KR20180048422A - Nanocomposite consisting of magnetic nanoparticles and gold nanoclusters and method for manufacturing the same - Google Patents

Abstract

The present invention relates to a nanocomposite in which magnetic nanoparticles and gold nanoclusters are joined, and to a preparation method for the same. More specifically, the present invention relates to a biosensor using high peroxidase mimetic activity of the nanocomposite in which magnetic nanoparticles and gold nanoclusters are joined by electrostatic attraction, and to a preparation method for the same. The nanocomposite in which magnetic nanoparticles and gold nanoclusters are joined according to the present invention has improved peroxidase activity; is synthesized by using electrostatic attraction; and is convenient to recover the composite. In addition, the nanocomposite is used in connection with an organic oxidase to detect the presence and concentration of glucose, which serves as a marker for diabetes, and accordingly, can be beneficially used in convenient color development and electrochemical detection of marker materials capable of providing information on various diseases (small molecule materials such as galactose and cholesterol).

Description

TECHNICAL FIELD The present invention relates to a nanocomposite comprising a magnetic nanoparticle and gold nanoclusters, and a method of manufacturing the nanocomposite.

The present invention relates to a nanocomposite comprising a magnetic nanoparticle and a gold nanocluster and a method of manufacturing the same. More particularly, the present invention relates to a nanocomposite comprising a magnetic nanoparticle and a gold nanocluster bonded by electrostatic attraction, And a method of manufacturing the same.

As society becomes more sophisticated and modernized, human desire to live longer and healthier is increasing. Especially, since the breakthrough of medical technology is making a society that can be cured if any disease is found early, disease diagnosis technology becomes more important. In addition, the development of diagnostic techniques that can be easily and easily used by the general public is more important than the disease diagnosis techniques that require expensive detection machines or skilled technicians. Techniques for detecting and quantifying small molecular substances such as glucose, galactose, and cholesterol, proteins, DNA, pathogens and the like, which are causative of diseases, with high sensitivity are very important economically and technically according to the necessity, It is a field that is being actively studied all over the world. Another important flow of disease diagnosis is the field of diagnostic technology called Point of care testing (POCT), which is a technique and device that can produce an immediate and easy diagnostic result in the field without a skilled analyst. . Recently, the newly formed world POCT market is demanding new technology that can diagnose diseases more quickly and easily.

For the development of diagnostic technology, biosensors and ELISA immunodiagnostic technology based on the activity of protein-based organic enzymes have been studied variously for several decades, and commercialization has already been made in various fields. These organic enzyme-based technologies have the advantage of being able to selectively and sensitively diagnose the target substance, but the activity of the enzyme changes depending on the reaction environment and storage time, and ultimately the diagnosis result may be changed. In order to solve these problems, researches on the introduction of nanostructures having enzymatic activity such as magnetic nanoparticles or gold nanoclusters into biosensors and immunoassays have been actively conducted (Korean Patent Publication No. 10-201300015634, No. 10-1311776 and No. 10-1096425). However, in order to use magnetic nanoparticles and gold nanoclusters as a more effective peroxide replacement, problems such as increased activity and sensitivity per individual must be solved.

The present inventors have made intensive efforts to solve the above problems. As a result, the present inventors have found that by preparing a nanocomposite in which magnetic nanoparticles having a peroxidase-mimicking activity and gold nanoclusters are easily combined with electrostatic attraction, Glucose, galactose, cholesterol, protein, and the like can be more effectively diagnosed and diagnosed, thereby completing the present invention.

It is an object of the present invention to provide a biosensor comprising a nanocomposite having peroxidase activity.

It is another object of the present invention to provide a method for detecting a small molecule substance using a biosensor including a nanocomposite having peroxidase activity.

In order to achieve the above object, the present invention provides a biosensor including a nanocomposite in which magnetic nanoparticles and gold nanoclusters are bonded with electrostatic attraction.

The present invention also provides a method for detecting a small molecule material using a biosensor including a nanocomposite in which magnetic nanoparticles and gold nanoclusters are bound by electrostatic attraction.

The magnetic nanoparticles and the gold nanocluster-bound nanocomposite according to the present invention have greatly enhanced peroxidase activity as compared with the magnetic nanoparticles and the gold nanoclusters, respectively, and are easily synthesized by electrostatic attraction, It is very easy. In addition, by detecting the presence or concentration of a substrate of a glucose enzyme, which is a marker of diabetes, by using it in conjunction with an organic enzyme, it is possible to detect the presence or absence of a marker substance (such as a small molecule substance such as galactose or cholesterol) And can be usefully used for color development detection.

FIG. 1 shows TEM and XPS patterns of magnetic nanoparticles, gold nanoclusters, magnetic nanoparticles, and gold nanoclusters bonded nanocomposites according to an embodiment of the present invention.
FIG. 2 is a graph showing the measured zeta potential before and after binding of a magnetic nanoparticle and a gold nanocluster-bound nanocomposite at pH 3.5 according to an embodiment of the present invention.
FIG. 3 is a graph showing binding / post-binding fluorescence characteristics, magnetic properties, and catalytic characteristics as a peroxidase of a nanocomposite comprising a magnetic nanoparticle and a gold nanocluster in accordance with an embodiment of the present invention.
FIG. 4 is a graph showing the Michaelis constant (Km) and the maximum rate constant (Vmax) of the peroxidase activity before and after binding of the magnetic nanoparticles and gold nanocluster-bound nanocomposite according to an embodiment of the present invention.
FIG. 5 is a graph showing the response curves and linear calibration curves for the detection of hydrogen peroxide using the peroxidase activity of the nanocomposite nanoparticles combined with the magnetic nanoparticles and the gold nanoclusters according to an embodiment of the present invention, stability with protein peroxidase (HRP) The results of the analysis of the reusability using magnetic characteristics and comparison are shown.
FIG. 6 is a diagram illustrating a method of detecting glucose using a nanocomposite in which magnetic nanoparticles and gold nanoclusters are combined according to an embodiment of the present invention.
FIG. 7 is a graph showing the results of measurement of a magnetic nanoparticle and a gold nanocluster-bonded nanocomposite according to an embodiment of the present invention
The response curve and linear calibration curve for glucose detection, the selectivity of glucose detection by measuring interference effects of molecules commonly found in human blood, the clinical availability in serum samples by gradually increasing the amount of human serum and measuring the target glucose concentration The results of the analysis are as follows.
FIG. 8 is a diagram illustrating a reaction strategy for electrochemically measuring and quantifying the presence or concentration of a phenol compound due to an enhanced peroxidase activity of a nanocomposite comprising a magnetic nanoparticle and gold nanoclusters according to an embodiment of the present invention .
FIG. 9 is a graph showing the results of measurement of the presence or absence of hydrogen peroxide by the cyclic voltammetry according to the peroxidase activity of the nanocomposite comprising the magnetic nanoparticles and gold nanoclusters according to an embodiment of the present invention. As a result, And linear calibration curves, phenol and phenol compounds, respectively.

The present invention can be all accomplished by the following description. It is to be understood that the following description is only illustrative of preferred embodiments of the invention, but the invention is not necessarily limited thereto. It is to be understood that the accompanying drawings are included to provide a further understanding of the invention and are not to be construed as limiting the present invention. The details of the individual components may be properly understood by reference to the following detailed description of the related description.

Unless otherwise defined, 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 is well known and commonly used in the art.

In one embodiment of the present invention, a nanocomposite in which magnetic nanoparticles having peroxidase activity and gold nanoclusters are bound by electrostatic attraction was prepared. In the nanocomposite, gold nanoclusters were bonded in the form of surrounding the magnetic nanoparticles . In addition, the zeta potential of the nanocomposite was slightly lower (+) than that of the gold nanoclusters, and it was confirmed that the gold nanoclusters surround the magnetic nanoparticles and contribute more to the surface zeta potential.

Accordingly, the present invention relates to a biosensor comprising a nanocomposite in which magnetic nanoparticles and gold nanoclusters are bound with electrostatic attraction in one aspect.

The magnetic nanoparticles of the present invention are characterized by having a peroxidase activity. The magnetic nanoparticles preferably have a particle size of 10 to 20 nm, and preferably have a zeta potential of -20 to -10 mV at pH 3.5. Particles.

The gold nanoclusters of the present invention preferably have a particle size of 1 to 10 nm and a zeta potential of 20 to 30 mV at pH 3.5.

In the nanocomposite in which the magnetic nanoparticles and the gold nanoclusters of the present invention are bound by the electrostatic attraction, it is preferable to mix the magnetic nanoparticles and the gold nanoclusters at a pH of from 2 to 5, most preferably at a pH of 3.5.

The nanocomposite in which the magnetic nanoparticles and gold nanoclusters of the present invention are bound by an electrostatic attraction can be applied to a color sensor for diagnosis of hydrogen peroxide due to its high peroxidase activity.

The nanocomposite in which the magnetic nanoparticles and the gold nanoclusters of the present invention are bound by the electrostatic attraction can be applied to an electrochemical biosensor based on its high peroxidase activity.

In another embodiment of the present invention, a nanocomposite is applied to a biosensor to detect glucose as a marker of diabetes and glucose as a target substance of glucose oxidase. In addition, easy color detection of marker substances (small molecules such as galactose, cholesterol and alcohol) and pollutants (phenol compounds such as phenol, cresol, catechol) related to industrial and agricultural fields that can provide information on various diseases in addition to glucose . ≪ / RTI >

Accordingly, the present invention relates to a method for detecting a small molecule material using a biosensor including the nanocomposite from another aspect.

The small molecule material is preferably glucose, galactose or cholesterol, but is not limited thereto.

In addition, when the hydrogen peroxide material is detected by using the biosensor including the nanocomposite of the present invention, compared to each case of using magnetic nanoparticles or gold nanoclusters, , And the rate constant Km for 5'-tetramethylbenzidine (TMB) decreased to 1/5. Km can be regarded as an indicator of the affinity of the enzyme for the substrate, and the lower the Km value, the higher the affinity with the substrate. Thus, the biosensor comprising the nanocomposite of the present invention has a greater affinity for the enzyme corresponding to the peroxidase enzyme than the magnetic nanoparticles or gold nanoclusters, which is about five times higher.

The nanocomposite comprising the magnetic nanoparticles and the gold nanoclusters according to the present invention can be used for analyzing a specific substance through a coupling reaction with various organic enzymes. That is, it can be used for colorimetric analysis of small molecules such as glucose, galactose and cholesterol.

[ Example ]

Hereinafter, the present invention will be described in more detail by way of examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the technical scope of the present invention is not limited to these embodiments.

Example  1: Manufacture of nanocomposites containing magnetic nanoparticles and gold nanoclusters

1-1. Synthesis of magnetic nanoparticles

Synthesis of magnetic nanoparticles was prepared by precipitation of FeCl ₃ and FeCl ₂ (Mehta et al., Biotechnology Techniques, 11 (7), 493-496, 1997). The uniformly precipitated magnetic nanoparticles were sufficiently dried in a vacuum oven, and then magnets and nanoparticles were identified through magnets and TEM images, respectively.

1-2. Synthesis of gold nanoclusters

Gold nanoclusters were synthesized using BSA (Bovine Serum Albumin) protein (Wang et al., Biosens. Bioelectron., 26, 3614-3619, 2011). The synthesized gold nanoclusters were separated by dialysis to remove unreacted salts, and the sizes of the fluorescence and nanoclusters were confirmed by UV light and TEM images, respectively.

1-3. Combination of magnetic nanoparticles and gold nanoclusters

The combination of magnetic nanoparticles and gold nanoclusters produced nanocomposites through electrostatic attraction. Magnetic nanoparticles prepared at 1-1 having opposing charge and gold nanoclusters made at 1-2 were mixed at pH 3.5, and nanocomposites bound with electrostatic attraction were obtained using magnets. The synthesized nanocomposites confirmed the binding of magnetic nanoparticles and gold nanoclusters by zeta potential change and TEM image.

FIG. 1 shows the TEM images of the magnetic nanoparticles, the gold nanoclusters, and the nanocomposites in which the magnetic nanoparticles and the gold nanoclusters are combined, and the morphology and the elements through the XPS pattern, The size of the magnetic nanoparticles is about 3 ~ 4 nm, and the size of the magnetic nanoparticles is about 12 ~ 13 nm. It is confirmed that the gold nanoclusters are bonded around the magnetic nanoparticles. In particular, it can be seen that gold nanoclusters (AuNC) are located around the magnetic nanoparticles (MNNC) in the complex, and only Au in the XPS analysis means that the magnetic nanoparticles are not surrounded by AuNC . In the XPS pattern, the illustration shows an enlarged pattern of the circled portion of each graph.

FIG. 2 shows the results of measurement of the zeta potential of the magnetic nanoparticles, gold nanoclusters, and gold nanoclusters combined with the magnetic nanoparticles and the gold nanoclusters prepared in this Example. The gold nanoclusters are positively charged and the magnetic nanoparticles are positively charged so that they can be electrostatically coupled and the zeta potential of the formed nanocomposite is slightly lower than that of gold nanoclusters It was. From this, it can be confirmed that gold nanoclusters surround the magnetic nanoparticles and contribute more to the surface zeta potential. Also, since the gold nanoclusters of the present invention have a zeta potential of (+) 23.84 mV at pH 3.5 and the magnetic nanoparticles have a zeta potential of (-) 16.33 mV, gold nanoparticles are formed around the magnetic nanoparticles It is possible to realize nanocomposite by defects with very simple static magic force.

FIG. 3 shows a) fluorescence characteristics, b) magnetic properties, and c) peroxidase characteristics before and after binding of the nanocomposite comprising the magnetic nanoparticles and the gold nanoclusters according to an embodiment of the present invention, The activity of peroxidase was found to be about three times higher than that of the complex.

The method using the present invention provides a method for developing a nanocomposite having peroxidase activity with high activity and sensitivity per individual. Although the peroxidase is inherently an organic enzyme, it has inevitable denaturation and decrease in activity over time, but the nanocomposite of the present invention is a more stable system because it uses inorganic nanoparticles and gold nanoclusters instead of peroxidase. In addition, a simple stirring method is used in the synthesis and a more simple separation method using magnetic force is used. In particular, the magnetic nanoparticle-gold nanocluster composite of the present invention exhibits about three times higher peroxidase activity than the case of using magnetic nanoparticles or gold nanoclusters alone, in the color development experiment using TMB as a substrate. Based on this enhanced peroxidase activity, the colorimetric detection method using the magnetic nanoparticle-gold nano cluster complex is very cost-effective because quantitative analysis of materials that are economically and industrially important can be easily performed by visual difference .

4 shows the Michaelis constant (Km) and the maximum rate constant (Vmax) of the peroxidase activity before and after binding of the magnetic nanoparticles and the gold nanocluster-bound nanocomposite according to an embodiment of the present invention , Km value about 5 times lower than TMB substrate and 5 times higher affinity for TMB than for gold nanocluster (AuNC) or magnetic nanoparticle (MNP) alone for substrate TMB System.

Example  2: MNP - AuNC  Containing nanocomposites Color  sensor

2-1. MNP - AuNC  Hydrogen peroxide containing nanocomposites Color  sensor

3,3 ', 5,5'-tetramethylbenzidine (TMB) was dissolved in sodium acetate buffer (0.1M, pH 4.0), and the concentration of 3,3', 5,5'-tetramethylbenzidine (TMB) was added to the nanocomposite bound to the magnetic nanoparticles and gold nanoclusters prepared in Example 1-3. After the addition at RT, the amount of H ₂ O _{2 in the} reaction was measured by the color reaction. As a result, it was confirmed that the degree of color development was proportional to the amount of H ₂ O ₂ .

FIG. 5 is a graph showing the reaction curves for the detection of hydrogen peroxide by the oxidation reaction of TMB, which is a chromogenic substrate due to the peroxidase activity of the nanocomposite comprising the magnetic nanoparticles and gold nanoclusters according to an embodiment of the present invention, and b) C) a comparison of stability with protein peroxidase (HRP) to temperature over a temperature range of 23-90 ° C, and d) reusability using the magnetic properties of the nanocomposite. From the above results, it was confirmed that the detection of hydrogen peroxide (H2O2) was detected in a sufficiently wide linear range of 150 ~ 5000 μM with LOD 100 μM, and a remarkable improvement in stability was confirmed when compared with protein peroxidase HRP. In the temperature stability experiments, HRP and nanocomposites were incubated for 2 hours at each temperature condition and their remaining activity was measured by TMB reaction.

2-2. MNP - AuNC  Glucose containing nanocomposites Color  sensor

When glucose oxidase was reacted with glucose and then reacted with the nanocomposite bound with the magnetic nanoparticles prepared in Example 1-3 and gold nanoclusters, when TMB was used as a chromogenic substrate, only when glucose was present Blue color, and the change in absorbance was observed. When other sugars such as lactose, arabinose, galactose and fructose were added, the color and absorbance did not change as in the case of adding buffer.

Therefore, it was confirmed that the glucose measurement with the glucose oxidase can be performed by color development, and the degree of color development is proportional to the concentration of glucose, so that glucose can be quantitatively measured. We also confirmed that glucose detection was detectable at a wide linear range of 150 ~ 750 μM with LOD 100 μM and confirmed the possibility of clinical measurement of glucose in actual blood samples.

FIG. 7 shows a) reaction curve for detecting glucose through oxidation of TMB, which is a chromogenic substrate due to peroxidase activity of nanoparticles bound with magnetic nanoparticles and gold nanoclusters according to an embodiment of the present invention, and b) C) the selectivity of glucose detection by measuring interference effects of molecules commonly found in human blood, and d) increasing the amount of human serum to measure the target glucose concentration, thereby indicating clinical availability in serum samples . The above results confirmed that glucose detection was detectable in a wide linear range of 150 to 750 μM with LOD 100 μM, and the clinical measurement of glucose in actual blood samples was confirmed.

Example 3: Electrochemical sensor comprising MNP-AuNC nanocomposite

3-1. Hydrogen peroxide electrochemical sensors including MNP-AuNC nanocomposites

The phenols in the sample were electrochemically detected using the enhanced peroxidase activity of the magnetic nanoparticle-gold nanocluster complex. As a result, it was confirmed that a large reduction current difference occurred depending on the amount of hydrogen peroxide, and the stepwise reduction current generation by the addition of hydrogen peroxide and the electrochemical analysis ability of phenol and cresol were confirmed.

8 is a graph showing a reaction strategy for electrochemically measuring and quantifying the presence and concentration of a phenol compound due to its enhanced peroxidase activity of a nanocomposite comprising a magnetic nanoparticle and a gold nanocluster in accordance with an embodiment of the present invention will be.

9 is a graph showing the results of measurement of the presence or absence of hydrogen peroxide by cyclic voltammetry according to the peroxidase activity of the nanocomposite comprising the magnetic nanoparticles and the gold nanoclusters according to an embodiment of the present invention, And c) Linear calibration curves through the current measurement reaction for phenol and phenol compounds, phenol and cresol, respectively. From the above results, it can be seen that the nanoparticles combined with the magnetic nanoparticles and the gold nanoclusters easily synthesized through the present invention can be effectively used for producing color or electrochemical biosensors due to their enhanced peroxidase activity there was.

MNP (magnetic nanoparticle): Magnetic nanoparticle
AuNC (gold nanocluster): Gold nanoclusters
MNP-AuNC: Nanocomposite with MNP and AuNC

Claims (4)

A biosensor comprising a nanocomposite wherein magnetic nanoparticles and gold nanoclusters are bound together by electrostatic attraction.
The biosensor according to claim 1, wherein the magnetic nanoparticles have a size of 10 to 20 nm and the gold nanoclusters have a size of 1 to 10 nm.
The biosensor according to claim 1, wherein the nanocomposite has a zeta potential of 10 to 20 mV.
A method for detecting a small molecule material using the biosensor including the nanocomposite of claim 1.

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