Molybdenum disulfide tandem enzyme and preparation method and application thereof

Abstract

The invention belongs to the field of analysis and detection, and particularly relates to molybdenum disulfide tandem enzyme and a preparation method and application thereof. According to the molybdenum disulfide tandem enzyme, a molybdenum disulfide nano material with a carboxyl functional group is prepared through a hydrothermal method and an ultrasonic method, and then bovine serum albumin modified gold nanoparticles with an amino functional group are prepared through a reduction method. Molybdenum disulfide and bovine serum albumin modified gold nanoparticles are combined by an amido bond cross-linking method to form the molybdenum disulfide tandem enzyme material. The molybdenum disulfide tandem enzyme can solve the problem that the activity of composite material glucose oxidase is inhibited after molybdenum disulfide and gold nanoparticles are combined, and can replace natural enzyme to realize colorimetric detection of glucose. The molybdenum disulfide tandem enzyme is convenient and rapid, a new scheme can be provided for synthesizing different types of mimic enzyme active materials from a large number of transition metal chalcogenide compounds, and the molybdenum disulfide tandem enzyme can realize colorimetric detection of glucose.

Description

Technical Field

[0001] The present invention belongs to the field of analysis and detection, and in particular relates to a molybdenum disulfide tandem enzyme and a preparation method and application thereof. Background Art

[0002] Nanozymes have widely replaced natural enzymes in medicine, chemical industry, food safety and other fields due to their high stability, adjustability, easy storage and low cost. In recent years, the use of nanozymes for colorimetric detection of glucose has received increasing attention. In the colorimetric detection of glucose, two enzymes, glucose oxidase and peroxidase, are required to complete it. However, the enzymes are unstable and easily affected by external temperature and pH. Purification is time-consuming and expensive.

[0003] Therefore, the preparation of enzyme nanomaterials with both glucose oxidase-like and peroxidase-like activities shows broad application prospects in colorimetric detection of glucose. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a molybdenum disulfide tandem enzyme and a preparation method and application thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a molybdenum disulfide tandem enzyme, comprising molybdenum disulfide with a surface-modified carboxyl functional group and gold nanoparticles modified with bovine serum albumin; wherein the molybdenum disulfide with a surface-modified carboxyl functional group and the bovine serum albumin-modified gold nanoparticles are linked via an amide bond; the molybdenum disulfide tandem enzyme exhibits both glucose oxidase-like and peroxidase-like activities. In the present invention, the gold nanoparticles modified with bovine serum albumin have amino groups on their surfaces; the molybdenum disulfide is surface-modified with a carboxyl functional group and linked to the bovine serum albumin-modified gold nanoparticles via an amide bond, thereby avoiding direct contact between the molybdenum disulfide and the gold nanoparticles. This results in the molybdenum disulfide tandem enzyme retaining both glucose oxidase-like and peroxidase-like activities, enabling non-enzymatic colorimetric detection of glucose.

[0007] In a second aspect, the present invention provides a method for preparing the aforementioned molybdenum disulfide tandem enzyme, comprising: preparing molybdenum disulfide with surface-modified carboxyl functional groups by a hydrothermal method and an ultrasonic method; preparing gold nanoparticles modified with bovine serum albumin by a reduction method; and combining the molybdenum disulfide with surface-modified carboxyl functional groups with the bovine serum albumin-modified gold nanoparticles by an amide bond crosslinking method to obtain the molybdenum disulfide tandem enzyme. The gold nanoparticles bind to 35 Cys residues in the bovine serum albumin to form Au-S bonds. The Au-S bonds formed here do not inhibit the mimetic enzyme activity of the gold nanoparticles due to the electronegativity difference between Au and S. Specifically, in the Au-S bonds formed when molybdenum disulfide and gold nanoparticles are combined, the electronegativity of Au is 10.9, while the electronegativity of Mo is 8.3, which is greater than that of Mo. Therefore, the resulting Au-S bond interaction is greater than the Mo-S interaction, causing the S ions to approach the gold nanoparticles, thereby competitively adsorbing them with the gold ions on the gold nanoparticle surface. In contrast, the Au-S bond formed by BSA with gold nanoparticles is formed with the sulfhydryl (-SH) groups in the gold nanoparticles. Since the electronegativity of H is 13.6, which is greater than that of Au, the resulting Au-S bond interaction is weaker than that of SH, making it difficult for S ions to approach the gold nanoparticles. Furthermore, BSA molecules can form a stable protective layer on the surface of the gold nanoparticles, thereby maintaining or enhancing the activity of the mimetic enzyme.

[0008] Furthermore, the step of preparing bovine serum albumin-modified gold nanoparticles by the reduction method specifically includes: preparing 4 mL of a bovine serum albumin solution with a concentration of 1 to 13 μM according to the following proportions; then adding 1 mL of a 24 mM aqueous chloroauric acid solution to the bovine serum albumin solution to obtain a first mixed solution; stirring the first mixed solution in the dark; preparing a sodium borohydride solution with a concentration of 1 mg / mL, taking 100 μL of the sodium borohydride solution and adding it to the first mixed solution after stirring, and stirring at room temperature; after completion, centrifuging and washing, collecting by centrifugation to obtain a first product, and freeze-drying the first product to obtain the bovine serum albumin-modified gold nanoparticles.

[0009] Furthermore, the first mixed solution was stirred in the dark for 30 minutes; 100 μL of the sodium borohydride solution was added to the first mixed solution after stirring, and the stirring time was 1 hour at room temperature.

[0010] Furthermore, the step of preparing molybdenum disulfide with surface modified carboxyl functional groups by hydrothermal method and ultrasonic method specifically includes: configuring each raw material according to the following proportions, taking 2.8 g thiourea, 1.76 g sodium molybdate and 0.6 g hexadecyltrimethylammonium bromide, adding them to 80 mL deionized water in sequence, stirring evenly, to obtain a second mixed solution; adjusting the pH of the second mixed solution to 8, and the total volume to 100 mL; transferring the second mixed solution to a high-pressure reaction vessel and drying it at 240 ° C for 48 hours; after the reaction is completed, cooling to room temperature, collecting the black precipitate product by centrifugation and washing it to obtain a second product; annealing the second product at 500 ° C for 1 hour in a single temperature zone tubular furnace under a nitrogen atmosphere to obtain molybdenum disulfide powder; configuring the molybdenum disulfide powder into a solution with a concentration of 2 mg / mL using deionized water, with a total volume of 15 mL, to obtain a molybdenum disulfide solution; adding 0.8~1.6 g sodium hydroxide and 1 g monochloroacetic acid, then ultrasonically treated, and finally centrifuged and washed to obtain the molybdenum disulfide with the surface modified with carboxyl functional groups.

[0011] Furthermore, the drying treatment time is 48 hours, the annealing treatment time is 1 hour, and the ultrasonic treatment parameters are 60° C. and 500 W power for 3 hours.

[0012] Furthermore, the step of combining the surface-modified carboxyl functional group-modified molybdenum disulfide with bovine serum albumin-modified gold nanoparticles using an amide bond cross-linking method specifically includes: configuring the raw materials according to the following proportions to prepare the surface-modified carboxyl functional group-modified molybdenum disulfide with a concentration of 1 mg / mL in a volume of 2 mL, then adding 500 μL of EDC and 500 μL of NHS, the concentrations of EDC and NHS are both 24 mM, reacting for 2 hours to obtain a third mixed solution; adding 2 mL of the bovine serum albumin-modified gold nanoparticles with a concentration of 1-5 mg / mL to the third mixed solution, reacting for 3 hours, and then dialyzing, centrifuging, washing and freeze-drying to obtain the molybdenum disulfide tandem enzyme.

[0013] In a third aspect, the present invention provides the use of the above-mentioned molybdenum disulfide tandem enzyme in the field of non-enzymatic glucose colorimetric detection.

[0014] Furthermore, the method for enzyme-free glucose colorimetric detection specifically includes: using a colorimetric detection method, the detection device is a UV-2450 ultraviolet-visible absorption spectrometer; taking 1.5 mL of Tris-HCl buffer with a pH of 3, adding 0.75 mL of the glucose solution to be tested at different concentrations and 0.5 mL of the molybdenum disulfide tandem enzyme with a concentration of 2 mg / mL to the buffer to obtain a colorimetric solution; adding 0.5 mL of a 5 mM chromogenic substrate TMB solution to the colorimetric solution, incubating in a 35°C water bath for 30 minutes, and then centrifuging at 5000 rpm for 10 minutes to obtain a reaction system; using UV-vis to measure the absorption spectrum of the reaction system at 652 nm (blue solution); adding 0.1 mL of dilute sulfuric acid to the reaction system to terminate the reaction, and measuring the absorption spectrum of the reaction system at 450 nm (yellow solution).

[0015] Furthermore, the concentration range of the glucose solution to be tested is 10 -6 ~10 -2 M.

[0016] The molybdenum disulfide tandem enzyme provided by the present invention has glucose oxidase-like activity. It first catalyzes a glucose solution into gluconic acid and hydrogen peroxide. Subsequently, its peroxidase-like activity catalyzes the hydrogen peroxide into hydroxyl radicals (·OH). The ·OH further oxidizes the chromogenic substrate TMB molecule, converting it from colorless to blue oxidized TMB, with an absorption peak observed at 652 nm. After the reaction is terminated by the dropwise addition of sulfuric acid, the blue oxidized TMB is oxidized to a yellow diimine, at which point an absorption peak is observed at 450 nm. As the glucose concentration increases, the hydrogen peroxide produced gradually increases, and the catalyzed chromogenic substrate also increases. The solubility of the glucose solution being tested is determined by the absorbance.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. In the present invention, gold nanoparticles are modified with bovine serum albumin and combined with molybdenum disulfide having surface-modified carboxyl functional groups via an amide bond, thereby avoiding direct contact between the gold nanoparticles and molybdenum disulfide, so that the molybdenum disulfide tandem enzyme has glucose oxidase-like activity and peroxidase-like activity;

[0019] 2. The introduction of bovine serum albumin-modified gold nanoparticles in the present invention enables the molybdenum disulfide composite material to have two enzyme activities. By comparing the glucose detection performance of bovine serum albumin-modified gold nanoparticles and molybdenum disulfide composite materials, the molybdenum disulfide tandem enzyme combines the respective enzymatic activities of the two, thereby increasing the activity of the composite nanoenzyme.

[0020] 3. The present invention combines molybdenum disulfide with surface-modified carboxyl groups and gold nanoparticles modified with bovine serum albumin, preventing the Au-S bonds formed by the molybdenum disulfide and gold nanoparticles from competing for active sites on the gold nanoparticle surface. This further imparts dual enzyme-mimicking activities to the composite, ultimately enabling colorimetric detection of glucose. This preparation method addresses the problem of conventional molybdenum disulfide composites lacking glucose oxidase-like activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] One or more embodiments are exemplarily described by the pictures in the corresponding drawings. These exemplifications do not constitute limitations on the embodiments. Unless otherwise stated, the pictures in the drawings do not constitute proportional limitations.

[0022] Figure 1 Comparison of the absorption spectra of conventional molybdenum disulfide / gold composite materials and the molybdenum disulfide tandem enzyme provided by the embodiments of the present invention for colorimetric detection of glucose;

[0023] Figure 2 This is a transmission electron microscopy image of gold nanoparticles modified with bovine serum albumin;

[0024] Figure 3 This is an infrared spectrum image of gold nanoparticles modified with bovine serum albumin;

[0025] Figure 4 This is a comparison of the tandem enzyme activity of gold nanoparticles modified with bovine serum albumin at concentrations ranging from 1 to 13 μM.

[0026] Figure 5 This is a scanning electron microscope image of molybdenum disulfide with surface modified carboxyl functional groups;

[0027] Figure 6 This is the infrared spectrum image of molybdenum disulfide with surface modified carboxyl functional groups;

[0028] Figure 7 The comparison of the activity of surface-modified carboxyl molybdenum disulfide peroxidases prepared with sodium hydroxide mass ranging from 0.8 to 1.6 g.

[0029] Figure 8 is a transmission electron microscope image of a molybdenum disulfide tandem enzyme provided in an embodiment of the present invention;

[0030] Figure 9 This is an infrared spectrum image of the molybdenum disulfide tandem enzyme provided in an embodiment of the present invention;

[0031] Figure 10 The comparison of the activity of MoS2 tandem enzymes prepared by combining gold nanoparticles modified with bovine serum albumin and MoS2 with surface modified carboxyl functional groups at a mass ratio ranging from 1:1 to 5:1.

[0032] Figure 11 The molybdenum disulfide tandem enzyme pair 10 provided in the embodiment of the present invention -6 M~10 -2 Absorption spectrum of glucose colorimetric detection at 450 nm at M concentration. DETAILED DESCRIPTION

[0033] The inventors have discovered that molybdenum disulfide, a typical transition metal chalcogenide, currently exhibits peroxidase-like catalytic activity. Its large surface area provides more active sites, and it has been effectively applied to the colorimetric detection of glucose. For example, combining molybdenum disulfide with glucose oxidase enables colorimetric detection of glucose. Another example is combining gold nanoparticles with molybdenum disulfide, similarly combined with glucose oxidase, to detect glucose in human serum. However, while gold nanoparticles possess tandem enzyme activity, the combination of gold nanoparticles and molybdenum disulfide exhibits only a single, peroxidase-like enzymatic activity, and true enzyme-free detection has not yet been achieved.

[0034] In view of this, the present invention provides a novel molybdenum disulfide tandem enzyme for realizing enzyme-free colorimetric detection of glucose, as well as its preparation method and application. The composite material (MoS2-COOH / BSA@Au) formed by combining molybdenum disulfide and gold nanoparticles, namely the molybdenum disulfide tandem enzyme described in the present invention, has both glucose oxidase-like activity and peroxidase-like activity to realize enzyme-free colorimetric detection of glucose.

[0035] The present invention provides a molybdenum disulfide tandem enzyme, which comprises molybdenum disulfide with a surface modified with carboxyl functional groups and gold nanoparticles modified with bovine serum albumin; wherein the molybdenum disulfide with a surface modified with carboxyl functional groups and the gold nanoparticles modified with bovine serum albumin are connected via an amide bond; and the molybdenum disulfide tandem enzyme has both glucose oxidase-like activity and peroxidase-like activity.

[0036] The present invention also provides a method for preparing a molybdenum disulfide tandem enzyme, comprising the following steps:

[0037] S1, prepare 4 mL of bovine serum albumin solution with a concentration of 1~13 μM, and then add 1 mL of 24 m A 100 μM aqueous solution of chloroauric acid was added to the bovine serum albumin solution, and the mixture was stirred in the dark for 30 minutes. Subsequently, a 1 mg / mL sodium borohydride solution was prepared and 100 μL of this solution was quickly added to the mixture. The solution turned dark brown and was stirred at room temperature for 1 hour. After the reaction, the precipitate was collected by centrifugation, washed, and lyophilized in a freeze dryer to obtain bovine serum albumin-modified gold nanoparticles.

[0038] S2. Weigh 2.8 g of thiourea, 1.76 g of sodium molybdate and 0.6 g of hexadecyltrimethylammonium bromide, add them to 80 mL of deionized water in sequence and stir evenly. Add NaOH or HCl to the mixed solution, adjust the pH of the solution to 8, and then adjust the total volume of the solution to 100 mL. Transfer the above mixed solution to a stainless steel high-pressure reactor with a polytetrafluoroethylene liner, place it in a blast drying oven, set the temperature to 240 ° C, and the reaction time is 48 hours. After the reaction is completed, cool it naturally to room temperature, collect the black precipitate product by centrifugation, wash it repeatedly with anhydrous ethanol and deionized water three times, and after drying, place the product in a single-temperature zone tubular furnace for annealing. The annealing temperature is set to 500 ° C for 1 hour to obtain molybdenum disulfide powder;

[0039] S3. To prepare the molybdenum disulfide tandem enzyme, molybdenum disulfide was carboxylated and cross-linked with amino groups on the surface of bovine serum albumin. Molybdenum disulfide powder was prepared into a 2 mg / mL solution with deionized water in a total volume of 15 mL. Then, 0.8–1.6 g of sodium hydroxide and 1 g of monochloroacetic acid were added to the molybdenum disulfide solution. The solution was sonicated at 60°C and 500 W for 3 hours. The molybdenum disulfide with surface modified carboxyl functional groups was obtained by centrifugation and washing.

[0040] S4. Molybdenum disulfide with surface-modified carboxyl functional groups is combined with gold nanoparticles modified with bovine serum albumin via an amide bond. A 1 mg / mL carboxyl molybdenum disulfide solution is prepared in a 2 mL volume. 500 μL of EDC and 500 μL of NHS, both at 24 mM concentrations, are then added. This step is to activate the carboxyl groups on the surface of the molybdenum disulfide with surface-modified carboxyl functional groups. The activation reaction lasts for 2 hours. Subsequently, 2 mL of gold nanoparticles modified with bovine serum albumin at a concentration of 1-5 mg / mL are added to connect the carboxyl and amino groups. The complex reaction lasts for 3 hours. After the reaction, the mixed solution is dialyzed, centrifuged, washed, and lyophilized to obtain the molybdenum disulfide tandem enzyme.

[0041] The present invention is further described in detail below with reference to the examples. It is particularly noted that if no specific conditions are specified in the following examples, the reactions were carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can be obtained from common commercial sources.

[0042] Example

[0043] (A) Effect of molybdenum disulfide tandem enzyme (hereinafter referred to as MoS2-COOH / BSA@Au) on colorimetric detection of glucose.

[0044] The detection performance of MoS2@Au and MoS2-COOH / BSA@Au were compared. MoS2@Au and MoS2-COOH / BSA@Au were added to the test solution containing 10 mM glucose, and then the colorimetric substrate TMB solution was added to the above solution. After incubation in a 35°C water bath for 30 minutes, the reaction was stopped by centrifugation. The absorption spectrum of the reaction system at 450 nm was measured by UV-vis.

[0045] The results are as follows Figure 1 As shown in the figure, there is no obvious absorption peak in the absorption spectrum of MoS2@Au after the reaction, while MoS2-COOH / BSA@Au shows a strong absorption peak, which proves that MoS2-COOH / BSA@Au has the ability to catalyze glucose and hydrogen peroxide.

[0046] (2) Verify whether bovine serum albumin can modify gold nanoparticles.

[0047] Acquire transmission electron microscopy images of gold nanoparticles modified with bovine serum albumin, e.g. Figure 2 As shown in Figure 2, it can be found that the size of the gold nanoparticles is about 11.58 nm. Figure 3 The Fourier transform infrared spectrum of the gold nanoparticles modified with bovine serum albumin showed that the -1 The characteristic peak at 1633 cm corresponds to the OH stretching vibration of -COOH. -1 The characteristic peak at 1549 cm is the C=O stretching vibration of -COOH. -1 The characteristic peaks are the bending vibrations of -NH, and the appearance of these characteristic peaks further proves the successful preparation of bovine serum albumin modified gold nanoparticles.

[0048] (3) Investigate the effect of BSA concentration on the enzyme activity of BSA-modified gold nanoparticles.

[0049] In order to explore the effect of BSA concentration on the enzyme activity of BSA-modified gold nanoparticles, five groups of BSA-modified gold nanoparticles prepared at different concentrations were set up.

[0050] Similarly, 4 mL of bovine serum albumin solution was prepared, with concentrations adjusted to 1, 4, 7, 10, and 13 μM, respectively. Subsequently, 1 mL of 24 mM chloroauric acid solution was added to the bovine serum albumin solution, and the mixture was stirred in the dark for 30 minutes. Subsequently, 100 μL of sodium borohydride solution was prepared at a concentration of 1 mg / mL and quickly added to the mixture. The solution turned dark brown and was stirred at room temperature for 1 hour. After the reaction, the precipitate was collected by centrifugation, washed, and lyophilized in a freeze dryer to obtain bovine serum albumin-modified gold nanoparticles.

[0051] Furthermore, the effect of the bovine serum albumin-modified gold nanoparticles on the colorimetric detection of glucose was verified: the detection device was a UV-2450 ultraviolet-visible absorption spectrometer; 1.5 mL of Tris-HCl buffer with a pH of 3 was taken, and 0.75 mL of a 10 mM glucose solution and 0.5 mL of bovine serum albumin-modified gold nanoparticles were added to the buffer to obtain a colorimetric solution; 0.5 mL of a 5 mM chromogenic substrate TMB solution was added to the colorimetric solution, incubated in a 35°C water bath for 30 minutes, and then centrifuged at 5000 rpm for 10 minutes to obtain a reaction system; 0.1 mL of dilute sulfuric acid was added dropwise to the reaction system to terminate the reaction, and the absorption spectrum of the reaction system at 450 nm (yellow solution) was measured using a UV-2450 ultraviolet-visible absorption spectrometer.

[0052] The reaction results showed that there was an obvious absorption peak at 450 nm, such as Figure 4 As shown, the BSA-modified gold nanoparticles exhibit tandem glucose oxidase-like and peroxidase-like enzymatic activities, achieving glucose catalysis. The absorption peak intensity gradually increased with increasing BSA concentration, demonstrating the increasing enzyme-mimicking activity of the BSA-modified gold nanoparticles. However, when the concentration exceeded 10 µM, the absorption peak intensity of the reaction system began to decrease. This is because BSA, as a natural biological carrier, forms a protective layer on the surface of the gold nanoparticles, enhancing their stability. As the BSA concentration increases, more BSA coats the gold nanoparticles, enhancing their stability and activity. However, when the BSA concentration is too high, the interaction between BSA molecules increases, leading to aggregation. This causes BSA to coat or shield the active sites on the gold nanoparticle surface, thereby weakening their enzyme-mimicking activity.

[0053] Therefore, the BSA-modified gold nanoparticles prepared at a BSA concentration of 10 μM have the best enzyme mimicking performance.

[0054] (4) Verify whether the preparation of carboxyl-modified molybdenum disulfide is achieved.

[0055] The results of scanning electron microscopy and Fourier transform infrared spectroscopy were as follows: Figure 5 and Figure 6 As shown in the figure, there are many nanosheets with different directions on the surface of MoS2, and the size is generally around 2 μm. Fourier infrared spectroscopy can be observed at 3438 cm -1 The peak at 1630 cm-1 is due to the OH stretching vibration of -COOH. -1 and 1387 cm-1 The characteristic peaks at 1068 cm-1 correspond to the C=O stretching vibration of -COOH and the bending vibration of -CH. −1 The peak at corresponds to the stretching vibration of C-OH, indicating the successful preparation of carboxyl-modified MoS2.

[0056] (V) Investigate the effect of the amount of sodium hydroxide on the activity of molybdenum disulfide simulated enzymes with surface modified carboxyl functional groups.

[0057] In order to explore the effect of the amount of sodium hydroxide on the activity of molybdenum disulfide simulated enzymes with surface modified carboxyl functional groups, 5 groups of molybdenum disulfide with surface modified carboxyl functional groups prepared under different sodium hydroxide masses were set up.

[0058] Similarly, weigh 2.8 g of thiourea, 1.76 g of sodium molybdate, and 0.6 g of hexadecyltrimethylammonium bromide, and add them to 80 mL of deionized water in sequence and stir evenly. Add NaOH or HCl to the mixed solution to adjust the pH of the solution to 8, and then adjust the total volume of the solution to 100 mL. The above mixed solution is transferred to a stainless steel high-pressure reactor with a polytetrafluoroethylene liner, placed in a blast drying oven, set the temperature to 240 ° C, and the reaction time is 48 hours. After the reaction is completed, it is naturally cooled to room temperature, and the black precipitate product is collected by centrifugation. It is repeatedly washed three times with anhydrous ethanol and deionized water. After drying, the product is placed in a single-temperature zone tubular furnace for annealing. The annealing temperature is set to 500 ° C for 1 hour to obtain molybdenum disulfide powder.

[0059] To prepare molybdenum disulfide with surface modified carboxyl functional groups, molybdenum disulfide powder was prepared into a solution with a concentration of 2 mg / mL using deionized water in a total volume of 15 mL. Then, 0.8-1.6 g of sodium hydroxide and 1 g of monochloroacetic acid were added to the molybdenum disulfide solution. The solution was ultrasonically treated at 60 °C and an ultrasonic power of 500 W for 3 h. The molybdenum disulfide with surface modified carboxyl functional groups was obtained by centrifugation and washing.

[0060] Further, the colorimetric detection effect of the surface-modified carboxyl functional group molybdenum disulfide on hydrogen peroxide was verified: 1.5 mL of Tris-HCl buffer with a pH of 3 was taken, 0.75 mL of a 10 mM glucose solution was added to the buffer, and then glucose oxidase was added to the above solution and incubated in a 35°C water bath for 30 minutes to allow glucose oxidase to catalyze glucose to produce hydrogen peroxide. Molybdenum disulfide with a surface-modified carboxyl functional group and 0.5 mL of a 5 mM chromogenic substrate TMB solution were added to the colorimetric solution and added to the above reaction system for a further 30 minutes of incubation, followed by centrifugation at 5000 rpm for 10 minutes to obtain a reaction system; 0.1 mL of dilute sulfuric acid was added dropwise to the reaction system to terminate the reaction, and the absorption spectrum of the reaction system at 450 nm (yellow solution) was measured using a UV-2450 ultraviolet-visible absorption spectrometer.

[0061] The reaction results are as follows Figure 7 As shown in the figure, there is an obvious absorption peak at 450 nm. When the mass of sodium hydroxide increases from 0.8 g to 1.4 g, the peroxidase-like activity of molybdenum disulfide with surface modified carboxyl functional groups gradually increases. When it increases to 1.6 g, it shows a weakening trend. This is because excessive sodium hydroxide will cause the reaction system to be overly alkaline, producing sodium salt of carboxylic acid, thereby inhibiting the peroxidase-like activity of molybdenum disulfide with surface modified carboxyl functional groups.

[0062] Therefore, the surface-modified carboxyl functional group molybdenum disulfide prepared under the condition of adding 1.4 g of sodium hydroxide has the best peroxidase-like activity performance.

[0063] (6) Verify whether the preparation of molybdenum disulfide composite material tandem enzyme is achieved.

[0064] The composite material was characterized by transmission electron microscopy and Fourier transform infrared spectroscopy. Figure 8 and Figure 9 As shown in the figure, it can be observed that the gold nanoparticles modified with bovine serum albumin are uniformly dispersed on the surface of the molybdenum disulfide modified with carboxyl functional groups, which preliminarily proves the successful synthesis of the molybdenum disulfide composite tandem enzyme. By Fourier infrared spectroscopy analysis, it was found that at 3234 cm -1 The characteristic peak observed at 1651 cm is due to the OH stretching vibration. -1 The peak at 1526 cm is the C=O stretching vibration of amide bond I, while the peak at 1526 cm -1 The peak at 1289 cm is the -NH bending vibration of the amide II bond. -1 The peak at 1046 cm corresponds to the CO stretching vibration. -1The peaks at correspond to the stretching vibration of C-OH. These characteristic peaks further prove that the synthesis of the molybdenum disulfide composite tandem enzyme is successful.

[0065] (7) To investigate the effect of the mass ratio of gold nanoparticles modified with bovine serum albumin and molybdenum disulfide with surface modified carboxyl functional groups on the activity of molybdenum disulfide tandem enzyme.

[0066] In order to investigate the effect of the mass ratio of bovine serum albumin-modified gold nanoparticles and molybdenum disulfide with surface modified carboxyl functional groups on the activity of molybdenum disulfide tandem enzyme, five groups of different mass ratios were set up to prepare molybdenum disulfide tandem enzyme.

[0067] Similarly, a 2 mL volume of molybdenum disulfide (MoS2) with a surface-modified carboxyl group was prepared at a concentration of 1 mg / mL. 500 μL of EDC and 500 μL of NHS, both at a concentration of 24 mM, were then added to activate the carboxyl groups on the surface of the MoS2. The activation reaction lasted for 2 hours. Subsequently, 2 mL of bovine serum albumin-modified gold nanoparticles (BSA) at a concentration of 1–5 mg / mL was added, ensuring a 1:1–5:1 mass ratio of the BSA-modified gold nanoparticles to the MoS2. The carboxyl and amino groups were linked for 3 hours. After the reaction, the mixed solution was dialyzed, centrifuged, washed, and lyophilized to obtain the MoS2 tandem enzyme.

[0068] Furthermore, the performance of molybdenum disulfide tandem enzyme for colorimetric detection of glucose was verified, and the detection device was a UV-2450 ultraviolet-visible absorption spectrometer; 1.5 mL of Tris-HCl buffer with a pH of 3 was taken, and 0.75 mL of the glucose solution of different concentrations and 0.5 mL of the molybdenum disulfide tandem enzyme with a concentration of 2 mg / mL were added to the buffer to obtain a colorimetric solution; 0.5 mL of a 5 mM chromogenic substrate TMB solution was added to the colorimetric solution, incubated in a 35°C water bath for 30 minutes, and then centrifuged at 5000 rpm for 10 minutes to obtain a reaction system; the absorption spectrum of the reaction system at 652 nm (blue solution) was measured using UV-vis; 0.1 mL of dilute sulfuric acid was added dropwise to the reaction system to terminate the reaction, and the absorption spectrum of the reaction system at 450 nm (yellow solution) was measured.

[0069] The reaction results are as follows Figure 10As shown in the figure, when the proportion of gold nanoparticles modified with bovine serum albumin increases, the enzymatic activity of the molybdenum disulfide tandem enzyme gradually increases. After the mass of the gold nanoparticles modified with bovine serum albumin increases to 3 mg, the intensity of the absorption peak tends to be stable and no longer changes significantly. This indicates that when the ratio of gold nanoparticles modified with bovine serum albumin to molybdenum disulfide with surface modified carboxyl functional groups is 3:1, the complexation of the amide bond reaches a saturated state. After the ratio is 3:1, the uncomplexed gold nanoparticles modified with bovine serum albumin are removed by washing, resulting in no significant change in the simulated enzyme activity.

[0070] Therefore, when the mass ratio of bovine serum albumin-modified gold nanoparticles to molybdenum disulfide with surface modified carboxyl functional groups is 3:1, the prepared molybdenum disulfide tandem enzyme has the best performance for colorimetric detection of glucose.

[0071] (8) A method for non-enzymatic colorimetric detection of glucose using molybdenum disulfide tandem enzyme. The specific implementation method is as follows.

[0072] Among them, the preparation of gold nanoparticles modified with bovine serum albumin includes: weighing 2.89 mg of bovine serum albumin, dissolving it in 4 mL of deionized water to prepare a bovine serum albumin solution with a concentration of 10 μM, adding 1 mL of a 24 mM aqueous solution of chloroauric acid to the bovine serum albumin solution, and stirring it in the dark for 1 hour. Subsequently, a sodium borohydride solution with a concentration of 1 mg / mL was prepared, and 100 μL was quickly added to the above mixed solution. The solution turned dark brown and stirred at room temperature for 1 hour. After the reaction, the product was centrifuged and washed to remove excess bovine serum albumin, and the product was collected by centrifugation and placed in a freeze dryer for freeze drying to obtain gold nanoparticles modified with bovine serum albumin, such as Figure 2 shown.

[0073] The preparation of molybdenum disulfide with surface-modified carboxyl functional groups includes: weighing 2.8 g of thiourea, 1.76 g of sodium molybdate, and 0.6 g of hexadecyltrimethylammonium bromide, adding them sequentially to 80 mL of deionized water and stirring evenly. NaOH or HCl is added to the mixed solution to adjust the pH of the solution to 8, and then the total volume of the solution is fixed to 100 mL. The mixed solution is transferred to a stainless steel autoclave with a polytetrafluoroethylene liner, placed in a forced air drying oven, and reacted at 240°C for 48 hours. After the reaction is completed, the mixture is naturally cooled to room temperature, and the black precipitate product is collected by centrifugation. It is repeatedly washed with anhydrous ethanol and deionized water. After drying, the product is placed in a single-temperature zone tube furnace for annealing. The annealing temperature is set to 500°C for 1 hour to obtain molybdenum disulfide powder. The molybdenum disulfide powder is prepared into a solution with a total volume of 15 mL and a concentration of 2 mg / mL using deionized water. Then, 1.4 g of sodium hydroxide and 2.0 g of monochloroacetic acid are added to the molybdenum disulfide solution. Ultrasonic treatment is performed using an ultrasonic machine at 60°C and an ultrasonic power of 500 W for 3 hours. Molybdenum disulfide with surface modified carboxyl functional groups is obtained by centrifugation and washing, as shown in FIG. Figure 4 shown.

[0074] The preparation of molybdenum disulfide tandem enzyme includes: combining gold nanoparticles modified with bovine serum albumin and molybdenum disulfide with surface modified carboxyl functional groups at a mass ratio of 3:1, first preparing 2 mL of molybdenum disulfide with surface modified carboxyl functional groups at a concentration of 1 mg / mL, then adding 500 μL of EDC and 500 μL of NHS, both at a concentration of 24 mM, and the reaction time is 2 hours. Subsequently, 2 mL of gold nanoparticles modified with bovine serum albumin at a concentration of 3 mg / mL are added to connect the carboxyl group and the amino group, and the complex reaction time is 3 hours. After the reaction is completed, the mixed solution is dialyzed, centrifuged, washed and freeze-dried to obtain the molybdenum disulfide tandem enzyme, such as Figure 6 shown.

[0075] In this example, in order to verify whether the molybdenum disulfide tandem enzyme can produce detection effects in glucose solutions with different concentrations, 5 groups of glucose solutions with different concentrations were selected to test its colorimetric sensing performance. The concentration of the glucose solution was 10 -6 M to 10 -2M. During the test, 1.5 mL of Tris-HCl buffer with a pH of 3 was taken, 0.75 mL of glucose solutions of different concentrations were added to the buffer, 0.5 mL of 2 mg / mL molybdenum disulfide tandem enzyme was added to the above reaction system solution, and then 0.5 mL of 5 mM chromogenic substrate TMB solution was added to the above reaction system solution. Finally, the mixed solution was placed in a 35°C water bath and incubated for 30 minutes. 0.1 mL of dilute sulfuric acid was added to the solution to terminate the reaction, and the absorption spectrum of the reaction system at 450 nm (yellow solution) was measured.

[0076] By testing the absorption spectrum of the reaction system solution before and after adding sulfuric acid, such as Figure 11 As shown, the reflected absorption peak intensity increases with the increase of glucose concentration, realizing the detection of glucose. This is because the molybdenum disulfide tandem enzyme catalyzes glucose and further catalyzes the product hydrogen peroxide, thereby causing the color of the chromogenic substrate to change. The regular change of color in the reaction solution can also be observed by the naked eye, thus further proving that the molybdenum disulfide tandem enzyme can effectively perform enzyme-free colorimetric detection on glucose solution. It can be seen that the molybdenum disulfide tandem enzyme provided by the present invention can solve the problem that the activity of the composite glucose oxidase is inhibited after molybdenum disulfide is combined with gold nanoparticles, and replaces the natural enzyme to realize colorimetric detection of glucose. The detection is convenient and fast, and can provide a new solution for the synthesis of a large number of transition metal sulfide compounds with different types of simulated enzyme activity materials.

[0077] Those skilled in the art will appreciate that the above-described embodiments are specific examples for implementing the present application, and that in actual applications, various changes in form and detail may be made thereto without departing from the spirit and scope of the present application. Any person skilled in the art may make changes and modifications without departing from the spirit and scope of the present application. Therefore, the scope of protection of the present application shall be subject to the scope defined in the claims.

Claims

1. A molybdenum disulfide tandem enzyme, characterized in that The molybdenum disulfide tandem enzyme comprises molybdenum disulfide with a surface modified carboxyl functional group and gold nanoparticles modified with bovine serum albumin; wherein the molybdenum disulfide with a surface modified carboxyl functional group and the gold nanoparticles modified with bovine serum albumin are connected via an amide bond; The molybdenum disulfide tandem enzyme has both glucose oxidase-like activity and peroxidase-like activity.

2. The method for preparing the molybdenum disulfide tandem enzyme according to claim 1, wherein The preparation method comprises: preparing molybdenum disulfide with surface modified carboxyl functional groups by hydrothermal method and ultrasonic method; preparing gold nanoparticles modified with bovine serum albumin by reduction method; combining the molybdenum disulfide with surface modified carboxyl functional groups with the gold nanoparticles modified with bovine serum albumin by amide bond cross-linking method to obtain the molybdenum disulfide tandem enzyme.

3. The method for preparing the molybdenum disulfide tandem enzyme according to claim 2, characterized in that: The step of preparing gold nanoparticles modified with bovine serum albumin by reduction method specifically comprises: preparing various raw materials according to the following proportions, preparing 4 mL of a bovine serum albumin solution with a concentration of 1-13 μM; then adding 1 mL of a 24 mM chloroauric acid aqueous solution to the bovine serum albumin solution to obtain a first mixed solution; stirring the first mixed solution in the dark; preparing a sodium borohydride solution with a concentration of 1 mg / mL, taking 100 μL of the sodium borohydride solution and adding it to the first mixed solution after stirring, and stirring at room temperature; after completion, centrifuging and washing, collecting by centrifugation to obtain a first product, and freeze-drying the first product to obtain the gold nanoparticles modified with bovine serum albumin.

4. The method for preparing the molybdenum disulfide tandem enzyme according to claim 3, characterized in that: The first mixed solution was stirred in the dark for 30 minutes; 100 μL of the sodium borohydride solution was added to the first mixed solution after stirring, and the stirring time was 1 hour at room temperature.

5. The method for preparing the molybdenum disulfide tandem enzyme according to claim 2, characterized in that: The step of preparing molybdenum disulfide with surface modified carboxyl functional groups by hydrothermal method and ultrasonic method specifically comprises: configuring each raw material according to the following proportion, taking 2.8 g thiourea, 1.76 g sodium molybdate and 0.6 g hexadecyltrimethylammonium bromide, adding them to 80 mL deionized water in sequence, stirring evenly, and obtaining a second mixed solution; adjusting the pH of the second mixed solution to 8, and the total volume to 100 mL; transferring the second mixed solution to a high-pressure reaction vessel, and drying it at 240° C. for 48 hours; cooling to room temperature after the reaction is completed, collecting the black precipitate product by centrifugation and washing it to obtain a second product; annealing the second product at 500° C. for 1 hour in a nitrogen atmosphere using a single-temperature zone tubular furnace to obtain molybdenum disulfide powder; The molybdenum disulfide powder is prepared into a solution with a concentration of 2 mg / mL using deionized water and a total volume of 15 mL to obtain a molybdenum disulfide solution; 0.8-1.6 g of sodium hydroxide and 1 g of monochloroacetic acid are added to the molybdenum disulfide solution, and then ultrasonic treatment is performed, and finally centrifugation and washing are performed to obtain the molybdenum disulfide with surface modified carboxyl functional groups.

6. The method for preparing the molybdenum disulfide tandem enzyme according to claim 5, characterized in that: The drying time is 48 hours, and the annealing time is 1 hour; The ultrasonic treatment parameters are 60° C. and 500 W of ultrasonic power for 3 hours.

7. The method for preparing the molybdenum disulfide tandem enzyme according to claim 2, characterized in that: The step of combining the molybdenum disulfide with surface modified carboxyl functional groups with gold nanoparticles modified with bovine serum albumin by using an amide bond cross-linking method specifically comprises: configuring the raw materials according to the following proportions to prepare the molybdenum disulfide with surface modified carboxyl functional groups with a concentration of 1 mg / mL and a volume of 2 mL, then adding 500 μL of EDC and 500 μL of NHS, the concentrations of EDC and NHS are both 24 mM, reacting for 2 h to obtain a third mixed solution; adding 2 mL of gold nanoparticles modified with bovine serum albumin with a concentration of 1-5 mg / mL to the third mixed solution, reacting for 3 h, and then dialyzing, centrifuging, washing and freeze-drying to obtain the molybdenum disulfide tandem enzyme.

8. Application of the molybdenum disulfide tandem enzyme as claimed in claim 1 in the field of enzyme-free glucose colorimetric detection.

9. The use according to claim 8, characterized in that: The method for non-enzymatic glucose colorimetric detection specifically comprises: utilizing a colorimetric detection method, wherein the detection device is a UV-2450 ultraviolet-visible absorption spectrometer; 1.5 mL of Tris-HCl buffer with a pH of 3 was taken, and 0.75 mL of glucose solutions of different concentrations and 0.5 mL of the molybdenum disulfide tandem enzyme with a concentration of 2 mg / mL were added to the buffer to obtain a colorimetric solution; 0.5 mL of a 5 mM colorimetric substrate TMB solution was added to the colorimetric solution, and the solution was incubated in a water bath at 35°C for 30 minutes, and then centrifuged at 5000 rpm for 10 minutes to obtain a reaction system; The absorption spectrum of the reaction system at 652 nm was measured by UV-vis; 0.1 mL of dilute sulfuric acid was added dropwise to the reaction system to terminate the reaction, and the absorption spectrum of the reaction system at 450 nm was measured.

10. The use according to claim 9, characterized in that: The concentration range of the glucose solution to be tested is 10 -6 ~10 -2 M.

Images