CN104759633A - Mimic enzyme, preparation method, application method and application of mimic enzyme - Google Patents

Abstract

The invention relates to mimic enzyme. The mimic enzyme is a molybdenum disulfide sheet layer of which the surface is loaded with platinum-copper alloy nanoparticles. The preparation method comprises the step of performing reduction reaction for a water solution in which the molybdenum disulfide sheet layer, platinum salt and copper salt to obtain the mimic enzyme that is the molybdenum disulfide sheet layer of which the surface is loaded with the platinum-copper alloy nanoparticles. The mimic enzyme has a plurality characteristics of activity of oxidase, peroxidase and catalase; different mimic enzyme activity can be used for creating different methods or applied to the unknown fields; the preparation method is simple and easy to be carried out; the conditions are mild; the one-step method can be carried out to reduce and prepare through a water-phase system.

Description

A mimetic enzyme, its preparation method, use method and application

technical field

The invention belongs to the field of nanotechnology and enzyme catalysis, and relates to a simulated enzyme, its preparation method, use method and application, and in particular to a kind of simulated enzyme activity (oxidase, peroxidase and catalase) which has three kinds of simulated enzyme activities simultaneously. The simulated enzyme, its preparation method, use method and application; the simulated enzyme is a composite material of single-layer molybdenum disulfide supported platinum-copper alloy nanostructure.

Background technique

Enzyme catalysis, as an important part of green chemistry, has become one of the most active research fields in the intersection of modern biology and chemistry. Mimetic enzymes are a kind of artificially synthesized catalysts with non-protein structure but similar catalytic activity to natural enzymes, which overcome the shortcomings of natural enzymes such as poor stability, variability and inactivation, difficult storage, complicated preparation process and high price. Nanomaterials have unique physical and chemical properties such as size, shape, and structure. Nanoparticle-mimicking enzymes have many advantages such as simple preparation, high catalytic efficiency, stability, economy, and stable properties. .

In recent years, materials such as nano-ferric oxide, nano-cobalt tetraoxide, nano-cerium oxide, nano-copper oxide, multi-walled carbon nanotubes, graphene oxide, iron sulfide nanosheets, nano-gold, and platinum nanocrystals have been found to have peroxide Biomimetic enzymes or oxidative mimetic enzyme activities greatly expand the application of related catalytic reactions under unfavorable conditions of natural enzymes. However, most of the above-mentioned nanomimetic enzymes are single components, and nanomimetic enzymes of composite materials are rarely reported. Nanoparticles are prone to agglomeration due to their small size and high surface energy. Although surface coating can reduce the agglomeration tendency of nanoparticles, it may also reduce the active sites on the particle surface, thereby reducing the catalytic activity of nanoparticles. The nano-mimetic enzyme of the multifunctional composite material can not only make the size of the nanomaterial controllable, uniform in shape, and present a monodisperse state, but also maintain high catalytic activity. This has important theoretical and practical significance for the in-depth development of nano-mimetic enzymes.

As an important class of two-dimensional layered nanomaterials, molybdenum disulfide has been widely used in lubricants, catalysis, energy storage, composite materials and other fields due to its unique "sandwich sandwich" layered structure. Molybdenum disulfide has many Mo-S facets, large specific surface area, and high reactivity; sulfur has strong adhesion to metals, so that molybdenum disulfide can be well attached to the metal surface. Therefore, how to develop a mimetic enzyme with controllable particle size, less agglomeration, more active sites, high catalytic activity, and various enzyme performances is a demand in this field.

Contents of the invention

Aiming at the problems of single performance of the simulated enzyme in the prior art, and easy aggregation of nanoparticles, and the reduction of active sites after coating the nanoparticles in order to prevent aggregation, the lack of high catalytic activity, tedious synthesis steps and harsh conditions of the simulated enzyme, the present invention One of the purposes of the invention is to provide a mimetic enzyme, which has the activities of oxidase, peroxidase and catalase, and is expected to replace related protein enzymes in the fields of immunoassay, biological detection and clinical diagnosis.

The simulated enzyme is a molybdenum disulfide sheet layer loaded with platinum-copper alloy nanoparticles on the surface.

Molybdenum disulfide has a large specific surface area and strong adsorption, and it is easy to react with modified molecules, so that the reduction of active sites on the surface of nanoparticles and the reduction of catalytic activity caused by surface modification can be avoided. In addition, molybdenum disulfide itself has weak peroxidase activity, so it can not only serve as a carrier but also play a synergistic catalytic role in the simulated enzyme of nanoparticle composites.

In the mimetic enzyme of the present invention, the number of atomic layers of the molybdenum disulfide sheet is 1-3 layers, preferably 1 layer.

Preferably, the molybdenum disulfide sheet is modified with chitosan molecules.

After the molybdenum disulfide sheets are modified by chitosan molecules, the obtained nanoparticle composite material has more excellent water solubility, dispersibility and biocompatibility.

Two of the purpose of the present invention is to provide a kind of preparation method of the mimetic enzyme as described in one of purpose, described method is: carry out reduction reaction with the aqueous solution that is dispersed with molybdenum disulfide lamellar, platinum salt and copper salt, obtain based on Mimic enzymes of molybdenum disulfide sheets loaded with platinum-copper alloy nanoparticles.

The mimetic enzyme of the present invention is obtained by in-situ loading platinum-copper alloy nanoparticles on the surface of a single-layer molybdenum disulfide in an aqueous phase system. The preparation method of the simulated enzyme provided by the invention is simple in process, and the prepared composite material (based on molybdenum disulfide sheets loaded with platinum-copper alloy nanoparticles) is easy to modify and has high catalytic activity.

Preferably, the molybdenum disulfide sheet is obtained by ultrasonically treating powdered molybdenum disulfide dispersed in an aqueous solution.

The powdery molybdenum disulfide can be obtained commercially.

Preferably, the ultrasonic power is 250-300W, and the ultrasonic time is 0.5-1.5h; further preferably, the ultrasonic power is 275W, and the ultrasonic time is 1h

Preferably, chitosan is added during the ultrasonic process to obtain a chitosan-modified molybdenum disulfide lamellar dispersion aqueous solution.

Preferably, the chitosan is added at a concentration of 0.01-0.1 wt%, preferably 0.05 wt%.

The modification of molybdenum disulfide sheets by chitosan molecules can improve the water solubility, dispersibility and biocompatibility of molybdenum disulfide sheets.

In the preparation method of the simulated enzyme of the present invention, in the aqueous solution dispersed with molybdenum disulfide sheets, platinum salts and copper salts, the concentration of molybdenum disulfide is 0.24-24mg/L, such as 0.3mg/L, 0.5mg /L, 0.9mg/L, 3mg/L, 7mg/L, 16mg/L, 18mg/L, 21mg/L, 23mg/L, etc.

Preferably, the platinum salt is chloroplatinic acid and/or platinum acetylacetonate.

Preferably, in terms of platinum element, in the aqueous solution dispersed with molybdenum disulfide sheet, platinum salt and copper salt, the concentration of platinum salt is 0.02-0.5mmol/L, such as 0.03mg/L, 0.05mg/L, 0.09mg/L, 0.12mg/L, 0.18mg/L, 0.22mg/L, 0.28mg/L, 0.33mg/L, 0.35mg/L, 0.42mg/L, 0.47mg/L, etc.

Preferably, the copper salt is any one or a combination of at least two of copper chloride, copper nitrate, or copper acetylacetonate; the combination typically but not limited includes a combination of copper acetate and copper acetylacetonate, nitric acid A combination of copper and copper chloride, a combination of copper chloride, copper nitrate and copper acetate, etc.

Preferably, in terms of copper element, in the aqueous solution dispersed with molybdenum disulfide sheet, platinum salt and copper salt, the concentration of copper salt is 0.02-0.5mmol/L, such as 0.03mg/L, 0.05mg/L, 0.09mg/L, 0.12mg/L, 0.18mg/L, 0.22mg/L, 0.28mg/L, 0.33mg/L, 0.35mg/L, 0.42mg/L, 0.47mg/L, etc.

The reducing agent of the reduction reaction of the present invention is sodium borohydride; the sodium borohydride is added in the form of an aqueous solution of sodium borohydride; the concentration of the aqueous solution of sodium borohydride is preferably 3 to 15 mmol/L, such as 4 mg/L, 6 mg/L L, 9mg/L, 11mg/L, 13mg/L, 14mg/L, etc.

Preferably, the aqueous solution of sodium borohydride is added in an amount of 1 mL in the aqueous solution dispersed with molybdenum disulfide sheets, platinum salts and copper salts.

Preferably, the sodium borohydride aqueous solution is added dropwise, and the dropwise addition rate is 30-60 μL/min, such as 35 μL/min, 38 μL/min, 42 μL/min, 44 μL/min, 47 μL /min, 53μL/min, 55μL/min, 59μL/min, etc.

Preferably, the temperature of the reduction reaction is 0° C.; the time is 1˜3 h, such as 1.2 h, 1.7 h, 2 h, 2.2 h, 2.7 h, 2.9 h, etc.

Preferably, the preparation method of the mimetic enzyme of the present invention comprises the following steps:

(1) Molybdenum disulfide sheets, platinum salts and copper salts are dispersed in water, stirred evenly, placed in an ice bath for cooling, and obtained are dispersed with molybdenum disulfide sheets, platinum salts and copper salts;

(2) In the aqueous solution obtained in step (1) dispersed with molybdenum disulfide sheets, platinum salts and copper salts, add a sodium borohydride solution with a concentration of 3 to 15 mmol/L dropwise, stir and continue to be placed in an ice bath carry out the reduction reaction;

(3) After the reaction is finished, separate and wash the precipitate to obtain a molybdenum disulfide sheet-layer nanocomposite material with platinum-copper alloy nanoparticles loaded on the surface.

Preferably, the washing solvent for the washing is water and/or ethanol.

The third object of the present invention is to provide a method for using the simulated enzyme according to the first object, and the simulated enzyme is used as any one of simulated oxidase, simulated peroxidase or simulated catalase.

Preferably, when used as a mimetic oxidase, capable of catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine, 2,2'-azino-bis-(3-ethylbenzothiazoline -6-sulfonic acid) diammonium salt or o-phenylenediamine to make the chromogenic substrate color;

Preferably, when used as a simulated peroxidase, capable of catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine, 2,2'-azino-bis-(3-ethane benzothiazoline-6-sulfonic acid) diammonium salt or o-phenylenediamine to make the chromogenic substrate develop color;

Preferably, when used as a catalase, it is capable of decomposing hydrogen peroxide to form water and oxygen.

As preferably, the method for using the simulated enzyme comprises the steps of:

Add the simulated enzyme described in one of the purposes into the buffer solution containing the chromogenic substrate to carry out the oxidation reaction of the chromogenic substrate, or catalyze the oxidation reaction of the catalyzed substrate by hydrogen peroxide, or catalyze the decomposition reaction of hydrogen peroxide ;

Preferably, the pH value of the buffer solution is 2.5-5.5;

Preferably, the reaction temperature during the oxidation reaction of the chromogenic substrate, or the oxidation reaction of the substrate by catalyzing hydrogen peroxide, or the decomposition reaction by catalyzing hydrogen peroxide is 30-60°C.

The fourth object of the present invention is to provide an application of the mimetic enzyme described in the first object, which is used in the fields of catalytic oxidation, chemical analysis, immunoassay, biological detection and clinical diagnosis.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The simulated enzyme provided by the present invention has multiple characteristics of oxidase, peroxidase and catalase activities at the same time, and can be used to construct different methods or be applied to unknown fields by utilizing different simulated enzyme activities;

(2) The simulated enzyme provided by the present invention is a nanocomposite material obtained by loading platinum-copper alloy nanoparticles on the surface of molybdenum disulfide sheets. In the nanocomposite material, molybdenum disulfide has a large specific surface area, strong adsorption, and is very It is easy to react with modified molecules, thereby avoiding the reduction of nanoparticle surface active sites and the reduction of catalytic activity caused by surface modification, which is conducive to maintaining high catalytic activity of nanocomposites; in said nanocomposites, molybdenum disulfide Not only can it be used as a carrier, but also can play a synergistic catalytic effect; the nanocomposite material has a regular nanostructure, which is conducive to increasing the contact area with the substrate and improving the reactivity of the simulated enzyme;

(3) The preparation method of the mimetic enzyme provided by the present invention is simple and easy, the conditions are mild, and it can be prepared by one-pot reduction in an aqueous phase system.

(4) The nanocomposite has good stability and has a wide range of pH and temperature applications. It is a novel multifunctional nanocomposite mimic enzyme that is extremely attractive in the fields of immune analysis, biological detection and clinical diagnosis. application prospects.

Description of drawings

Fig. 1 is a transmission electron microscope image of the nanocomposite material prepared in Example 1 of the present invention.

Fig. 2 is an x-ray diffraction diagram of the nanocomposite material prepared in Example 1 of the present invention.

Fig. 3 is the ultraviolet-visible spectrogram of test example 1-1 and test example 1-2;

Fig. 3 shows, no matter whether hydrogen peroxide exists, the mimetic enzyme provided by embodiment 1 all has very high catalytic activity to the oxidation of 3,3',5,5'-tetramethylbenzidine;

Fig. 4 is test example 1-3 after adding simulated enzyme, the variation relation of the absorption value of hydrogen peroxide at 240nm place with time;

Fig. 4 shows that the simulated enzyme provided by embodiment 1 can decompose hydrogen peroxide, reflecting the performance of catalase;

Fig. 5 is the response curve of test example 1-4 simulated enzyme catalytic temperature to reaction activity;

Fig. 6 is the response curve of the pH value of the buffer solution to the reaction activity when the simulated enzyme is catalyzed by test examples 1-5.

Fig. 7 is a comparison chart of the color change of the system before and after the reaction of the simulated enzymes to TMB, ABTS, and OPO provided by Test Example 1-1, Test Example 1-6, and Test Example 1-7.

Detailed ways

In order to facilitate understanding of the present invention, the present invention enumerates the following examples. It should be clear to those skilled in the art that the embodiments are only for helping to understand the present invention, and should not be regarded as specific limitations on the present invention.

Example 1

A mimetic enzyme based on a single-layer molybdenum disulfide-loaded platinum-copper alloy nanocomposite, obtained by the following method:

(1) Disperse the powdery molybdenum disulfide in the aqueous solution, and add 0.05wt% chitosan, and ultrasonically peel off to obtain the single-layer molybdenum disulfide aqueous solution modified by chitosan; the above-mentioned single-layer molybdenum disulfide, platinum chloride Disperse acid and copper acetate in 10mL of water to make the concentrations 2.4mg/L, 0.1mmol/L and 0.03mmol/L respectively, stir evenly, and place in an ice bath to cool for 20min to obtain molybdenum disulfide flakes, platinum Aqueous solutions of salts and copper salts;

(2) Add 1 mL of 9mmol/L sodium borohydride solution dropwise to the aqueous solution obtained in step (1) that is dispersed with molybdenum disulfide sheets, platinum salts and copper salts, the rate of addition is 60 μL/min, stir and continue to place Carry out reduction reaction in ice bath for 2h;

(3) After the reaction is over, remove the supernatant by centrifugation, wash the precipitate with water, disperse the precipitate in 10 mL of water after centrifugation, and obtain simulated enzymes (molybdenum disulfide sheet-layer nanocomposites with platinum-copper alloy nanoparticles loaded on the surface) of aqueous solution.

Morphological characterization:

The simulated enzyme obtained in Example 1 (i.e., the molybdenum disulfide sheet nanocomposite material loaded with platinum-copper alloy nanoparticles on the surface) is scanned by transmission electron microscope, and the scanning results are as shown in Figure 1 (Figure 1 is the simulated enzyme obtained in Example 1 As can be seen from Figure 1, on the simulated enzyme prepared in Example 1, platinum-copper alloy nanoparticles are distributed on the surface of the molybdenum disulfide sheet;

The simulated enzyme obtained in Example 1 (i.e., the molybdenum disulfide sheet nanocomposite material loaded with platinum-copper alloy nanoparticles on the surface) is carried out by x-ray diffraction, and the results are as shown in Figure 2 (Fig. 2 is the simulated enzyme obtained in Example 1). As shown in the x-ray diffraction diagram), it can be seen from Figure 2 that on the simulated enzyme prepared in Example 1, the nanoparticles distributed on the surface of the molybdenum disulfide sheet are platinum-copper alloy nanoparticles.

Test example 1-1

Add 50 μL of the aqueous solution prepared in Example 1 dispersed with simulated enzyme (molybdenum disulfide lamellar nanocomposite material with platinum-copper alloy nanoparticles on the surface) to 1 mL of phosphate buffer solution (pH=3.5), shake well, and place Incubate in a constant temperature water tank at 30°C for 15 minutes, then add 10 μL of 50 mmol/L chromogenic substrate 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB) solution to the solution, shake well, and set at 30°C Incubate for 15 minutes; then observe the color change of the system and scan the 400-800nm UV-Vis spectrum;

The experimental results show that the system is colorless when the simulated enzyme aqueous solution is not added, and after adding the simulated enzyme aqueous solution, the system turns blue; the ultraviolet-visible spectrum results are as shown in Figure 3 (Figure 3 is Test Example 1-1 and Test Example 1-2 As shown in the ultraviolet-visible spectrogram), the absorption value at 652nm is significantly increased; it shows that the nanocomposite can simulate the oxidation of oxidase to catalyze the oxygen oxidation of 3,3',5,5'-tetramethylbenzidine, and has oxidase activity.

Test example 1-2

Add 50 μL of the aqueous solution prepared in Example 1 dispersed with simulated enzymes (molybdenum disulfide lamellar nanocomposites with platinum-copper alloy nanoparticles on the surface) to 1 mL of phosphate buffer solution (pH=3.5), shake well, and fill Deoxygenate with nitrogen, and place in a constant temperature water bath at 30°C for 30 minutes, then add 10 μL of 100 mmol/L hydrogen peroxide solution and 10 μL of 50 mmol/L chromogenic substrate 3,3',5,5'-Tetra Methylbenzidine hydrochloride solution, shake well, and incubate at 30°C for 15 minutes; then observe the color change of the system, and scan the 400-800nm ultraviolet-visible spectrum;

The experimental results show that the system is colorless when the simulated enzyme aqueous solution is not added, and after adding the simulated enzyme aqueous solution, the system turns blue; the ultraviolet-visible spectrum results are as shown in Figure 3 (Figure 3 is Test Example 1-1 and Test Example 1-2 As shown in the UV-Vis Spectrum), the absorption value at 652nm was significantly increased; indicating that the nanocomposite can simulate peroxidase to catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine with peroxidase. Oxidase activity.

Test case 1-3

Prepare 1mL of 10mmol/L hydrogen peroxide phosphate buffer solution (pH=3.5), place it in a cuvette, record the absorbance at 240nm and observe the generation of bubbles on the cuvette wall; then add 50 μL of the preparation prepared in Example 1 to it The obtained aqueous solution dispersed with simulated enzyme (molybdenum disulfide sheet nanocomposite material loaded with platinum-copper alloy nanoparticles on the surface), recorded the absorbance value at 240nm, and observed the generation of bubbles on the wall of the cuvette;

Experimental result such as Fig. 4 (Fig. 4 is test case 1-3 after adding simulated enzyme, hydrogen peroxide absorbance value at 240nm place changes with time), the result shows, hydrogen peroxide absorbance value at 240nm place is in the absence of nanocomposite When the material exists, it basically remains unchanged, and there is no visible bubble formation on the cuvette wall; the absorbance value of hydrogen peroxide at 240nm decreases gradually with the prolongation of the nanocomposite existence time, and obvious bubbles can be seen on the cuvette wall Generate; the above results show that the mimetic enzyme can mimic catalase to catalyze hydrogen peroxide to generate water and oxygen, and has the activity of catalase;

Test case 1-4

To 1mL of phosphate buffer solution (pH=3.5), add 50 μL of the aqueous solution prepared in Example 1 dispersed with simulated enzymes (molybdenum disulfide sheet nanocomposites with platinum-copper alloy nanoparticles loaded on the surface), shake well; prepare the same 9 parts of the solution were placed in constant temperature water baths at different temperatures for 15 minutes (the temperatures were 0°C, 10°C, 20°C, 25°C, 30°C, 35°C, 40°C, 50°C, 60°C), Add 10 μL of 50 mmol/L chromogenic substrate 3,3',5,5'-tetramethylbenzidine hydrochloride solution to each solution, shake well, and continue warming for 15 minutes; measure the absorbance of the system at 652 nm.

The test result is shown in Figure 5 (Figure 5 is the response curve of test example 1-4 simulated enzyme catalytic temperature to reaction activity), as can be seen from Figure 5, when the reaction temperature is higher than 30°C, it is beneficial to simulate the catalytic activity of the enzyme; When the temperature is as high as 60 DEG C, the catalytic activity of the nanocomposite material can still maintain more than 97% of the highest catalytic activity; compared with the horseradish peroxidase, the nanocomposite material of the present invention has a wider temperature application range.

Test case 1-5

To 1 mL of phosphate buffer solution with different pH values (pH values are 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, 9.0, 10.0) were added 50 μL of the prepared dispersion Shake the aqueous solution of simulated enzyme (molybdenum disulfide sheet nanocomposite material loaded with platinum-copper alloy nanoparticles on the surface); place it in a constant temperature water bath at 30°C for 15 minutes, and then add 10 μL of 50 mmol/L chromogenic substrate to the solution respectively 3,3',5,5'-Tetramethylbenzidine hydrochloride solution, shake well, incubate at 30°C for 15 minutes; measure the absorbance of the system at 652nm.

Test result is shown in Figure 6 (Figure 6 is the response curve of buffer solution pH value to reactivity when test example 1-5 simulates enzyme catalysis), as can be seen from Figure 6, in the solution of partial acidic pH value, nanocomposite material has High catalytic activity; its simulated enzyme catalytic activity reaches its maximum at pH 3.5.

Test case 1-6

The difference with Test Example 1-1 is: replace TMB with 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS); test result and test example The same as 1-1: the system is colorless without adding the simulated enzyme aqueous solution, and the system turns dark green after adding the simulated enzyme aqueous solution; it shows that the nanocomposite material can simulate the oxidase to catalyze the oxygen oxidation of 2,2-azino-bis-( 3-ethylbenzothiazoline-6-sulfonic acid), which has oxidase activity.

Test case 1-7

The difference with Test Example 1-1 is that TMB is replaced by o-phenylenediamine (OPD); the test result is the same as Test Example 1-1: the system is colorless when the simulated enzyme aqueous solution is not added, and the system changes after adding the simulated enzyme aqueous solution. It is orange yellow; it shows that the nanocomposite can imitate oxidase to catalyze the oxidation of o-phenylenediamine with oxygen, and has oxidase activity.

Fig. 7 is the contrast diagram of system color change before and after the simulated enzyme that test example 1-1, test example 1-6, test example 1-7 provides to TMB, ABTS, OPO reacts; As can be seen from Fig. 7, the present invention provides The substrates of mimic enzymes are diverse.

Example 2

A mimetic enzyme based on a single-layer molybdenum disulfide-loaded platinum-copper alloy nanocomposite, obtained by the following method:

(1) Disperse powdered molybdenum disulfide in aqueous solution, and ultrasonically peel off to obtain a single-layer molybdenum disulfide aqueous solution; disperse single-layer molybdenum disulfide, chloroplatinic acid and copper chloride in 10mL water to make the concentrations 5mg respectively /L, 0.1mmol/L and 0.1mmol/L, stir evenly, place in an ice bath to cool for 20 minutes, and obtain an aqueous solution dispersed with molybdenum disulfide sheets, platinum salts and copper salts;

(2) Add 1mL of 12mmol/L sodium borohydride solution dropwise to the aqueous solution obtained in step (1) that is dispersed with molybdenum disulfide sheets, platinum salts and copper salts, the rate of addition is 30 μL/min, stir and continue to place Carry out reduction reaction in ice bath for 1h;

(3) After the reaction is over, remove the supernatant by centrifugation, wash the precipitate with water, disperse the precipitate in 10 mL of water after centrifugation, and obtain simulated enzymes (molybdenum disulfide sheet-layer nanocomposites with platinum-copper alloy nanoparticles loaded on the surface) of aqueous solution.

Example 3

A mimetic enzyme based on a single-layer molybdenum disulfide-loaded platinum-copper alloy nanocomposite, obtained by the following method:

(1) Disperse powdered molybdenum disulfide in aqueous solution, and ultrasonically peel off to obtain a single-layer molybdenum disulfide aqueous solution; disperse single-layer molybdenum disulfide, chloroplatinic acid and copper acetate in 10mL water to make the concentrations respectively 4.5mg /L, 0.05mmol/L and 0.15mmol/L, stir evenly, place in an ice bath to cool for 15min, and obtain an aqueous solution dispersed with molybdenum disulfide sheets, platinum salts and copper salts;

(2) Add 10mmol/L sodium borohydride solution 1mL dropwise to the aqueous solution obtained in step (1) that is dispersed with molybdenum disulfide sheets, platinum salts and copper salts, the rate of addition is 50 μL/min, stir and continue to place Carry out reduction reaction in ice bath for 1h;

(3) After the reaction, remove the supernatant by centrifugation, wash the precipitate with water, and disperse the precipitate in 10 mL of water after centrifugation to obtain simulated enzymes (molybdenum disulfide sheet-layer nanocomposites with platinum-copper alloy nanoparticles loaded on the surface) of aqueous solution.

Example 4

The difference from Example 1 is: in the aqueous solution dispersed with molybdenum disulfide sheets, platinum salts and copper salts, the concentrations of molybdenum disulfide, platinum salts and copper salts are respectively: 24mg/L, 0.5mmol/L and 0.15 mmol/L;

The concentration of sodium borohydride solution is 15mmol/L.

Example 5

The difference with Example 1 is: in the aqueous solution dispersed with molybdenum disulfide sheets, platinum salts and copper salts, the concentrations of molybdenum disulfide, platinum salts and copper salts are respectively: 0.24mg/L, 0.02mmol/L and 0.02mmol/L;

The concentration of sodium borohydride solution is 15mmol/L.

The applicant declares that the present invention illustrates the detailed process equipment and process flow of the present invention through the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed process equipment and process flow, that is, it does not mean that the present invention must rely on the above-mentioned detailed process equipment and process flow process can be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (10)

1. an analogue enztme, is characterized in that, described analogue enztme is the molybdenum bisuphide lamella that area load has Mock gold nano particle.

2. analogue enztme as claimed in claim 1, it is characterized in that, described analogue enztme has oxidizing ferment, peroxidase and catalatic activity simultaneously;

Preferably, the atom number of plies of described molybdenum bisuphide lamella is 1 ~ 3 layer, preferably 1 layer;

Preferably, described molybdenum bisuphide lamella is modified with chitosan molecule.

3. the preparation method of an analogue enztme as claimed in claim 1 or 2, it is characterized in that, described method is: the aqueous solution being dispersed with molybdenum bisuphide lamella, platinum salt and mantoquita is carried out reduction reaction, obtains the analogue enztme of the molybdenum bisuphide lamella having Mock gold nano particle based on area load.

4. method as claimed in claim 3, is characterized in that, described molybdenum bisuphide lamella is obtained by ultrasonic process dispersion powdery molybdenum bisuphide in aqueous;

Preferably, described ultrasonic power is 250 ~ 300W, and ultrasonic time is 0.5 ~ 1.5h; Further preferably, described ultrasonic power is 275W, and ultrasonic time is 1h;

Preferably, in described ultrasonic procedure, add shitosan, obtain chitosan-modified molybdenum bisuphide lamella aqueous dispersion.

5. the method as described in claim 3 or 4, is characterized in that, described in be dispersed with in the aqueous solution of molybdenum bisuphide lamella, platinum salt and mantoquita, the concentration of molybdenum bisuphide is 0.24 ~ 24mg/L;

Preferably, described platinum salt is chloroplatinic acid and/or acetylacetone,2,4-pentanedione platinum;

Preferably, in platinum element, described in be dispersed with in the aqueous solution of molybdenum bisuphide lamella, platinum salt and mantoquita, the concentration of platinum salt is 0.02 ~ 0.5mmol/L;

Preferably, described mantoquita is the combination of any a kind or at least 2 kinds in copper chloride, copper nitrate, Schweinfurt green or acetylacetone copper;

Preferably, in copper, described in be dispersed with in the aqueous solution of molybdenum bisuphide lamella, platinum salt and mantoquita, the concentration of mantoquita is 0.02 ~ 0.5mmol/L.

6. the method as described in one of claim 3 ~ 5, is characterized in that, the reducing agent of described reduction reaction is sodium borohydride; Described sodium borohydride adds with the form of sodium borohydride aqueous solution; The concentration preferably 3 ~ 15mmol/L of described sodium borohydride aqueous solution;

Preferably, described in be dispersed with in the aqueous solution of molybdenum bisuphide lamella, platinum salt and mantoquita, the addition of sodium borohydride aqueous solution is 1mL;

Preferably, the feed postition of described sodium borohydride aqueous solution is for dropwise to add, and the described rate of addition dropwise added is 30 ~ 60 μ L/min;

Preferably, the temperature of described reduction reaction is 0 DEG C; Time is 1 ~ 3h.

7. the method as described in one of claim 3 ~ 6, is characterized in that, described method comprises the steps:

(1) molybdenum bisuphide lamella, platinum salt and mantoquita are scattered in water, stir, be placed in ice bath and cool, obtain the aqueous solution being dispersed with molybdenum bisuphide lamella, platinum salt and mantoquita;

(2) dropwise add to being dispersed with in the aqueous solution of molybdenum bisuphide lamella, platinum salt and mantoquita of obtaining of step (1) sodium borohydride solution that concentration is 3 ~ 15mmol/L, stir and continue to be placed in ice bath and carry out reduction reaction;

(3), after reaction terminates, washing, centrifugally obtains nano composite material;

Preferably, the cleaning solvent of described washing is water and/or ethanol.

8. a using method for analogue enztme as claimed in claim 1 or 2, is characterized in that, described analogue enztme is used as any a kind in simulation oxidizing ferment, Mimetic enzyme or simulation catalase;

Preferably, when being used as simulation oxidizing ferment, can catalytic oxidation TMB, 2,2 '-Lian nitrogen base-bis--(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) diamino salt or o-phenylenediamine, make chromogenic substrate develop the color;

Preferably, when being used as Mimetic enzyme, can be oxidized TMB, 2 by catalyzing hydrogen peroxide, 2 '-Lian nitrogen base-bis--(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) diamino salt or o-phenylenediamine, make chromogenic substrate develop the color;

Preferably, when being used as catalase, peroxide decomposition can be made to generate water and oxygen.

9. using method as claimed in claim 8, it is characterized in that, described method comprises the steps:

Analogue enztme described in claim 1 or 2 is added in the cushioning liquid containing chromogenic substrate, carry out the oxidation reaction of chromogenic substrate, or catalyzing hydrogen peroxide carries out the oxidation reaction of catalytic substrate, or catalyzing hydrogen peroxide carries out decomposition reaction;

Preferably, the pH value of described cushioning liquid is 2.5 ~ 5.5;

Preferably, the oxidation reaction of described chromogenic substrate, or catalyzing hydrogen peroxide carries out the oxidation reaction of catalytic substrate, or the catalyzing hydrogen peroxide reaction temperature of carrying out in decomposition reaction process is 30 ~ 60 DEG C.

10. a purposes for analogue enztme as claimed in claim 1 or 2, is characterized in that, described analogue enztme is used for catalytic oxidation, chemical analysis, immunoassay, biological detection and clinical diagnosis field.