1. The gold nanoparticle with the core-shell structure is characterized by comprising a gold nanoparticle inner core modified by 3-butenylamine hydrochloride and a gel shell layer coated on the surface of the gold nanoparticle inner core, wherein the gel shell layer is a copolymer of acrylamide and N-isopropylacrylamide.

2. The core-shell structure gold nanoparticles according to claim 1, wherein the size of the gold nanoparticles is 3 to 30 nm;

preferably, the gel shell layer is a reticular hydrogel structure, and the thickness of the gel shell layer is 1-10 nm.

3. The method for preparing gold nanoparticles having a core-shell structure according to claim 1 or 2, comprising: and carrying out polymerization reaction on the 3-butenyl amine acid modified gold nanoparticles, acrylamide, N-isopropylacrylamide, a cross-linking agent and an initiator to obtain the gold nanoparticles.

4. The method for preparing gold nanoparticles having a core-shell structure according to claim 3, wherein the method for preparing 3-butenylamine hydrochloride-modified gold nanoparticles comprises: stirring and centrifuging the gold nanoparticles and the 3-butenyl amine hydrochloride ethanol solution, and collecting precipitates;

preferably, the concentration of the 3-butenyl amine hydrochloride in the 3-butenyl amine hydrochloride ethanol solution is 2.0-3.0mmol/L, preferably 2.88 mmol/L;

preferably, the concentration of the gold nano particle aqueous solution is 10 mmol/L;

preferably, the stirring speed is 800-;

preferably, the centrifugation parameters are 6000rpm, 30 min.

5. The method for preparing gold nanoparticles having a core-shell structure according to claim 3, comprising: firstly, dispersing acrylamide, N-isopropyl acrylamide and a cross-linking agent in water, removing air in a device, heating and stirring, then adding 3-butenyl amine acid modified gold nanoparticles, stirring, and then adding an initiator for reaction;

preferably, the mass ratio of the acrylamide to the N-isopropylacrylamide to the cross-linking agent is as follows: 5-8:50-60:8-15, preferably 7:56: 10;

preferably, the acrylamide concentration is 0.5 g/L;

preferably, the heating temperature is 60-80 ℃, and the heating and stirring time is 0.5-2h, preferably 70 ℃, 1 h;

preferably, the 3-butenylamine acid-modified gold nanoparticles are added and stirred for 10-30min, preferably 15 min.

6. The method for preparing gold nanoparticles with core-shell structures according to claim 5, wherein the initiator is dissolved in water to prepare a solution of 15-20g/L, and the solution is added into the reaction system, wherein the concentration of the initiator solution is preferably 17 g/L;

preferably, the volume ratio of the initiator solution to the mixed solution of the 3-butenyl amine salt acid-modified gold nanoparticles, acrylamide, N-isopropyl acrylamide and the cross-linking agent is 1: 14;

preferably, the initiator is added and then the stirring reaction is carried out for 1.5 to 2.5 hours, preferably 2 hours;

preferably, the cross-linking agent is selected from N, N' -methylene-bisacrylamide, and the initiator is selected from ammonium persulfate, persulfuric acid or 2-hydroxy-2-methyl-1-phenyl-1-acetone;

preferably, the preparation method further comprises the steps of cooling, centrifuging and purifying after the reaction is finished.

7. The method for preparing gold nanoparticles having a core-shell structure according to claim 3, wherein the method for preparing gold nanoparticles comprises reducing chloroauric acid with sodium citrate;

preferably, the chloroauric acid aqueous solution is stirred and heated to boiling, then the preheated sodium citrate aqueous solution is added, the mixture is continuously stirred for 20 to 30min under the boiling state, then the mixture is cooled to room temperature, and the chloroauric acid aqueous solution is obtained after separation;

preferably, a surfactant, preferably an anionic surfactant, is added during the reaction;

preferably, the anionic surfactant is selected from sodium lauryl sulfate;

preferably, the concentration of the chloroauric acid aqueous solution is 5 x 10^-4mol/L, the concentration of the sodium citrate aqueous solution is 1 wt%, and the volume ratio of the chloroauric acid aqueous solution, the sodium citrate aqueous solution and the anionic surfactant is 50:2.433: 0.291.

8. The use of core-shell structure gold nanoparticles according to claim 1 or 2 in the field of detecting hydrogen peroxide and/or glucose.

9. A hydrogen peroxide detection reagent or detector comprising the core-shell structure gold nanoparticles according to claim 1 or 2.

10. A glucose detecting reagent or detector comprising the core-shell structure gold nanoparticles according to claim 1 or 2.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The enzyme (enzyme) is an organic substance produced by living cells, and the composition of the enzyme is a large amount of protein and a small amount of genetic material RNA, so that the enzyme has high specific recognition function and catalytic efficiency on a substrate. Enzymes belong to biological macromolecules, and their catalytic action is highly dependent on the integrity of the primary and steric structure of the molecule, which can be inactivated if the enzyme molecule is denatured or depolymerised.

With the intensive research on the structure and function of enzyme molecules, the kinetics of enzymatic reactions, etc., some serious disadvantages of natural enzymes have been gradually revealed, such as unstable catalytic activity, dependence on structural integrity, susceptibility to environmental influences during the reaction process, and complicated and expensive preparation, purification and storage of natural enzymes. The artificial simulation of enzyme has come into play and become a new research hotspot. To date, it has been found that over 40 types of nanomaterials have intrinsic enzyme-like activity, including Fe₃O₄、Co₃O₄、CeO₂、BiFeO₃、MnFe₂O₄Peroxidase activity of CdS, etc.; au, Pt, CoFe₂O₄、ZnFe₂O₄Etc. oxidase activity; MnO₂The activity of the halogenated peroxidase of the vanadium pentoxide nanoparticles; superoxide dismutase activity of nano platinum, fullerene derivatives and nano cerium oxide. Among them, the noble metal-based nanoenzyme-gold nanoenzyme has attracted great interest to researchers due to its advantages of simple preparation process, adjustable size, strong stability, easy surface modification, etc.

The inventor researches and discovers that although the gold particles have certain activity, the gold particles still have inevitable defects, and the special size can also cause the naked gold nanoparticles to easily and rapidly aggregate in normal saline and a special pH environment to lose the characteristics of the naked gold nanoparticles while showing advantages. To prevent this, some stabilizers (surfactants) are usually added to the nanogold solution, but this also introduces some other substances that have a more or less influence on the catalytic action of the mimic enzyme.

Disclosure of Invention

In order to solve the problems that the gold particles in the prior art have poor stability when used as enzyme, are very easy to quickly aggregate in normal saline and special pH environment to lose the characteristics of the gold particles and have limited catalytic performance, the invention provides the gold nanoparticles with the core-shell structure, and the material not only has the biological mimic enzyme activity of the gold nanoparticles and has the effects of resisting glucose and H₂O₂Has selectivity; and the aggregation of the active carbon can be prevented, and the active carbon can be well dispersed in the solution. In addition, the invention also provides a preparation method of the gold nano-particles with the core-shell structure, the method is simple and easy to implement, the raw materials are easy to obtain, and the method has good prospects when being applied to production.

Specifically, the invention is realized by the following technical scheme:

the invention provides a gold nanoparticle with a core-shell structure, which comprises a gold nanoparticle inner core modified by 3-butenylamine hydrochloride and a gel shell layer coated on the surface of the gold nanoparticle inner core, wherein the gel shell layer is a copolymer of acrylamide and N-isopropyl acrylamide.

In a second aspect of the present invention, a method for preparing gold nanoparticles with a core-shell structure is provided, which comprises: and carrying out polymerization reaction on the 3-butenyl amine acid modified gold nanoparticles, acrylamide, N-isopropylacrylamide, a cross-linking agent and an initiator to obtain the gold nanoparticles.

The third aspect of the invention provides an application of gold nanoparticles with a core-shell structure in the field of detection of hydrogen peroxide and/or glucose.

The fourth aspect of the invention provides a hydrogen peroxide detection reagent or detector, which comprises core-shell structure gold nanoparticles.

In a fifth aspect of the invention, a glucose detection reagent or detector is provided, which comprises core-shell structure gold nanoparticles.

One or more technical schemes of the invention have the following beneficial effects:

1) the gold nanoparticles with the core-shell structure comprise a gold nanoparticle inner core modified by 3-butenyl amine hydrochloride and a gel shell layer coated on the surface of the gold nanoparticle inner core, wherein the gel shell layer is a copolymer of acrylamide and N-isopropyl acrylamide. The gold nanoparticles are modified by 3-butenyl amine hydrochloride acid, amine groups on the 3-butenyl amine hydrochloride acid are adsorbed on the surfaces of the gold nanoparticles through electrostatic action, and the gold nanoparticles indirectly have vinyl functionality, so that the vinyl can be directly polymerized and encapsulated to generate a gel shell layer.

2) The stability of the bare gold nanoparticles is improved by using the sodium dodecyl sulfate in the preparation process.

3) The invention discovers for the first time that the gold nanoparticles with the core-shell structure have catalytic oxidation capacity on peroxidase substrate Tetramethylbenzidine (TMB) and the catalytic oxidation capacity is H₂O₂In the coexistence, the gold-core nanogel can accelerate the oxidation of TMB (Ox-TMB) by a blue product generated by the oxidation reaction of the TMB.

4) Glucose can be decomposed by glucose oxidase into hydrogen peroxide and gluconic acid, and the amount of hydrogen peroxide is generally more than gluconic acid. During the glucose assay, H is still used₂O₂Color reaction with TMB.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a schematic diagram of preparation of gold nanoparticles of core-shell structure according to example 1;

FIG. 2 is a transmission electron microscope photograph of gold nanoparticles of core-shell structure of example 1;

FIG. 3 is H in the presence of TMB developer in example 2₂O₂Detecting ultraviolet absorption spectrogram and color development diagram;

FIG. 4 is a chart of an ultraviolet absorption spectrum and a color development of glucose detection in the presence of TMB color development agent in example 3;

FIG. 5 shows bare gold prepared in comparative example 1 and Au @ PNIPAm prepared in example 1 at 25mmol/LH₂O₂Comparing catalytic efficiency in the solution;

FIG. 6 is UV absorption spectra of AuNPs prepared in comparative example 1, AuNPs-BA prepared in comparative example 2, and Au @ PNIPAm prepared in example 1;

fig. 7 shows the uv absorption of AuNPs prepared in comparative example 1 and Au @ PNIPAm prepared in example 1 in a salt environment.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In order to solve the problems that the gold particles in the prior art have poor stability when used as enzyme, are very easy to quickly aggregate in normal saline and special pH environment to lose the characteristics of the gold particles and have limited catalytic performance, the invention provides the gold nanoparticles with the core-shell structure, and the material not only has the biological mimic enzyme activity of the gold nanoparticles and has the effects of resisting glucose and H₂O₂Has selectivity; and the aggregation of the active carbon can be prevented, and the active carbon can be well dispersed in the solution. In addition, the invention also provides a preparation method of the gold nano-particles with the core-shell structure, the method is simple and easy to implement, the raw materials are easy to obtain, and the method has good prospects when being applied to production.

Specifically, the invention is realized by the following technical scheme:

the invention provides a gold nanoparticle with a core-shell structure, which comprises a gold nanoparticle inner core modified by 3-butenylamine hydrochloride and a gel shell layer coated on the surface of the gold nanoparticle inner core, wherein the gel shell layer is a copolymer of acrylamide (AAM) and N-isopropylacrylamide (NIPAM).

In the invention, the gold nanoparticles are modified by 3-butenyl amine hydrochloride acid, amine groups on the 3-butenyl amine hydrochloride acid are adsorbed on the surfaces of the gold nanoparticles through electrostatic action, and the gold nanoparticles indirectly have the functionality of vinyl groups, so that the vinyl groups can be directly polymerized and encapsulated to generate a gel shell layer.

In one or more embodiments of the present invention, the gold nanoparticles have a size of 3 to 30 nm. The gold nanoparticles have overlarge size and small specific surface area, are not beneficial to surface modification and formation of a later-stage coating layer, and simultaneously, the overlarge size influences the detection sensitivity of hydrogen peroxide and the stability of the composite material. If the size of the gold nanoparticles is too small, blocking and agglomeration are easily generated.

Preferably, the gel shell layer is a reticular hydrogel structure, and the thickness of the gel shell layer is 1-10 nm. The network hydrogel structure is derived from a copolymer of acrylamide, N-isopropyl acrylamide and surface modified vinyl of gold nanoparticles.

In a second aspect of the present invention, a method for preparing gold nanoparticles with a core-shell structure is provided, which comprises: and carrying out polymerization reaction on the 3-butenyl amine acid modified gold nanoparticles, acrylamide, N-isopropylacrylamide, a cross-linking agent and an initiator to obtain the gold nanoparticles.

Acrylamide and N-isopropyl acrylamide are subjected to polymerization reaction with vinyl under the action of a cross-linking agent and an initiator, and a net-shaped shell layer is formed on the surface of the gold nano-particle.

The 3-butenyl amine salt acid modification is to make the surface of the gold nano-particle have C ═ C through physical adsorption of ammonium groups and the gold nano-particle, and if no C ═ C exists, copolymerization can not occur on the surface of the gold nano-particle. In addition, if the gold nanoparticles are not modified by 3-butenylamine hydrochloride acid, and the gold nanoparticles are directly subjected to polymerization reaction with acrylamide and N-isopropylacrylamide, the two monomers are only polymerized in solution, and a gel shell layer cannot be directly generated on the surface of the gold.

In order to generate a gel shell layer by directly carrying out polymerization reaction on the nano surface, 3-butenylamine hydrochloride (BA) is dropwise added in the preparation process. In one or more embodiments of the present invention, a method for preparing 3-butenylamine hydrochloride-modified gold nanoparticles includes: and (3) stirring and centrifuging the gold nanoparticle aqueous solution and the 3-butenylamine hydrochloride ethanol solution, and collecting the precipitate. The amino group on the 3-butenyl amine hydrochloride is adsorbed on the surface of the nanogold through electrostatic action, indirectly has the functionality of vinyl, and can directly polymerize and encapsulate the vinyl.

The concentration of 3-butenyl amine hydrochloride in the 3-butenyl amine hydrochloride ethanol solution directly influences the surface modification condition of the gold nanoparticles, and the 3-butenyl amine hydrochloride concentration is too high or too low, and is in poor contact with the gold nanoparticles, or is aggregated, or cannot be modified uniformly. Thus in some embodiments, the 3-butenylamine acid concentration in the 3-butenylamine amine acid ethanolic solution is from 2.0 to 3.0mmol/L, preferably 2.88 mmol/L.

Preferably, the concentration of the gold nano particle aqueous solution is 10 mmol/L. When the gold nanoparticle aqueous solution and the 3-butenyl amine hydrochloride ethanol solution react, 3-butenyl amine hydrochloride is excessive in order to ensure that the gold nanoparticles are completely modified, and unreacted 3-butenyl amine hydrochloride is removed by centrifugation.

Preferably, the stirring rate is 800-. The 3-butenyl amine hydrochloride is connected with the gold nanoparticles through amine groups, and if the stirring speed is too high, physical adsorption can be damaged, so that the loading of the 3-butenyl amine hydrochloride on the surfaces of the gold nanoparticles is reduced, and the polymerization reaction with monomers is influenced. If the stirring rate is too slow, the modifier and gold nanoparticles are liable to aggregate or precipitate, and the loading is not uniform.

Preferably, the centrifugation parameters are 6000rpm, 30 min.

The centrifugation process is carried out in water in order to remove unreacted monomers, and after removal of the supernatant, the coagulated product is redispersed in water and stored.

In one or more embodiments of the present invention, the preparation method comprises: firstly, dispersing acrylamide, N-isopropyl acrylamide and a cross-linking agent in water, removing air in a device, heating and stirring, then adding 3-butenyl amine acid modified gold nanoparticles, stirring, and then adding an initiator for reaction.

Different from the traditional scheme of simultaneously reacting gold nanoparticles, a monomer, a cross-linking agent and an initiator, the method provided by the invention has the advantages that the monomer and the cross-linking agent are mixed and uniformly dispersed to prepare for the next polymerization reaction. The purpose of the air removal is to remove oxygen from the reaction system by using an inert gas such as nitrogen, argon or helium to avoid the inhibition. After the core material, the monomer and the cross-linking agent are uniformly mixed, the initiator is finally added, so that the coating layer can be uniformly formed on the surface of the gold nano-particle.

The monomer concentration and the raw material ratio directly influence the coating effect, and in some embodiments, the mass ratio of the acrylamide to the N-isopropylacrylamide to the cross-linking agent is as follows: 5-8:50-60:8-15, preferably 7:56:10, and the concentration of the acrylamide is 0.5 g/L.

In some embodiments, the heating temperature is 60-80 ℃, the heating stirring time is 0.5-2h, preferably 70 ℃, 1h, and the heating is carried out for better polymerization reaction on the surface of the gold nanoparticles to form a uniform coating layer.

In some embodiments, the 3-butenylamine acid-modified gold nanoparticles are added and stirred for 10-30min, preferably 15 min.

In order to further improve the dispersion uniformity of raw materials and enable the polymerization reaction to uniformly occur in the system, in one or more embodiments of the invention, the initiator is firstly dissolved in water to prepare a solution of 15-20g/L, and the solution is added into the reaction system, wherein the concentration of the initiator solution is preferably 17 g/L.

Preferably, the volume ratio of the initiator solution to the mixed solution of the 3-butenyl amine salt acid-modified gold nanoparticles, acrylamide, N-isopropyl acrylamide and the cross-linking agent is 1: 14;

in order to ensure the crosslinking and polymerization reaction, the initiator is added and then stirred to react for 1.5 to 2.5 hours, preferably 2 hours, the reaction time is too long, excessive polymerization is carried out, the coating layer completely covers the characteristics of the gold nanoparticles, and the detection sensitivity is reduced.

Preferably, the cross-linking agent is selected from N, N' -methylenebisacrylamide (Bis), and the initiator is selected from Ammonium Persulfate (APS), persulfuric acid, or 2-hydroxy-2-methyl-1-phenyl-1-propanone;

preferably, the preparation method further comprises the steps of cooling, centrifuging and purifying after the reaction is finished.

The gold nanoparticles are the prior art, and the protection method can be used as long as the gold nanoparticles meeting the size requirement can be prepared. In one or more embodiments of the present invention, the gold nanoparticle preparation method includes reducing chloroauric acid with sodium citrate;

preferably, the chloroauric acid aqueous solution is stirred and heated to boiling, then the preheated sodium citrate aqueous solution is added, the mixture is continuously stirred for 20 to 30min under the boiling state, then the mixture is cooled to room temperature, and the chloroauric acid aqueous solution is obtained after separation;

further stabilizing the bare gold nanoparticles, adding a surfactant, preferably an anionic surfactant, at room temperature during the reaction process before adding 3-butenylamine amine hydrochloride;

preferably, the anionic surfactant is selected from sodium lauryl sulfate.

Preferably, the concentration of the chloroauric acid aqueous solution is 5 x 10^-4mol/L, the concentration of the sodium citrate aqueous solution is 1 wt%, and the volume ratio of the chloroauric acid aqueous solution, the sodium citrate aqueous solution and the anionic surfactant is 50:2.433: 0.291.

Even more preferably, 50mL of 5X 10^-4The mol chloroauric acid aqueous solution was filled into a three-necked flask, stirred and heated to boiling, then 2.433mL (1 wt%) of an aqueous solution of sodium citrate preheated in advance was added, and the solution was cooled to room temperature (25 ℃) after stirring for 20min under boiling. To stabilize the nanogold, 0.291mL of 0.1mmol/L Sodium Dodecyl Sulfate (SDS) solution was added thereto, and after stirring for 20min, 0.953mL of 2.88mmol of 3-butenylamine hydrochloride (BA) ethanol was addedThe solution was stirred for another 20 min. Centrifuging the obtained gold nanoparticle water dispersion solution at 6000rpm for 30min to obtain precipitate, dispersing in 20mL of water, performing ultrasonic dispersion, and storing in a refrigerator for later use.

The third aspect of the invention provides an application of gold nanoparticles with a core-shell structure in the field of detection of hydrogen peroxide and/or glucose.

The fourth aspect of the invention provides a hydrogen peroxide detection reagent or detector, which comprises core-shell structure gold nanoparticles.

In a fifth aspect of the invention, a glucose detection reagent or detector is provided, which comprises core-shell structure gold nanoparticles.

The invention provides a hydrogen peroxide detection method, which comprises the steps of adding gold nanoparticles with a core-shell structure into an acetic acid-acetate buffer solution, then adding a Tetramethylbenzidine (TMB) ethanol solution, then adding a hydrogen peroxide solution, incubating the mixed solution, and finally collecting an ultraviolet-visible absorption spectrum on a spectrophotometer.

In the seventh aspect of the present invention, there is provided a glucose detection method comprising adding core-shell structure gold nanoparticles and a glucose solution to a PBS buffer solution, incubating the mixture in a 37 ℃ warm water bath continuously and in the absence of light, centrifuging the mixture to obtain a supernatant, and then verifying H produced in the supernatant (100. mu.L) in HAc-NaAc buffer solution by HRP (5. mu.g/mL, 20. mu.L) -TMB (10mM, 100. mu.L) -based color reaction₂O₂. Finally, the uv-vis absorption spectra were collected on a spectrophotometer.

The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.

Example 1

1. Preparation of gold nanoparticles with surface modified double bonds

Will contain 50mL of 5X 10^-4A three-necked flask (100mL) of the aqueous solution of the chloroauric acid was placed in an electric heating mantle, magnetically stirred and heated to boiling, then 2.433mL (1 wt%) of the aqueous solution of sodium citrate preheated in advance was rapidly added, and stirring was continued for 20m under boilingAfter in the solution was cooled to room temperature. To stabilize the nanogold, 0.291mL of 0.1mmol/L Sodium Dodecyl Sulfate (SDS) solution was added thereto, and after stirring for 20min, 0.953mL of 2.88 mmol/L3-butenylamine hydrochloride (BA) ethanol solution was added dropwise, and stirring was continued for 20 min. Centrifuging the obtained gold nanoparticle water dispersion solution at 6000rpm for 30min to obtain precipitate, dispersing in 20mL of water, performing ultrasonic dispersion, and storing in a refrigerator for later use.

2. Preparation of gold nanoparticle gel with core-shell structure

As shown in FIG. 1, the preparation method comprises the following steps:

monomer NIPAM (0.056g), AAM (0.007g) and crosslinking agent Bis (0.01g) are put into a three-necked bottle, added with 14mL of ultrapure water for dissolving, placed in an oil bath pot for magnetic stirring and heated to 70 ℃. A reflux condenser was added to the three-necked flask and nitrogen was continuously introduced. One hour later, 10mL of the functionalized gold particles obtained in the first step (aqueous precipitate) were added dropwise and incubated, stirred for 15min, and 1mL of 0.017g/mL aqueous solution of initiator APS was added rapidly to start the polymerization. After two hours, the dispersion was allowed to cool to room temperature and the reaction was stopped. Centrifuging the obtained core-shell hybrid particles at 6000rpm for 60min, performing primary centrifugation to obtain core-shell structure nanoparticles, precipitating unreacted monomers and impurities in the solution in a supernatant, pouring out the supernatant, and adding 20mL of ultrapure water again to the precipitate to obtain the redispersion.

And (3) dropwise adding the prepared gold nanoparticle gel solution with the core-shell structure on a copper net, and placing the gold nanoparticle gel solution in a constant-temperature incubator for 12 hours. Fig. 2 is a transmission electron microscope image of the gold nanoparticle gel with the core-shell structure prepared in example 1, and it can be seen from the image that the gold nanoparticle gel with the core-shell structure is of an obvious core-shell structure, and has uniform coating and no uncoated and agglomeration phenomena.

Example 2

Sensitive detection of hydrogen peroxide by nanogels

0.1mL of the dispersed nanogel solution prepared in example 1 (theoretical concentration 25mmol/L) was added to acetic acid-acetate (Hac-NaAc 1.5mL of 0.1mol/L, pH 4.0) buffer, followed by 0.1mL of tetramethylbenzidine (TMB 10mmol/L) ethanol solution, and then 0.3mL of peroxylHydrogen (H)₂O₂10mmol/L) solution, and the mixture is incubated for 10 min. Finally, the UV-visible absorption spectra were collected on a UV-2500 spectrophotometer.

As shown in FIG. 3, at H₂O₂In the environment of (2), the Au @ PNIPAm gold core nanogel is found to accelerate the oxidation reaction of TMB to generate a blue product (the right side cell in the figure 3 inset shows dark gray color) to oxidize TMB (Ox-TMB), and the maximum ultraviolet absorption peak of the oxidized TMB is shown at 655+ nm. In contrast, the Au @ PNIPAm and TMB system (point-dashed line in fig. 3) does not produce a significant absorption band at 652nm, where a slight absorption peak may result from the auto-oxidation of TMB and not from the Au @ PNIPAm nanogel effect. When Au @ PNIPAm and H₂O₂The Au @ PNIPAm gold core nanogel can be used as a hydrogen peroxide mimic enzyme, and the reaction is greatly accelerated by the existence of the Au @ PNIPAm.

Example 3

Sensitive detection of nanogels for glucose

First, 0.1mL of nanogel and 0.5mL of glucose (100m mol) solution were added to PBS (1.4mL of 0.1mol/L, pH 9.0) buffer and incubated continuously in a warm water bath at 37 ℃ for 30min in the absence of light, the mixture was centrifuged (10000rpm 10min) to take the supernatant, and then H produced in the supernatant (100. mu.L) was verified by a color reaction based on horseradish peroxidase HRP (5. mu.g/mL, 20. mu.L) -TMB (10mmol/L, 100. mu.L) in HAc-NaAc buffer (0.1mol/L of 500. mu.L, pH 4.0)₂O₂. Finally, the UV-visible absorption spectra were collected on a UV-2500 spectrophotometer.

As shown in fig. 4, the HRP-TMB color reagent system added to the supernatant obtained after the incubation of glucose and Au @ PNIPAm nanogel exhibited better ultraviolet characteristic absorption (ultraviolet absorption of oxidized TMB), however, no obvious ultraviolet characteristic absorption peak appeared in the control experiment using only glucose or glucose added with TMB color reagent, which indicates that Au @ PNIPAm has similar effect to natural glucose oxidase and that H was indeed generated in the glucose oxidation reaction catalyzed by Au @ PNIPAm₂O₂。

Comparative example 1 preparation of gold nanoparticles (AuNPs)

Will contain 50mL of 5X 10^-4A three-necked flask (100mL) of an aqueous solution of chloroauric acid was placed in an electric heating mantle, magnetically stirred and heated to boiling, then 2.433mL (1 wt%) of an aqueous solution of sodium citrate preheated in advance was rapidly added, and the solution was cooled to room temperature after stirring for 20min under boiling.

As shown in FIG. 5, the bare gold prepared in comparative example 1 and the Au @ PNIPAm prepared in example 1 were at 25mmol/L H₂O₂In the solution, 0.1mL of AuNPs prepared in comparative example 1 and 0.1mL of Au @ PNIPAm nanogel prepared in example 1 are compared in catalytic efficiency, the test conditions are the same as those of example 2, the figure shows that the AuNPs are slightly influenced by the polymer before and after being wrapped, and the naked AuNPs are slightly stronger than the Au @ PNIPAm nanogel under the same concentration, which means that the naked AuNPs are easier to contact H₂O₂And the result is that.

Comparative example 2 preparation of gold nanoparticles (AuNPs-BA) surface-modified with double bonds

Will contain 50mL of 5X 10^-4A three-necked flask (100mL) of an aqueous solution of chloroauric acid was placed in an electric heating mantle, magnetically stirred and heated to boiling, then 2.433mL (1 wt%) of an aqueous solution of sodium citrate preheated in advance was rapidly added, and the solution was cooled to room temperature after stirring for 20min under boiling. To stabilize the nanogold, 0.291mL of 0.1mmol/L Sodium Dodecyl Sulfate (SDS) solution was added thereto, and after stirring for 20min, 0.953mL of 2.88 mmol/L3-butenylamine hydrochloride (BA) ethanol solution was added dropwise, and stirring was continued for 20 min. Centrifuging the obtained gold nanoparticle water dispersion solution at 6000rpm for 30min to obtain precipitate, dispersing in 20mL of water, performing ultrasonic dispersion, and storing in a refrigerator for later use.

FIG. 6 shows UV absorption spectra of AuNPs prepared in comparative example 1, AuNPs-BA prepared in comparative example 2 and Au @ PNIPAm prepared in example 1, 0.1mL of the dispersed nanogel solution (theoretical concentration of 25mmol/L) prepared in example 1, the BA-modified AuNPs-BA solution (theoretical concentration of 25mmol/L) and the bare gold nanoparticle (AuNPs) solution of the same concentration were dissolved in 1.9mL of ultrapure water, respectively, and finally, UV-visible absorption spectra were collected on a UV-2500 spectrophotometer.

Due to surface plasmon resonance of the bare spherical gold nanoparticles, the maximum absorption band was about 523 nm. The amino group in the 3-butenamide hydrochloride was adsorbed on the surface of the bare spherical gold nanoparticles by electrostatic interaction and created a double bond on the surface that could continue to be functionalized, and fig. 6 shows that the maximum uv absorption of the gold nanoparticles AuNPs-BA improved by this functionalization resulted in a slight red shift of about 526 nm. In addition, the synthesized Au @ PNIPAm nanogel is easily characterized by uv-vis spectroscopy due to the presence of a gold core. Comparing the ultraviolet absorption spectra of the gold nanoparticles before and after gel shell synthesis, the maximum infrared absorption of the Au @ PNIPAm nanogel occurs at about 528 nm. The plasmon band appears slightly red-shifted due to the environmental change around the gold nanoparticles caused by the polymer shell. Furthermore, there was no sign of gold colloid aggregation after the polymer shell was synthesized. These plasmon absorption bands all reflect the presence of an isolated gold nanoparticle core surrounded by a polymer shell, rather than multiple gold particles trapped in a polymer gel, or simply physical doping.

Fig. 7 shows the uv absorption of AuNPs prepared in comparative example 1 and Au @ PNIPAm prepared in example 1 in a salt environment. 0.1mL of the dispersed nanogel solution (theoretical concentration of 25mmol/L) prepared in example 1 and 1.9mL of a 0.2M NaCl solution were added to 1.9mL of water and 1.9mL of the solution, respectively, of bare gold nanoparticles having the same concentration, and the mixture was allowed to stand for 10min, and then an ultraviolet-visible absorption spectrum was collected on a UV-2500 spectrophotometer.

Gold nanoparticles stabilized by citrate are easy to aggregate after NaCl is added, so that the absorption band of the nanoparticles is red-shifted, and gold nanoparticles protected by protein or polymer can resist salt-induced aggregation. FIG. 7 shows that the naked gold nanoparticles rapidly aggregate in NaCl solutions at concentrations as high as 0.2M, with their maximum UV absorption followed by a large red-shift that loses its own characteristic properties. The Au @ PNIPAm gold core nanogel with the same concentration has the action of resisting salt aggregation, and shows the maximum ultraviolet absorption at about 523nm in a large-concentration NaCl solution, which indicates that the formed polymer gel shell layer protects the naked gold nanoparticles.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.