KR20220132271A - Method for producing gold nanoparticles having increased glucose oxidase and peroxidase activities and gold nanoparticles by the method - Google Patents

Abstract

The present invention relates to a preparation method of gold nanoparticles with increased glucose oxidase and peroxidase activities, and more specifically, to a preparation method of gold nanoparticles using a Galla chinensis extract to prepare gold nanoparticles having both glucose oxidase and peroxidase activities with substrate selectivity from a gold precursor.

Description

Method for producing gold nanoparticles with increased glucose oxidase and peroxidase activity, and gold nanoparticles prepared accordingly

The present invention relates to a method for producing gold nanoparticles with increased glucose oxidase and peroxidase activity, wherein the gold nanoparticles have both increased glucose oxidase and peroxidase activity along with substrate selectivity from a gold precursor using a galnut extract. It relates to a method for preparing gold nanoparticles, for preparing nanoparticles.

Glucose detection is playing an important role in improving quality of life in the medical and food fields. With the development of biosensor technology, more convenient glucose measurement methods have been reported, and among them, a colorimetric signal readout strategy using a linkage reaction between glucose oxidase and peroxidase is being studied for its ease of use and measurement.

In the above study, glucose oxidase and/or peroxidase are metal nanoparticles, metal oxide nanoparticles, carbon-based nanoparticles, etc. that are expected to secure economic feasibility through simple mass production using stable activity and chemical synthesis methods. Research on nanozyme enzyme mimics is in progress.

Among the nanozymes, Fe ₃ O ₄ NPs, Au NPs, and the like have been reported as metal nanozymes capable of mimicking glucose oxidase or peroxidase activity. As an example, in "Fe ₃ O ₄ magnetic nanoparticles as peroxidase mimetics and their applications in H ₂ O ₂ and glucose detection (Wei, H. and E. Wang (2008) Anal. Chem. 80: 2250-2254.)", Disclosed is a new sensing platform that can detect glucose using both the peroxidase activity and glucose oxidase activity of Fe ₃ O ₄ NPs (MNPs), showing high substrate specificity for glucose in glucose diagnosis using nanozyme. Glucose oxidase (GOx) is used. GOx oxidizes glucose and induces H ₂ O ₂ generation. Depending on the concentration of H ₂ O ₂ generated, the intensity of generation of the substrate (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid),ABTS) reacted with Fe ₃ O ₄ MNPs appears differently. Quantitatively analyze the concentration of glucose.

However, since the Au NPs nanozyme including the Fe ₃ O ₄ MNPs is essentially a nanostructure that does not have an active site characteristically, it lacks substrate selectivity such as an organic enzyme, and its activity is still significantly lower than that of an organic enzyme. has a disadvantage. In addition, in the case of an enzyme-mimicking active material, storage stability that does not change even when stored for a long period of time is required.

Therefore, in order to compensate for the above problems and to improve the enzyme mimicking activity, research on manufacturing nanozymes by various methods such as microorganisms, plant extracts, and seaweeds is in progress.

As an example, there is a method for preparing gold nanoparticles using a plant extract. "Green Synthesis of Gold Nanoparticles Using Aqueous Extract of Garcinia mangostana Fruit Peels, Volume 2016, 7 page, Journal of Nanomaterials states that gold nanoparticles can be prepared using the bark extract of Garcinia mangostana, and the method is environmentally friendly, so this method In the case of gold nanoparticles prepared with , it is disclosed that they can be applied to biomedical and other applications requiring non-toxicity.

However, since the above document does not disclose the glucose oxidase or peroxidase mimic activity of gold nanoparticles, the degree of enzymatic mimetic activity of gold nanoparticles prepared by the above method cannot be predicted. Gold nanoparticles have differences in the shape, size, active site, etc. of the manufactured particles depending on the plant extract used, which affects the properties of gold nanoparticles including enzyme-mimicking activity.

Accordingly, as a result of repeated research on a method for preparing gold nanoparticles having both improved glucose oxidase and peroxidase mimic activity, the present inventors have conducted research on a method for preparing gold nanoparticles using a galnut plant extract. It was found to have substrate selectivity and improved enzyme mimicking activity, and the present invention was completed.

Wei, H. and E. Wang, "Fe3O4 magnetic nanoparticles as peroxidase mimetics and their applications in H2O2 and glucose detection", Anal. Chem. 80, 2008, pp2250-2254. Kar Xin Lee, Karyar Shameli, Mikio Miyake, et al., "Green Synthesis of Gold Nanoparticles Using Aqueous Extract of Garcinia mangostana Fruit Peels", Journal of Nanomaterials, Volume 2016(2), 2016, pp1-7

The present invention provides a method for preparing gold nanoparticles using a galnut plant extract to have both improved glucose oxidase and peroxidase mimic activity with high substrate selectivity.

The present invention provides gold nanoparticles prepared by the above method.

The present invention provides a sensor for detecting glucose and a sensor for detecting hydrogen peroxide, including the prepared gold nanoparticles.

In addition, the present invention provides a method for preparing flower-shaped gold nanoparticles using a galnut plant extract and flower-shaped gold nanoparticles prepared according to the preparation method.

In addition, the present invention provides a catalyst for the reduction reaction of 4-nitrophenol (4-NP), including the flower-shaped gold nanoparticles.

In order to solve the above problems, the present invention includes the steps of (1) preparing a gold precursor solution: (2) adding a galnut extract to the gold precursor solution and stirring until the reaction is completed. It provides a method for producing gold nanoparticles having increased glucose oxidase and peroxidase activity.

The present invention also provides gold nanoparticles having increased glucose oxidase and peroxidase activity, which are prepared by the above method, and in one embodiment, the gold nanoparticles are rhombic dodecahedron, decahedron, and rhombic dodecahedron. It has at least one shape selected from the cone shape, and may have a particle size of 1 to 100 nm.

In addition, the present invention provides a sensor for detecting glucose and a sensor for detecting hydrogen peroxide, which comprises the gold nanoparticles.

The present invention also includes the steps of (1) preparing a mixture by adding a gold precursor solution to the galnut extract while maintaining constant stirring; and (2) stirring the mixture to prepare flower-shaped gold nanoparticles; It provides a method for producing flower-shaped gold nanoparticles, characterized in that.

In one embodiment, the stirring of the mixture in step (2) includes the steps of: (a) reacting phytochemicals of the galnut extract in the mixture with a gold precursor to form a primary crystal of Au; (b) the Au invitation and phytochemicals are aggregated, preparing aggregated nanoparticles; and (c) subjecting the aggregated nanoparticles to Ostwald Ripening to prepare flower-shaped gold nanoparticles; may include.

In addition, it provides a flower-shaped gold nanoparticles, characterized in that produced according to the method.

In addition, it provides a catalyst for the reduction reaction of 4-nitrophenol (4-NP), characterized in that it contains the flower-shaped gold nanoparticles.

In the case of the gold nanoparticles prepared with the gal nut extract according to the present invention, both the glucose enzyme and peroxidase mimic activity improved by the active substance or active site provided from the gal nut extract, and only the gold nanoparticles without the addition of other enzymes or H ₂ O ₂ It can be used to detect glucose in a sample. It can also be used to detect H ₂ O ₂ in a sample.

In addition, the gold nanoparticles prepared from the galnut extract synthesized in the present invention have high selectivity for glucose and H ₂ O ₂ with low K _m values of 0.089 mM and 0.120 mM, respectively, and have excellent storage stability.

Therefore, the gold nanoparticles according to the present invention can mimic natural enzymes that can catalyze multiple enzymatic cascade reactions in various biotechnology applications such as colorimetric biosensors and diagnostic kits.

In addition, according to the present invention, as gold nanoparticles are prepared by using the gal nut extract, it is not only eco-friendly and non-toxic, but also can be mass-produced, thereby securing economic feasibility.

1 shows a method of manufacturing gold nanoparticles according to an embodiment of the present invention.
2 illustrates a method of manufacturing flower-shaped gold nanoparticles according to an embodiment of the present invention.
3 shows (a) UV-Vis absorbance spectrum, (b) TEM image, (c) FTIR and (d) EDS and element mapping of GNE-based AuNPs according to an embodiment of the present invention.
Figure 4 is a UV-Vis spectrum showing the GOx enzyme mimic activity of GNE-based AuNPs, SB and SC according to an embodiment of the present invention.
5 is a photograph showing the GOx enzyme mimic activity of GNE-based AuNPs, SB and SC according to an embodiment of the present invention.
6 is a UV-Vis spectrum showing the POx enzymatic mimic activity of GNE-based AuNPs, SB and SC according to an embodiment of the present invention.
7 shows a GNE-based AuNPs cascade catalyst system according to an embodiment of the present invention.
8 is a UV-Vis spectrum showing GOx activity according to catalytic parameters (a) pH, (b) temperature, (c) glucose concentration, and (d) sugar selectivity of GNE-AuNPs according to an embodiment of the present invention. to be.
9 is a UV-Vis spectrum showing the POx activity according to the catalytic parameters (a) H ₂ O ₂ concentration, (b) AuNPs concentration of GNE-AuNPs according to an embodiment of the present invention.
10 is a graph showing the storage stability of GNE-based AuNPs according to an embodiment of the present invention.
11 is a graph showing the catalytic activity of GNE-based AuNPs and GNE-based flower AuNPs 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 is those well known and commonly used in the art.

In the present specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

The present invention relates to a method for preparing gold nanoparticles having improved glucose oxidase and peroxidase activity by reducing Au from a gold precursor using a galnut extract.

According to one aspect of the present invention, the method comprising the steps of (1) preparing a gold precursor solution: (2) adding a galnut extract to the gold precursor solution and stirring until the reaction is completed; , provides a method for preparing gold nanoparticles with increased glucose oxidase and peroxidase activity.

In the present invention, the gal nut extract refers to a mixed solution obtained by eluting the active ingredient from the gal nut by heating or non-heating by putting it in an extraction solvent, without grinding or not grinding the gal nut (Termia chebula). Polyphenols such as acid, Pentagalloyl glucose, Egallic acid, Digallic acid, and Tannic acid are included as phytochemicals, and the extraction solvent is water, methanol, ethanol, propanol, octanol, glycerol, or a mixture thereof. can be

In the present invention, step (1) is a step of preparing a gold precursor solution, and the gold precursor may be a water-soluble gold salt, a water-soluble gold oxide salt, or a combination thereof. For a more specific example, AuCl, AuCl ₃ , AuBr ₃ , AuCl ₄ H ₄ N, HAuCl ₄ , KAuCl ₄ , KAuBr ₄ , NaAuCl ₄ , may be NaAuBr ₄ , preferably HAuCl ₄ .

In the present invention, step (2) is a step of adding a galnut extract to the gold precursor solution and stirring until the reaction is completed.

Specifically, step (2) is a step of stirring until the formation of Au nanoparticles (AuNPs) is completed while adding the galnut extract to the gold precursor solution. It is preferable to make it in a uniform reaction environment, and the temperature at the time of addition and stirring can be adjusted according to the size of the Au particles to be manufactured, the process speed, etc., so it is not limited to a specific temperature range, but preferably 0 to 100 ℃ can This is because when the temperature at the time of addition and stirring in step (2) is less than 0 ° C, the reaction rate is slow, and it is difficult to liberate Au ions from the gold precursor, so mixing with the added galnut extract may not be done properly. If it exceeds 100 ° C, the reaction rate is fast, but the average diameter of the nanoparticles Au is reduced, and there is a risk of decomposition of the active ingredient of the galnut extract added dropwise. More preferably, it may be 20 to 95 °C.

In addition, the amount of the gal nut extract added dropwise to the metal precursor solution in step (2) is not limited thereto, but preferably, 10 g of gal nut powder is added to 1 L of the extraction solvent and the extract is extracted with about 1 to 3 ml per 100 ml of the metal precursor solution. can be added in a proportion of

In step (2) performed under these conditions, when the gal nut extract is added dropwise to the gold precursor solution, polyphenols such as Gallic acid, Pentagalloyl glucose, Egallic acid, Digallic acid, and Tannic acid of the gal nut extract are added. The Au ³⁺ ions of the Au metal compound are capped to form a stabilized colloid.

In addition, when the Au nanoparticles (AuNPs) formed in step (2) are stirred while adding the galnut extract to the gold precursor solution, Au nucleus crystal growth (Nucleation), Au cluster formation (cluster formation) and Au nanoparticles It can be prepared through the growth (AuNPs Growth) step, and it can be confirmed through UV-Vis absorbance measurement that the Au nanoparticles have been prepared.

The Au nanoparticles prepared according to the above method have a shape such as a rhombic dodecahedron, a decahedron, a conical shape, and a particle size of 1 to 100 nm, and as they contain C and O element components together with Au, the substrate selectivity is It shows high and improved glucose enzyme activity and peroxidase activity. Specifically, in the case of Au nanoparticles prepared by the above method, as they are supplied with C and O components as well as capped by the polyphenol component of the gal nut extract, the Au nanoparticles containing C and O on the surface It is predicted that by increasing the active site, it can exhibit both substrate selectivity and glucose enzyme activity and peroxidase activity. In addition, gold nanoparticles prepared according to the method of the present invention may have improved glucose enzyme activity and peroxidase activity than gold nanoparticles prepared by other methods, and by having a zeta potential more negative than -30 mV, , has high storage stability.

Therefore, the gold nanoparticles according to the present invention can be used as a biosensor such as a sensor for detecting glucose and a sensor for detecting hydrogen peroxide, and it is shown that the gold nanoparticles according to the present invention can also be used in various bio fields including the same.

In the present invention, gold nanoparticles are prepared using a gal nut extract, but the gold nanoparticles may also be prepared in a flower shape.

The method for preparing flower-shaped gold nanoparticles according to the present invention includes the steps of (1) preparing a mixture by adding a gold precursor solution to a gal nut extract; and (2) stirring the mixture to obtain flower-shaped gold nanoparticles. It provides a method for producing flower-shaped gold nanoparticles, comprising the steps of:

Step (1) is a step of preparing a mixture by adding an aqueous gold precursor solution to the galnut extract solution.

The amount of the gold precursor added may be such that the gold precursor concentration in the mixed solution of the galnut extract and the gold precursor solution is 0.1 to 1 mM. This is because, when gold particles are included in less than 0.1 mM, gold nanoparticles may not be produced, and when it exceeds 1 mM, the content of gold precursors that can react with the galnut extract is exceeded, so a large amount of gold precursors is not This is because economic feasibility is reduced by being in a reactive state, and as the gold precursor, a water-soluble gold salt, a water-soluble gold oxide salt, or a combination thereof may be used. For a more specific example, AuCl, AuCl ₃ , AuBr ₃ , AuCl ₄ H ₄ N, HAuCl ₄ , KAuCl ₄ , KAuBr ₄ , NaAuCl ₄ , may be NaAuBr ₄ , preferably HAuCl ₄ .

In addition, it is preferable that the addition of the aqueous gold precursor solution is carried out in a dropwise manner in order to prevent uniform reaction conditions and large crystal growth.

Next, step (2) is a step of preparing flower-shaped gold nanoparticles by stirring the mixture at 0 to 100° C. Specifically, (a) phytochemicals and gold of the galnut extract in the mixture reacting the precursor to form a primary crystal of Au; (b) the Au primary and phytochemicals are aggregated to prepare aggregated nanoparticles; and (c) subjecting the aggregated nanoparticles to Ostwald Ripening to prepare flower-shaped gold nanoparticles.

In step (2), step (a) is a step of forming a primary crystal of Au by reacting the components of the galnut extract constituting the mixture, that is, phytochemicals and Au ³⁺ ions of the metal compound including Au As a result, the Au primary crystal formed includes not only Au but also C and O element components supplied from phytochemicals of the galnut extract, and the phytochemicals include polysaccharides such as Gallic acid, Pentagalloyl glucose, Egallic acid, Digallic acid, and Tannic acid. Contains polyphenols.

In step (2), step (b) is a step of preparing aggregated nanoparticles by aggregating the Au primary crystals and phytochemicals, wherein the Au primary crystals are aggregated with a plurality of Au primary crystals and phytochemicals, multipad-like It forms loose nanoparticles.

In step (2), step (c) is a step in which the agglomerated nanoparticles undergo Ostwald Ripening to prepare flower-shaped gold nanoparticles.

Ostwald ripening (Ostwald Ripening) is a phenomenon in which the surface energy of the particles becomes a driving force and the smaller particles in the dispersion system become smaller or disappear, so that larger particles grow. Thus, the number of particles was reduced, while the average particle size was 10 to 100 nm to prepare flower-shaped gold nanoparticles.

The prepared flower-shaped gold nanoparticles can be used as catalysts as the gold nanoparticles are grown by agglomeration and Ostwald aging, and have sufficient active sites including C and O on the surface.

For example, the flower-shaped gold nanoparticles may also be used as a catalyst for a 4-nitrophenol (4-NP) reduction reaction.

Hereinafter, examples will be given for a more detailed description of the present invention. However, the following examples are only preferred examples of the present invention, and the present invention is not limited thereto.

<Example>

1. AuNPs Synthesis

(1) Synthesis of gold nanoparticles (AuNPs) using gal nut extract

i) Preparation of gal nut extract

1 g of gallnut powder was placed in a round bottom flask containing 100 mL of distilled water and boiled under continuous reflux for 1 hour. The extract was cooled and centrifuged at 13000 rpm for 20 minutes to remove solid components. Thereafter, the supernatant was filtered using a 0.45 μm syringe filter to remove impurities.

Accordingly, a Gallnut extract (GNE) was prepared, and diluted to 1% (v/v) for use in the synthesis of gold nanoparticles (AuNPs) through reduction of HAuCl ₄ ·3H ₂ O.

ii) Synthesis of GNE-based AuNPs

AuNPs were synthesized through reduction of HAuCl ₄ ·3H ₂ O using galnut extract (GNE).

An aqueous HAuCl ₄ ·3H ₂ O solution (0.01% w/v 250 mL) dissolved in distilled water was boiled at 95° C. in a round bottom flask equipped with a condenser to preheat the HAuCl ₄ ·3H ₂ O solution. A heating mantle was used for the heating. Thereafter, galnut extract (1%, 3.75 mL) was added dropwise to the preheated HAuCl ₄ ·3H ₂ O solution to form a mixture, and then the resulting mixture was stirred for 10 minutes. It was kept under continuous boiling.

After that, the heating source was removed and the resulting colloidal solution was stirred for 35 minutes to completely reduce gold ions.

The purple galnut extract (GNE)-based AuNPs prepared accordingly are denoted as GNE-based AuNPs. By the absorption peak at 541 nm of the UV-visible spectrophotometer and transmission electron microscopy (TEM) analysis, it was confirmed that the GNE-based AuNPs prepared above were AuNPs with an average size of 27.5 nm, and the actual concentration of AuNPs was the inductively coupled plasma mass. It was estimated to be 57.9 μg/mL by using a spectrophotometer (ICP).

(2) Synthesis of GNE-based Au-Nanoflowers

1 mL of 1 mM HAuCl _4· 3H ₂ O aqueous solution was added dropwise to the beaker containing the 50 mL galnut extract prepared above under constant stirring at 200 rpm. Nanoflowers synthesized by reacting at room temperature for 60 minutes were centrifuged at 10000 rpm for 20 minutes. Thereafter, the precipitated nanoflowers were washed with distilled water and centrifuged again. The final product was dispersed in 10 mL distilled water for further use. Accordingly, the prepared gold nanoparticles will be referred to as GNE-based Au-Nanoflowers.

(3) "SB" - NaBH ₄ (SB) of gold nanoparticles (AuNPs) using chemical synthesis

A colloidal dispersion of AuNPs was prepared by treating 0.125 mM HAuCl _4· 3H ₂ O aqueous solution with NaBH ₄ (SB: Au = 5: 1 mol/mol) under a nitrogen atmosphere in the presence of an excess of glucose (0.35 mol/L). .

Accordingly, the prepared AuNPs are denoted as "SB", and from the TEM image, it was confirmed that the prepared AuNPs were spherical particles with an average diameter of 24.5 nm.

(4) "SC" - of gold nanoparticles (AuNPs) using sodium citrate (SC) chemical synthesis

In a 500 mL round bottom flask equipped with a condenser, 250 mL of 0.01% HAuCl ₄ 3H ₂ O was boiled with stirring. To this solution was added 3.75 mL of 1% sodium citrate. After maintaining boiling for another 10 minutes, the heating source was removed and the colloidal solution was stirred for an additional 15 minutes.

Accordingly, the prepared AuNPs are denoted as "SC", and from the TEM image, it was confirmed that the prepared AuNPs were aggregated particles having an average size of 4.43 nm.

<Analysis Example>

1. Characterization of GNE-based AuNPs

(i) UV-Vis absorbance spectrum

Figure 3(a) shows the UV-Vis absorbance spectrum of AuNPs synthesis using GNE as a function of time. Looking at this, the absorbance starts to appear from 30 seconds at 541 nm, and the absorbance is rapidly increased in the initial stage, and after 25 minutes It can be seen that is kept constant.

From the above results, in the synthesis of AuNPs using GNE, AuNPs start to form from 30 seconds, and it is confirmed that the stepwise nanoparticle formation process including Au ³⁺ reduction, crystal nucleation growth, and nanocluster formation is achieved in 35 minutes. can

(ii) TEM (Transmission Electron Microscope) image

Figure 3 (b) is a TEM image showing the nanoparticle size and shape of GNE-based AuNPs.

Referring to this, AuNPs synthesized using GNE had rhombic dodecahedron, decahedron, and conical shapes, and the average particle size was 27.5 nm.

(iii) Fourier Transform Infrared Spectrometry (FT-IR)

Figure 3(c) is FTIR spectra for GNE, HAuCl ₄ and GNE-based AuNPs. FTIR spectroscopy was performed to identify the GNE functional groups responsible for Au ³⁺ reduction and AuNPs capping.

Referring to this, the FTIR spectrum of GNE-based AuNPs can confirm the same characteristic peak as the phytochemicals detected in GNE, which means that GNE phytochemicals are present on the AuNPs surface.

(iv) EDS and element mapping

3(d) shows EDS and elemental mapping for GNE-based AuNPs, and shows a peak at 2.3 keV in EDS analysis, suggesting the formation of pure AuNPs.

In addition, in EDS mapping, GNE-based AuNPs appeared to contain C (11.05%) and O (1.62%) along with Au (87.34%), indicating that a total of 12.67% of GNE phytochemicals were immobilized on AuNPs. suggest

2. Glucose oxidase (GOx) and peroxidase (POx) mimic activity analysis of AuNPs

(1) Sample preparation and analysis method

(i) Glucose detection (GOx mimic activity) sample preparation and analysis method

In order to analyze the glucose oxidase (GOx) mimic activity for the prepared GNE-based AuNPs, SB and SC, GOx mimicking activity samples for each AuNPs were prepared according to the following method, and analyzed in FIGS. 4 and 5 . was indicated.

<GOx mimic activity sample preparation and UV-Vis analysis method>

After mixing 200 µL of glucose and 150 µL of the prepared AuNPs (57.9 µg/mL) in 200 µL phosphate buffer (10 mM, pH 7.5), it was maintained at 37 °C for 35 minutes. To this solution, 200 μL of 12 mM 3,3′,5,5′-tetramethylbenzidine (TMB) and 300 μL of acetate buffer (200 mM, pH 4.2) were added.

The mixture was maintained at 37° C. for 20 minutes, and after centrifugation, the absorbance at 652 nm was measured using a UV-visible spectrophotometer. Glucose concentrations were varied from 0.05 to 60 mM.

(ii) H ₂ O ₂ Methods for preparing and analyzing colorimetric (POx mimicking activity) samples

In order to analyze the POx (peroxidase) mimic activity for the prepared GNE-based AuNPs, SB and SC, POx mimicking activity samples for each AuNPs were prepared according to the following method, and analyzed in FIGS. 4 and 7 , indicated.

<H ₂ O ₂ colorimetric measurement (POx mimic activity) sample preparation and UV-Vis analysis method>

The peroxidase mimic activity assay was performed in a 1.5 mL Eppendorf tube by sequentially adding 200 µL of 12 mM TMB, 300 µL of acetate buffer (200 mM, pH 4.2), 100 µL of AuNPs (57.9 µg/mL), and 100 µL H ₂ O ₂ . carried out. The prepared mixture was maintained at 37° C. for 20 minutes, and absorbance at 652 nm was measured using a UV-visible spectrophotometer. The H ₂ O ₂ concentration was varied from 0.008-30 mM.

(2) Analysis result

(i) GOx mimic activity assay

UV-Vis spectral analysis - GOx mimic activity

4 is a UV-Vis spectrum showing the GOx enzyme mimic activity of GNE-based AuNPs, SB and SC.

Looking at this, GNE-based AuNPs showed significantly higher GOx activity than SB, and SC showed little GOx activity.

Analysis of GOx-mimicking activity using the glucose oxidase assay kit

In order to analyze the glucose oxidase (GOx) mimic activity against the prepared GNE-based AuNPs, SB and SC, using a commercially available glucose oxidase assay kit (MAK097-1KT, Sigma-Aldrich), GOx according to the product manual The mimic activity was measured and shown in FIG. 5 .

In Figure 5, GNE-based AuNPs are discolored pink, whereas SB and SC are not discolored, so it can be seen that only GNE-based AuNPs have significant GOx mimic activity.

(ii) POx mimic activity assay

UV-Vis spectrum - POx mimic activity

6 is a UV-Vis spectrum showing the POx enzyme mimic activity of GNE-based AuNPs, SB and SC. Through this, it can be seen that GNE-based AuNPs exhibit significantly higher POx activity compared to SB and SC.

(iii) GNE-based AuNPs cascade catalyst system

7 shows a GNE-based AuNPs cascade catalyst system, and the following reaction formula shows a catalytic reaction according to the system.

Referring to this, it can be seen that GNE-based AuNPs exhibit both GOx and POx enzyme mimic activity.

That is, glucose and oxygen are produced as gluconic acid and H ₂ O ₂ by the catalytic activity of GNE-based AuNPs, and after the TMB absorbance peak at 652 nm disappears, the color of the solution changes to yellow ( 7(a) and Scheme (1)), TMB is oxidized to oxTMB by H ₂ O ₂ generated by the catalytic activity of GNE-based AuNPs, and changes from colorless to blue (Fig. 7(b) and Reaction Scheme) (2)) can be confirmed.

From the above results, as GNE-based AuNPs have both GOx and POx enzyme mimic activity, it was confirmed that POx activity could be exhibited with H ₂ O ₂ prepared by the GOx activity after GOx was formed. In the catalyst system, the oxidation process was stopped or controlled by adding H ₂ SO ₄ .

3. Catalytic parameters for the enzymatic mimic activity of GNE-AuNPs

In order to evaluate the effect of catalytic parameters on the enzymatic mimic activity of GNE-AuNPs through the UV-Vis absorption spectrum as described above, the effect of reaction time on GOx and POx activity was first analyzed.

When analyzing GOx and POx activity through the UV-Vis absorption spectrum of the GNE-AuNPs, after a reaction time of 35 minutes, almost no change in peak absorbance at 652 nm was observed, so this time was used to evaluate the effect of the following catalytic parameters. applied.

(1) Catalytic parameters for GOx mimicry

(i) pH

FIG. 8(a) shows the analysis of GOx activity of GNE-AuNPs according to pH using a series of phosphate buffers ranging from pH 4 to 9. FIG.

From this, it can be seen that GNE-AuNPs exhibit optimal GOx activity performance in the pH range of 6 to 8, and GOx activity is sensitive to pH.

(ii) temperature

FIG. 8(b) shows the GOx activity of GNE-AuNPs according to temperature by analyzing it at pH 7.5 and a temperature range of 10 to 80°C.

Referring to FIG. 8(b), the absorbance significantly increased while the temperature was increased from 10 °C to 40 °C, but as the absorbance decreased at a temperature of 40 °C or higher, a temperature of 40 °C or higher had a negative effect on GOx activity. You can check the blemishes.

Considering the protein properties, enzymes are reported to be very sensitive to thermal changes, and the optimum temperature for most human enzymes is 37 °C, so it is appropriate that GOx and POx mimic activity assays be performed at 37 °C.

(iii) glucose concentration

Figure 8(c) shows the GOx activity of GNE-AuNPs according to the glucose concentration (0.05-60 mM).

In FIG. 8(c) , no change in absorbance appears after the glucose concentration is increased to a certain level or more. This means that when the glucose concentration is increased above a certain concentration, the activity of GNE-AuNPs does not increase as the gold nanoparticles and glucose bonds reach saturation.

(iv) sugar selectivity

Figure 8(d) and the inset (TMB color change) show the sugar selectivity of GNE-AuNPs. (d-Fructose), d-galactose (galactose), lactose (lactose) and saccharose (saccharose) as it exhibits a significantly higher absorbance compared to, it can be seen that the selectivity to glucose of GNE-AuNPs is very large, , it can be seen that selective analysis for glucose detection can be carried out through this.

(2) catalytic parameters for POx mimic activity

(i) H ₂ O ₂ concentration

Figure 9 (a) shows the POx mimic activity of GNE-AuNPs according to the H ₂ O ₂ concentration.

Referring to this, when the H ₂ O ₂ concentration is increased from 0 to 10 mM, the absorbance at 652 nm is greatly increased, whereas when the H ₂ O ₂ concentration is increased to 10 mM or more, the absorbance slightly increases and TMB is completely oxidized. it can be seen that

(ii) AuNPs concentration

Figure 9 (b) shows the analysis of POx mimic activity according to AuNPs concentration, and was analyzed using GNE-AuNPs solutions (0-57.9 μg/mL) of various concentration ranges.

In FIG. 9(b), it can be seen that the absorbance value at 652 nm increases as the AuNPs concentration increases, and the TMB color darkens to blue (FIG. 9(b) inset). Through this, it can be seen that as the concentration of AuNPs increases, more active sites exist, and thus the reaction rate increases.

4. Comparison of enzyme-mimicking catalytic properties of GNE-based AuNPs

The GOx and POx mimetic activities of GNE-based AuNPs were analyzed by TMB oxidation to blue oxTMB with a characteristic absorption peak at 652 nm. The kinetic parameters of the catalytic reaction were determined based on the change in absorbance by varying the glucose or H ₂ O ₂ concentration and keeping the other concentrations constant. The reaction rate was calculated as the change in absorbance per unit time using the molar extinction coefficient of oxTMB in the Beer Lambert's law equation (3) below.

where A ₆₅₂ is the absorbance at 652 nm, ε _oxTMB is the molar extinction coefficient (39000 M ⁻¹ cm ⁻¹ ) and l is the sample path length (1 cm) of the cuvette used for absorbance measurement. Taking into account the reaction stoichiometry (1 mol of glucose yields 0.5 mol oxTMB), V(reaction rate, M/s) was calculated as c/2t. where t is the reaction time (s).

As a result of plotting the change in reaction rate as a function of substrate concentration [s], AuNPs catalysis followed the Michaelis-Menten equation for a specific range of glucose and H ₂ O ₂ concentrations, and the double reciprocal Lineweaver-Burk curve was plotted to The kinetic parameters Km and Vmax were determined. Km values were estimated to be 0.089 and 0.118 mM for glucose and H ₂ O ₂ , respectively.

The catalytic properties of the synthesized GNE-based AuNPs are shown in Table 1 below by comparing them with natural enzymes and SC with kinetic parameters.

The data for glucose oxidase and peroxidase in Table 1 below are the values described in References [1] to [3], and are also measured and shown for other materials listed in Table 1 in the same manner.

Material Substrate AuNPs
average particle
Size (nm) K _m
(mM) V _max
(μM/s) Linear
range
(mM) Reference
literature glucose oxidase glucose - 4.87 0.69 - [One] glucose oxidase glucose - 2.7 0.006 - [2] peroxidase H ₂ O ₂ - 0.47 2.667 - [3] peroxidase H ₂ O ₂ - 3.7 0.087 - [3] SC-based AuNPs glucose 13-50 6.97 0.63 - [One] GNE-based AuNPs glucose 27.5 0.089 0.133 0.05-10 - GNE-based AuNPs H ₂ O ₂ 27.5 0.120 0.147 0.05-1 -

[1] W. Luo, C. Zhu, S. Su, D. Li, Y. He, Q. Huang, C. Fan, Self-catalyzed, self-limiting growth of glucose oxidase-mimicking gold nanoparticles, ACS Nano 4 (2010) 7451 - 7458

[2] S. Ate㎧, N. Ivli, Properties of immobilized glucose oxidase and enhancement of enzyme activity, Artif. Cells Nanomed. Biotechnol. 41 (2013) 264 - 268

[3] H. Ikemoto, Q. Chi, J. Ulstrup, Stability and catalytic kinetics of horseradish peroxidase confined in nanoporous SBA-15, J. Phys. Chem. C. 114 (2010) 16174 - 16180

In Table 1, the affinity between the substrate and AuNPs can be confirmed through the Km value. That is, the lower the Km value, the higher the affinity between the substrate and AuNPs.

In the case of the GNE-based AuNPs, as the Km values for both glucose and H ₂ O ₂ appear lower than that of the native enzyme and SC, GNE-based AuNPs have higher affinity between the substrate and AUNPs than the natural enzyme and SC and thus It can be seen that it has improved enzymatic performance.

In addition, the proposed colorimetric method exhibited excellent sensitivity for glucose detection with a linear detection range of 0.05-10 mM.

Diabetes is usually diagnosed after blood or urine tests, and normal glucose levels in human blood and urine have been reported to be in the range of 4.4-7.8 mM and 0-0.8 mM, respectively. GNE-based AuNPs showed excellent sensitivity to glucose oxidation in the range of 0.05–60 mM, demonstrating their potential as a nanosensor for glucose detection in blood and urine samples.

6. Zeta Potential of GNE-based AuNPs

Zeta potential values for the synthesized GNE-based AuNPs were measured . The zeta potential provides information about the stability and surface charge of colloidal nanoparticle systems. The zeta potential was measured at 25° C. using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). A gold-plated electrode capillary cuvette was filled with 0.75 ml of a gold nanoparticle sample and stored in the instrument's cuvette holder. The zeta potential for the particles was then measured using the instrument's software.

The GNE-based AuNPs have an average zeta potential value of -32.2 mV, indicating excellent physical stability of the resulting nanosuspension due to particle-particle repulsion. The negative value of the zeta potential is mostly due to the negative charge of the GNE biomolecule. In general, a zeta potential more positive than +30 mV or more negative than -30 mV indicates a stable colloidal nanoparticle system. The resulting GNE-based AuNPs colloidal nanosuspension did not show color change or aggregate formation for more than one month, whereas SB and SC-based nanoparticles showed precipitation after one week.

7. Long-term performance stability of GNE-based AuNPs

Long-term performance stability evaluation was performed for the synthesized GNE-based AuNPs, and is shown in FIG. 10 .

The evaluation was carried out by storing the GNE-based AuNPs samples in sealed dark bottles at 4 °C and room temperature (20 °C) for 0.5 mM glucose concentration at 5-day intervals.

Figure 10 shows the stability of GNE-based AuNPs stored at 4 °C and 20 °C based on the absorbance at 652 nm. The GNE-based AuNPs stored at 4 °C showed excellent reproducibility and long-term stability for 10 days, GNE-based AuNPs stored at 20 °C showed a slight decrease in stability after storage for 5 days, but maintained high stability for relatively 10 days.

Through this, it can be seen that GNE-based AuNPs exhibit improved long-term performance stability.

8. Comparison of catalytic activity for 4-NP of GNE-based AuNPs and Au-Nanoflowers

The catalytic activity of the synthesized GNE-based AuNPs and Au-Nanoflowers was compared with a model reduction reaction of 4-nitrophenol (4-NP) to 4-aminophenol using NaBH ₄ , and is shown in FIG. 11 .

The catalytic reduction of 4-NP was carried out in a quartz cuvette by adding 500 μL of 4-NP aqueous solution, 200 μL of AuNF aqueous solution and 500 μL of NaBH ₄ solution, and the reduction of 4-NP was visualized by UV-visible spectroscopy. was decided. The effect of 4-NP concentration (0.1-0.25 mM), NaBH ₄ concentration (0.1-0.25 M) and temperature (298.15-313.15 K) on 4-NP reduction was studied.

Referring to FIG. 11 , the first-order reaction rate constant and % removal rate of 4-NP for gold nanoparticles and gold nanoparticles are 2.37x10 ^-3 s ^-1 , 81.8% and 1.51x10 ^-3 s ^-1 , 71.6%, respectively. It can be confirmed that gold nanoflowers are more effective in catalytic reduction than gold nanoparticles.

Claims (9)

(1) preparing a gold precursor solution:
(2) adding a galnut extract to the gold precursor solution and stirring until the reaction is completed;
Gold nanoparticles with increased glucose oxidase and peroxidase activity, characterized in that prepared by the method of claim 1 .
3. The method of claim 2,
The gold nanoparticles have at least one shape selected from a rhombic dodecahedron, a decahedron, and a dipyramid, and have a particle size of 1 to 100 nm. Gold nanoparticles with increased glucose oxidase and peroxidase activity .
A sensor for glucose detection comprising the gold nanoparticles of any one of claims 2 to 3.
A sensor for detecting hydrogen peroxide comprising the gold nanoparticles of any one of claims 2 to 3.
(1) preparing a mixture by adding a gold precursor solution to the galnut extract while maintaining constant stirring; and
(2) stirring the mixture to prepare flower-shaped gold nanoparticles; A method for producing flower-shaped gold nanoparticles, comprising:
7. The method of claim 6,
Stirring the mixture in step (2),
(a) forming a primary crystal of Au by reacting phytochemicals of the galnut extract with a gold precursor in the mixture;
(b) the Au invitation and phytochemicals are aggregated, preparing aggregated nanoparticles; and
(c) subjecting the aggregated nanoparticles to Ostwald Ripening to prepare flower-shaped gold nanoparticles; A method for producing flower-shaped gold nanoparticles, comprising:
Claims 6 and 7, characterized in that produced according to any one of the method, flower-shaped gold nanoparticles.
A catalyst for the reduction reaction of 4-nitrophenol (4-NP), characterized in that it comprises the flower-shaped gold nanoparticles of claim 8.

Images