 9 with an antibody.
The magnetic nanoparticles are fixed on the surface of the graphene oxide,
Platinum nanoparticles are fixed on the surfaces of the magnetic nanoparticles and the oxidized graphene,
An oxidase-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid with an oxidized enzyme integrated on the surface of the oxidized graphene.
17. The method of claim 16,
Wherein the oxidizing enzyme is selected from the group consisting of a glucose oxidase, a cholesterol oxidase, a galactose oxidase, a pyruvate oxidase and an alcohol oxidase, wherein the oxidase-platinum nanoparticle-magnetic nanoparticle-oxidized graphene Hybrid.
17. The method of claim 16,
Platinum nanoparticle-magnetic nanoparticle-oxide graphene hybrid characterized in that the magnetic nanoparticle has a particle diameter of 4 nm to 6 nm and the platinum nanoparticle has a particle diameter of 3 nm to 5 nm.
17. The method of claim 16,
Wherein the concentration of the oxidizing enzyme and the platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid are 1 to 5 mg / mL and 1 to 2 mg / mL, respectively, wherein the oxidase-platinum nanoparticle-magnetic nanoparticle- Pin hybrid.

17. The method of claim 16,
Wherein the magnetic nanoparticles are iron oxide or ferrite. The oxidized enzyme-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid according to claim 1, wherein the magnetic nanoparticle is iron oxide or ferrite.
21. The method of claim 20,
The iron oxide is Fe ₂ O ₃ or Fe ₃ O ₄ , and the ferrite is CoFe ₂ O ₄ Or MnFe ₂ O ₄ is an oxidase-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid.
A method for producing an oxidase-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid according to claim 1, comprising the step of accumulating an oxidase in the platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid of claim 1.
23. The method of claim 22,
Wherein said oxidizing enzyme is selected from the group consisting of glucose oxidase, cholesterol oxidase, galactose oxidase, pyruvate oxidase and alcohol oxidase.
A method for color development diagnosis comprising the step of reacting the oxidase-platinum nanoparticle-magnetic nanoparticle-oxidized graphene hybrid according to claim 16 with a chromogenic substrate.
25. The method of claim 24,
The coloring substrate may be selected from the group consisting of ABTS, TMB, o-phenylenediamine dihydrochloride (OPD), 3,3'-diaminobenzidine (DAB) and 10- (10-acetyl-3, 7-dihydroxyphenoxazine). &Lt; / RTI &gt;

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