A molybdenum disulfide tandem enzyme and its preparation method and application

Abstract

The present invention belongs to the field of analysis and detection, and specifically relates to a molybdenum disulfide tandem enzyme and its preparation method and application. The molybdenum disulfide tandem enzyme is prepared by a hydrothermal method and an ultrasonic method to prepare a molybdenum disulfide nanomaterial with a carboxyl functional group, and then a reduction method is used to prepare gold nanoparticles modified with bovine serum albumin with an amino functional group, and the molybdenum disulfide and the bovine serum albumin modified gold nanoparticles are combined by an amide bond cross-linking method to form a molybdenum disulfide tandem enzyme material. The molybdenum disulfide tandem enzyme can solve the problem that the activity of composite glucose oxidase is inhibited after molybdenum disulfide is combined with gold nanoparticles, and replaces the natural enzyme to realize the colorimetric detection of glucose. The invention is convenient and fast, and can provide a new solution for synthesizing a large number of transition metal sulfide compounds with different types of simulated enzyme activity materials. The molybdenum disulfide tandem enzyme can realize the colorimetric detection of glucose.

Description

Technical Field

[0001] The invention belongs to the field of analysis and detection, and specifically 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 purpose, the technical solution adopted by the present invention is as follows:

[0006] In the first aspect, the present invention provides a molybdenum disulfide tandem enzyme, 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 is connected to the gold nanoparticles modified with bovine serum albumin via an amide bond; the molybdenum disulfide tandem enzyme has both glucose oxidase-like activity and peroxidase-like activity. In the present invention, the gold nanoparticles modified with bovine serum albumin have amino groups on their surfaces; the carboxyl functional group is modified on the surface of the molybdenum disulfide, and the gold nanoparticles modified with bovine serum albumin are connected via an amide bond, thereby avoiding direct contact between the molybdenum disulfide and the gold nanoparticles, thereby achieving the retention of glucose oxidase-like activity and peroxidase-like activity in the molybdenum disulfide tandem enzyme, and can be used for non-enzyme colorimetric detection of glucose.

[0007] In a second aspect, the present invention provides a method for preparing the above-mentioned molybdenum disulfide tandem enzyme, which 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 gold nanoparticles modified with bovine serum albumin by amide bond crosslinking method to obtain the molybdenum disulfide tandem enzyme. The gold nanoparticles are combined with 35 Cys residues in bovine serum albumin to form Au-S bonds, and the Au-S formed here will not inhibit the simulated enzyme activity of the gold nanoparticles because of the difference in electronegativity between Au and S. Specifically, in the Au-S bond formed when molybdenum disulfide and gold nanoparticles are combined, the electronegativity of Au is 10.9, and the electronegativity of Mo is 8.3, which is greater than the electronegativity of Mo, so the interaction of the Au-S bond formed will be greater than the interaction of Mo-S, resulting in the S ions being closer to the gold nanoparticles, thereby competitively adsorbing with the gold ions on the surface of the gold nanoparticles. In contrast, the Au-S bond formed by bovine serum albumin and gold nanoparticles is formed with the thiol (-SH) in the gold nanoparticles, and the electronegativity of H is 13.6, which is greater than the electronegativity of Au. Therefore, the Au-S bond interaction formed is smaller than that of SH, making it difficult for S ions to approach the gold nanoparticles. In addition, bovine serum albumin molecules can form a stable protective layer on the surface of gold nanoparticles, thereby maintaining or enhancing the activity of the simulated enzyme.

[0008] Furthermore, the step of preparing gold nanoparticles modified with bovine serum albumin by reduction method specifically includes: preparing 4 mL of bovine serum albumin solution with a concentration of 1-13 μM according to the following proportions; then adding 1 mL of 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.

[0009] Furthermore, the first mixed solution was stirred for 30 minutes in the dark; 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] Further, 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 of deionized water in turn, 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; 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 nitrogen atmosphere using a single temperature zone tubular furnace to obtain molybdenum disulfide powder; configuring the molybdenum disulfide powder into a solution with a concentration of 2 mg / mL with deionized water, and the total volume is 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 surface modified 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 for 3 hours.

[0012] Furthermore, 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 includes: configuring the raw materials according to the following proportions to prepare the molybdenum disulfide with surface modified carboxyl functional groups at 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 h to obtain a third mixed solution; adding 2 mL of the gold nanoparticles modified with bovine serum albumin at a concentration of 1-5 mg / mL to the third mixed solution, reacting for 3 h, and then performing dialysis, centrifugation, washing and freeze-drying to obtain the molybdenum disulfide tandem enzyme.

[0013] In a third aspect, the present invention provides the use of the molybdenum disulfide tandem enzyme as described above in the field of enzyme-free 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 of different concentrations to be tested 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 min 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] Further, 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, first catalyzes a glucose solution into gluconic acid and hydrogen peroxide, and then its peroxidase-like activity catalyzes hydrogen peroxide into hydroxyl radicals (·OH), and ·OH further oxidizes the color-developing substrate TMB molecule, from colorless to blue oxidized TMB, and an absorption peak can be observed at 652 nm. After sulfuric acid is added to terminate the reaction, the blue oxidized TMB is oxidized to yellow diimine, and an absorption peak can be observed at 450 nm. When the glucose concentration increases, the hydrogen peroxide generated gradually increases, and the catalyzed color-developing substrate also increases accordingly, and the solubility of the measured glucose solution is judged by 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 by bovine serum albumin and combined with molybdenum disulfide with surface modified carboxyl functional groups through amide bonds, thereby avoiding direct contact between 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 test of glucose detection performance of bovine serum albumin-modified gold nanoparticles and molybdenum disulfide composite materials, the molybdenum disulfide tandem enzyme will combine the enzyme activities of the two and increase the activity of the composite nanoenzyme;

[0020] 3. In the present invention, since the molybdenum disulfide with surface modified carboxyl functional groups is composited with gold nanoparticles modified with bovine serum albumin, the Au-S bonds formed by the molybdenum disulfide and the gold nanoparticles are prevented from competing for the active sites on the surface of the gold nanoparticles, and the composite material is further endowed with two simulated enzyme activities, and finally the colorimetric detection of glucose is realized. The preparation method of the present invention solves the problem that conventional molybdenum disulfide composite materials do not have glucose oxidase-like activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] One or more embodiments are exemplarily described by pictures in the corresponding drawings, and 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 It is a comparison of the absorption spectra of conventional molybdenum disulfide / gold composite materials and the molybdenum disulfide tandem enzyme provided by the embodiment 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 is the comparison of the tandem enzyme activity of gold nanoparticles modified with bovine serum albumin prepared in the range of bovine serum albumin concentration from 1 to 13 μM;

[0026] Figure 5 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 MoS2 with carboxyl functional groups modified on the surface;

[0028] Figure 7 The comparison of the activity of surface-modified carboxyl functional groups of molybdenum disulfide peroxidase prepared with the mass of sodium hydroxide 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] Fig. 9 is an infrared spectrum image of a molybdenum disulfide tandem enzyme provided in an embodiment of the present invention;

[0031] Fig.10 The comparison of the activity of the MoS2 tandem enzyme prepared by the composite mass ratio of gold nanoparticles modified with bovine serum albumin and MoS2 with surface modified carboxyl functional groups ranging from 1:1 to 5:1;

[0032] Fig.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 found that at present, molybdenum disulfide, as a typical transition metal sulfide, has peroxidase-like catalytic activity, and its large specific surface area brings more active sites, and has been effectively applied to the colorimetric detection of glucose. For example, molybdenum disulfide is combined with glucose oxidase to achieve colorimetric detection of glucose; for another example, gold nanoparticles are combined with molybdenum disulfide, and glucose oxidase is also combined to achieve the detection of glucose in human serum. However, although gold nanoparticles have tandem enzyme activity, the combination of gold nanoparticles and molybdenum disulfide only exhibits a peroxidase-like enzyme 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 non-enzyme glucose colorimetric detection and a preparation method and application thereof, so that a composite material (MoS 2 -COOH / BSA@Au), i.e., the molybdenum disulfide tandem enzyme described in the present invention, has both glucose oxidase-like activity and peroxidase-like activity to achieve enzyme-free colorimetric detection of glucose.

[0035] The invention provides a molybdenum disulfide tandem enzyme, which comprises molybdenum disulfide with a surface modified with a carboxyl functional group and gold nanoparticles modified with bovine serum albumin; wherein the molybdenum disulfide with a surface modified with a carboxyl functional group and the gold nanoparticles modified with the 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 1-13 μM bovine serum albumin solution, then add 1 mL of 24 m M aqueous chloroauric acid solution was added to the bovine serum albumin solution, and the mixed solution was stirred for 30 minutes in the dark. Then, a sodium borohydride solution with a concentration of 1 mg / mL was prepared, and 100 μL of the sodium borohydride solution was quickly added to the mixed solution. The solution turned dark brown and stirred at room temperature for 1 hour. After the reaction, the precipitate was collected by centrifugation and the product was washed and placed in a freeze dryer for freeze drying to obtain gold nanoparticles modified with bovine serum albumin;

[0038] S2. Weigh 2.8 g thiourea, 1.76 g sodium molybdate and 0.6 g hexadecyltrimethylammonium bromide, add them to 80 mL of deionized water in turn 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 to 48 hours. After the reaction is completed, cool 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. In order to prepare the molybdenum disulfide tandem enzyme, the molybdenum disulfide was carboxylated and cross-linked with the amino groups on the surface of bovine serum albumin. The molybdenum disulfide powder was prepared into a solution with a concentration of 2 mg / mL using deionized water, and the total volume was 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 hours using an ultrasonic machine. The molybdenum disulfide with surface modified carboxyl functional groups was obtained by centrifugation and washing.

[0040] S4, MoS2 with surface modified carboxyl functional groups and gold nanoparticles modified with bovine serum albumin are combined through amide bonds. MoS2 with carboxyl functional groups is prepared with a concentration of 1 mg / mL and a volume of 2 mL. Then 500 μL of EDC and 500 μL of NHS are added, both at a concentration of 24 mM. The purpose of this step is to activate the carboxyl groups on the surface of MoS2 with surface modified carboxyl functional groups. The activation reaction time is 2 hours. Then 2 mL of gold nanoparticles modified with bovine serum albumin at a concentration of 1-5 mg / mL are added to connect the carboxyl groups with the amino groups. The composite reaction time is 3 hours. After the reaction, the mixed solution is dialyzed, centrifuged, washed and freeze-dried to obtain the MoS2 tandem enzyme.

[0041] The present invention is further described in detail below in conjunction with the examples. It is particularly noted that if no specific conditions are specified in the following examples, the reactions are carried out according to conventional conditions or 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] (I) Molybdenum disulfide tandem enzyme (hereinafter referred to as MoS 2 -COOH / BSA@Au) on the colorimetric detection of glucose.

[0044] For MoS 2 @Au and MoS2 -COOH / BSA@Au detection performance was compared to MoS 2 @Au and MoS 2 -COOH / BSA@Au were added to the test solution system containing 10 mM glucose, and then the color 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, and 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, MoS 2 @Au has no obvious absorption peak in the absorption spectrum after the reaction, while MoS 2 -COOH / BSA@Au exhibits a strong absorption peak, proving that MoS 2 -COOH / BSA@Au has the ability to catalyze glucose and hydrogen peroxide.

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

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

[0048] (III) To investigate the effect of BSA concentration on the enzyme activity of BSA-modified gold nanoparticles.

[0049] In order to investigate 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, and the concentrations were adjusted to 1, 4, 7, 10, and 13 μM, respectively. Then, 1 mL of 24 mM chloroauric acid aqueous solution was added to the bovine serum albumin solution, and the mixed solution was stirred for 30 minutes in the dark. Then, a sodium borohydride solution with a concentration of 1 mg / mL was prepared, and 100 μL of sodium borohydride solution was quickly added to the mixed solution. The solution turned dark brown and stirred at room temperature for 1 hour. After the reaction was completed, the precipitate was collected by centrifugation and the product was washed, and it was placed in a freeze dryer for freeze drying to obtain gold nanoparticles modified with bovine serum albumin.

[0051] Further, the effect of the gold nanoparticles modified with bovine serum albumin 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 gold nanoparticles modified with bovine serum albumin 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 water bath at 35°C for 30 minutes, and then centrifuged at 5000 rpm for 10 min 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 an obvious absorption peak at 450 nm, such as Figure 4 As shown, it is shown that the gold nanoparticles modified with bovine serum albumin have tandem enzyme activity similar to glucose oxidase and peroxidase, and realize the catalysis of glucose. It is observed that the absorption peak intensity gradually increases with the increase of bovine serum albumin concentration, proving that the simulated enzyme activity of gold nanoparticles modified with bovine serum albumin gradually increases, but when the concentration is greater than 10 µM, the absorption peak intensity of the reaction system shows a decreasing trend. This is because bovine serum albumin, as a natural biological carrier, forms a protective layer on the surface of gold nanoparticles to enhance its stability. When the concentration of bovine serum albumin increases, more bovine serum albumin will wrap the gold nanoparticles, enhancing their stability and activity. However, when the concentration of bovine serum albumin is too high, the interaction between bovine serum albumin molecules will increase, which will lead to aggregation, so that the active sites on the surface of gold nanoparticles are wrapped or shielded by bovine serum albumin, thereby weakening its simulated enzyme activity.

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

[0054] (iv) 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 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) To investigate the effect of the amount of sodium hydroxide on the activity of molybdenum disulfide mimetic enzymes with surface modified carboxyl functional groups.

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

[0058] Similarly, weigh 2.8 g thiourea, 1.76 g sodium molybdate and 0.6 g hexadecyltrimethylammonium bromide, add them to 80 mL deionized water 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 mixed solution to a stainless steel autoclave 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 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.

[0059] In order to prepare molybdenum disulfide with surface modified with carboxyl functional groups, molybdenum disulfide powder was prepared into a solution with a concentration of 2 mg / mL using deionized water and 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 hours using an ultrasonic machine. Molybdenum disulfide with surface modified with carboxyl functional groups was obtained by centrifugation and washing.

[0060] Further, the colorimetric detection effect of the molybdenum disulfide with surface modified carboxyl functional groups 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 water bath at 35°C for 30 minutes to allow the glucose oxidase to catalyze glucose to produce hydrogen peroxide, and 1 mg / mL of molybdenum disulfide with surface modified carboxyl functional groups 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 continued incubation for 30 minutes, followed by centrifugation at 5000 rpm for 10 min 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 was measured using a UV-2450 ultraviolet-visible absorption spectrometer (yellow solution).

[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 and produce 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 molybdenum disulfide with carboxyl functional groups prepared under the condition of adding 1.4 g of sodium hydroxide has the best peroxidase-like activity performance.

[0063] (VI) 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 Fig. 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. Through Fourier infrared spectroscopy analysis, it was found that the -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 MoS2 composite tandem enzyme is successful.

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

[0066] In order to explore the effect of the mass ratio of gold nanoparticles modified with bovine serum albumin and molybdenum disulfide with surface modified with carboxyl functional groups on the activity of molybdenum disulfide tandem enzyme, 5 groups of different mass ratios were set up to prepare molybdenum disulfide tandem enzyme.

[0067] Similarly, a 1 mg / mL surface-modified carboxyl molybdenum disulfide was prepared in a volume of 2 mL, followed by the addition of 500 μL of EDC and 500 μL of NHS, both at a concentration of 24 mM. The purpose of this step is to activate the carboxyl groups on the surface of the surface-modified carboxyl molybdenum disulfide, and the activation reaction time is 2 hours. Subsequently, 2 mL of bovine serum albumin-modified gold nanoparticles at a concentration of 1-5 mg / mL was added to ensure that the mass ratio of the bovine serum albumin-modified gold nanoparticles to the surface-modified carboxyl molybdenum disulfide was 1:1-5:1, and the carboxyl group and the amino group were connected, and the composite reaction time was 3 hours. After the reaction, the mixed solution was dialyzed, centrifuged, washed and freeze-dried to obtain the molybdenum disulfide tandem enzyme.

[0068] Further, 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 water bath at 35°C for 30 minutes, and then centrifuged at 5000 rpm for 10 min to obtain a reaction system; the absorption spectrum of the reaction system at 652 nm was measured by UV-vis (blue solution); 0.1 mL of dilute sulfuric acid was added to the reaction system to terminate the reaction, and the absorption spectrum of the reaction system at 450 nm was measured (yellow solution).

[0069] The reaction results are as follows Fig.10As shown in the figure, when the specific gravity of gold nanoparticles modified with bovine serum albumin increases, the enzymatic activity of molybdenum disulfide tandem enzyme gradually increases. After the mass of 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, which 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 amide bonds 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 gold nanoparticles modified with bovine serum albumin and 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] (VIII) 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, preparing a bovine serum albumin solution with a concentration of 10 μM, adding 1 mL of 24 mM chloroauric acid aqueous solution to the bovine serum albumin solution, and stirring it in the dark for 1 hour. Then prepare a sodium borohydride solution with a concentration of 1 mg / mL, take 100 μL and quickly add it to the above mixed solution, the solution turns dark brown, and stir the reaction at room temperature for 1 hour. After the reaction is completed, centrifuge and wash the product to remove excess bovine serum albumin, collect the product by centrifugation, and place it 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 to 80 mL of deionized water in sequence and stirring 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 mixed solution to a stainless steel autoclave with a polytetrafluoroethylene liner, place it in a blast drying oven, and react 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. The product is repeatedly washed 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. The molybdenum disulfide powder is configured with deionized water to form a solution with a total volume of 15 mL and a concentration of 2 mg / mL. Then, 1.4 g of sodium hydroxide and 2.0 g of monochloroacetic acid are added to the molybdenum disulfide solution. An ultrasonic machine is used to perform ultrasonic treatment 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, such as 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. Then, 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 composite 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 of different concentrations, five groups of glucose solutions of 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 pH = 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, and finally the mixed solution was placed in a 35 ° C water bath for incubation 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, Fig.11 As shown, the reflected absorption peak intensity increases with the increase of glucose concentration, and the detection of glucose is realized. 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 the 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 material glucose oxidase is inhibited after the combination of molybdenum disulfide and gold nanoparticles, and replace the natural enzyme to realize the colorimetric detection of glucose. The detection is convenient and fast, and can provide a new scheme for synthesizing a large number of transition metal sulfide compounds with different types of simulated enzyme activity materials.

[0077] Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in practical applications, various changes can be made to them in form and detail without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application, so the scope of protection of the present application shall be based on the scope defined in the claims.

Claims

1. An application of a molybdenum disulfide tandem enzyme in the field of enzyme-free glucose colorimetric detection, 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; The preparation method of the molybdenum disulfide tandem enzyme 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.

2. The use according to claim 1, characterized in that: The step of preparing gold nanoparticles modified with bovine serum albumin by reduction method specifically comprises: preparing each raw material according to the following proportions, preparing 4 mL of bovine serum albumin solution with a concentration of 1-13 μM; then adding 1 mL of 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.

3. The use according to claim 2, 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.

4. The use according to claim 1, 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.

5. The use according to claim 4, 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.

6. The use according to claim 1, 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 at a concentration of 1 mg / mL in a volume of 2 mL, then adding 500 μL of EDC and 500 μL of NHS, wherein 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 at a concentration of 1-5 mg / mL to the third mixed solution, reacting for 3 h, and then performing dialysis, centrifugation, washing and freeze-drying to obtain the molybdenum disulfide tandem enzyme.

7. The use according to claim 1, 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.

8. The use according to claim 7, characterized in that: The concentration range of the glucose solution to be tested is 10 -6 ~10 -2 M.

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