Nanoprotease assembly film material and preparation method thereof

Abstract

This invention provides a nanozyme assembled membrane material and its preparation method, belonging to the field of biosensing technology. The nanozyme assembled membrane material comprises a base membrane and an Au1Cu single-atom nanozyme; the Au1Cu single-atom nanozyme is dispersed on the surface of the base membrane; the Cu nanocubes in the Au1Cu single-atom nanozyme have a side length of 36-84 nm, and the surface of the Cu nanocubes has atomically dispersed Au single atoms, with an Au mass fraction of 2%-3%; the thickness of the nanozyme assembled membrane material is 2-5 μm. This invention develops a strategy for constructing nanozyme assembled membrane materials, which greatly improves the catalytic activity and selectivity of the nanozymes. The prepared Au1Cu assembled membrane material simultaneously possesses catalytic capabilities similar to glucose oxidase and peroxidase, enabling low-cost, rapid, and accurate blood glucose detection via a flow-through method.

Description

Technical Field

[0001] This invention relates to the field of biosensing, and more particularly to a nanoenzyme assembly membrane material and its preparation method. Background Technology

[0002] Abnormal fluctuations in blood glucose levels are closely related to the occurrence and development of various metabolic diseases, with diabetes being the most typical. Developing accurate, rapid, and reliable blood glucose testing technologies is of great practical significance for clinical diagnosis, disease monitoring, and treatment planning. Currently, the glucose oxidase method remains the most widely used technical route in clinical and commercial blood glucose testing. This method relies on the synergistic catalytic action of natural enzymes. Glucose oxidase is responsible for oxidizing glucose to gluconic acid and producing hydrogen peroxide, while horseradish peroxidase further catalyzes the reaction of hydrogen peroxide with a chromogenic substrate, thereby amplifying and converting the signal. Because both enzymes possess highly specific substrate recognition capabilities and efficient catalytic reaction rates, this detection system has advantages such as high sensitivity, excellent selectivity, and stable results, making it one of the standard methods for blood glucose testing today. Summary of the Invention

[0003] In view of this, in order to at least partially solve the aforementioned technical problems, the present invention provides a nanoenzyme assembly membrane material and a method for preparing the same.

[0004] According to one aspect of the present invention, a nanozyme assembly membrane material is provided, comprising a base membrane and an Au1Cu single-atom nanozyme; the Au1Cu single-atom nanozyme is dispersed on the surface of the base membrane; the side length of the Cu nanocube in the Au1Cu single-atom nanozyme is 36-84 nm, and the surface of the Cu nanocube has atomically dispersed Au single atoms, with a mass fraction of Au of 2%-3%; the thickness of the nanozyme assembly membrane material is 2-5 μm.

[0005] According to one embodiment of the present invention, the pore size of the base film is 50 nm.

[0006] According to another aspect of the present invention, a method for preparing the above-mentioned nanoenzyme assembled membrane material is provided, comprising: sequentially adding Au1Cu single-atom nanoenzyme dispersion to the surface of a base membrane, and adding the dispersion one time after the dispersion evaporates, for a total of 10-25 times, to obtain Au1Cu nanoenzyme assembled membrane material.

[0007] According to one embodiment of the invention, the concentration of the dispersion is 0.5-1 mg / mL.

[0008] According to one embodiment of the invention, the single drop volume of the dispersion is 200 μL.

[0009] According to another aspect of the present invention, a method for detecting blood glucose is provided, which utilizes the above-described nanozyme-assembled membrane material for detection.

[0010] According to one embodiment of the present invention, the above-mentioned method for detecting blood glucose includes: passing a reaction solution through a nanozyme assembled membrane material, detecting the absorbance of the product solution at 652 nm, and substituting the absorbance value into a standard curve to obtain the glucose concentration in the test solution; the reaction solution is a mixture of the test solution and an ethanol solution of 3,3',5,5'-tetramethylbenzidine.

[0011] According to another aspect of the present invention, a method for preparing Au1Cu single-atom nanozymes is provided, comprising:

[0012] a. Dissolve cuprous bromide and tri-zinc-based phosphorus oxide in oleylamine, heat to 70-90℃ under inert gas protection and hold for 10-20 minutes, then continue heating the solution to 250-270℃ at a heating rate of 10-15℃ / min and hold for 1 hour to generate Cu nanocubes.

[0013] b. Cool the reaction solution in a to 200-220℃, then inject an oleylamine solution of chloroauric acid into the reaction solution and maintain it at 200-220℃ for 15-30 minutes to obtain Au1Cu single-atom nanozyme.

[0014] According to another aspect of the present invention, a method for detecting blood glucose using the above-described Au1Cu single-atom nanozyme is provided.

[0015] According to embodiments of the present invention, the present invention has at least one of the following technical effects:

[0016] (1) After assembling Au1Cu single-atom nanozymes to obtain nanozyme assembled membrane materials, they simultaneously possess the catalytic capabilities of glucose oxidase and peroxidase, enabling rapid oxidation of glucose molecules and efficient utilization of hydroxyl radicals, thereby achieving one-step detection of glucose concentration.

[0017] (2) Improved catalytic selectivity of nanozymes: After assembling Au1Cu single-atom nanozymes into nanozyme-assembled membrane materials, thanks to the selectivity of Au active sites for glucose oxidation and the size selectivity of channels in the membrane material, the nanozyme-assembled membrane materials exhibit excellent glucose selectivity, which can ensure the accuracy of detection results.

[0018] (3) Rapid detection of glucose: When using the glucose oxidase method to detect glucose content, traditional nanozymes usually require a long incubation process (about 30-60 minutes), while the present invention uses nanozyme-assembled membrane materials to achieve rapid glucose detection within 1-3 minutes through the "flow method".

[0019] (4) Low-cost detection of glucose: The nanoenzyme assembly membrane material used in this invention has the catalytic ability of both glucose oxidase and peroxidase, which can realize one-step detection of glucose concentration without the participation of biological enzymes, thus significantly reducing the cost of glucose detection. Attached Figure Description

[0020] Figure 1 The images shown are transmission electron microscopy (TEM) and aberration electron microscopy (AEM) images of the Au1Cu single-atom nanozyme in Example 1 of this invention, where a is a TEM image with a scale bar of 200 nm and b is an AEM image with a scale bar of 2 nm.

[0021] Figure 2 The image shows a scanning electron microscope image of the nanozyme assembly membrane material in Example 3 of this invention. The scale bar is 5 μm in a and 20 μm in b.

[0022] Figure 3 This is a schematic diagram illustrating the working process of the nanoenzyme assembly membrane material of the present invention;

[0023] Figure 4 The images show the absorbance curve and standard curve of the nanozyme assembled membrane material used for glucose detection in Example 6 of the present invention, where a is the absorbance curve and b is the standard curve.

[0024] Figure 5 This is a graph showing the performance results of the nanozyme assembled membrane material in Example 7 of the present invention when used for actual blood glucose detection.

[0025] Figure 6 This figure shows a performance comparison of the typical incubation catalytic process of Au1Cu single-atom nanozymes and the "flow-through" catalytic process of nanozyme assembly membrane materials for glucose detection.

[0026] Figure 7 This is a diagram showing the signal response results of the nanoenzyme assembly membrane material of the present invention to different biomolecules;

[0027] Figure 8 This is a transmission electron microscope image of the Cu nanocubic structure prepared in Example 2 of the present invention, with a scale bar of 50 nm;

[0028] Figure 9 The image shown is a scanning electron microscope (SEM) image of the nanozyme assembly membrane material prepared in Example 5 of this invention. The scale bar is 20 nm. Detailed Implementation

[0029] Natural enzymes face significant limitations in practical applications of blood glucose detection, including source dependence, high preparation costs, strong environmental sensitivity, and stringent storage and transportation conditions. To overcome these shortcomings, nanozymes, as nanomaterials with enzyme-like activity, have attracted widespread attention in recent years. Nanozymes possess significant advantages such as low synthesis cost, high structural stability, resistance to inactivation, and the ability to operate over a wider pH and temperature range, making them potential alternatives to biological enzymes. The application of nanozymes in blood glucose detection is expected to significantly reduce detection costs while improving the durability and stability of the detection system.

[0030] Developing nanozymes with excellent catalytic performance is a key prerequisite for their practical application in blood glucose detection, especially bifunctional nanozymes that simultaneously possess glucose oxidase-like and peroxidase-like catalytic capabilities. Currently, most research focuses on the fine-tuning of the active sites of nanozymes to enhance their catalytic performance. Among these, single-atom nanozymes, with their well-defined geometric configurations and tunable electronic structures, can simulate the metal coordination environment and electron distribution characteristics of the active center of natural enzymes with atomic-level precision, thus reproducing, to some extent, the highly evolved catalytic functions of natural enzymes. Compared to traditional nanoparticles, single-atom nanozymes not only have the advantage of maximizing atom utilization but also enable precise control over catalytic pathways and reaction selectivity by regulating metal-support interactions, coordination number, and ligand environment. Therefore, single-atom nanozymes show greater potential in improving enzyme-like activity and selectivity and have become one of the important research directions in nanozyme design and optimization.

[0031] However, existing bifunctional nanozymes currently exhibit significantly lower catalytic efficiency and substrate specificity compared to natural enzymes, which greatly limits their practical application in blood glucose detection and other scenarios requiring high sensitivity and selectivity. The main reason for this is that nanozymes lack the highly evolved local microenvironment surrounding the active site of natural enzymes, such as precise distribution of hydrophobic / hydrophilic regions, specific charge regulation, fine conformational changes, and substrate-guided channels. These factors collectively determine the superior catalytic activity, selectivity, and substrate adaptability of natural enzymes, but are difficult to fully replicate in nanozymes.

[0032] Based on this, the present invention provides a nanoenzyme assembly membrane material, comprising a base membrane and an Au1Cu single-atom nanoenzyme; the Au1Cu single-atom nanoenzyme is dispersed on the surface of the base membrane; the side length of the Cu nanocube in the Au1Cu single-atom nanoenzyme is 36-84 nm, and the surface of the Cu nanocube has atomically dispersed Au single atoms, with a mass fraction of 2%-3% for Au; the thickness of the nanoenzyme assembly membrane material is 2-5 μm.

[0033] The side length of Cu nanocubes affects their specific surface area; a smaller side length results in a larger specific surface area and correspondingly better catalytic activity. However, considering the pore size of the substrate membrane, excessively small side lengths of Cu nanocubes can cause them to permeate through the membrane. Au is the active site for glucose oxidation during the catalytic process; a higher Au mass fraction leads to better catalytic activity, but excessively high Au mass fractions can cause Au single atoms to aggregate. A thicker nanozyme assembly membrane material contains more nanozymes, resulting in better catalytic activity, but it reduces membrane flux, leading to increased detection time. Therefore, the side length of the Cu nanocubes, the Au mass fraction, and the thickness of the nanozyme assembly membrane material all need to be within appropriate ranges.

[0034] According to embodiments of the present invention, a strategy for constructing nanozyme assembled membrane materials is proposed, which greatly improves the catalytic activity and selectivity of nanozymes. The prepared Au1Cu assembled membrane material simultaneously possesses catalytic capabilities similar to glucose oxidase and peroxidase, enabling low-cost, rapid, and accurate blood glucose detection via a flow-through method.

[0035] Specifically, in related technologies, nanozyme-based detection involves dispersing nanozymes in a solution for catalysis, with the nanozyme surface in contact with the bulk solution. In this invention, after assembling nanozymes into a membrane material, confined spaces are formed between the nanozyme particles. When the solution flows through these nanoscale confined spaces, a catalytic reaction occurs, which is similar to the catalytic process of natural enzymes. Thus, the nanozymes of this invention have a highly evolved local microenvironment similar to that around the active center of natural enzymes.

[0036] The synthesis methods of Au1Cu single-atom nanozymes in nanozyme assembly membrane materials include:

[0037] a. Dissolve cuprous bromide and tri-zinc-based phosphorus oxide in oleylamine. Under an argon atmosphere, heat the solution to 70-90℃ and hold for 10-20 minutes. Then, continue heating the solution to 250-270℃ at a heating rate of 10-15℃ / min and hold for 1 hour to generate Cu nanocube structures.

[0038] b. Cool the reaction solution from step a to 200-220°C, then inject a solution of chloroauric acid in oleylamine into the solution and maintain the temperature at 200-220°C for 10-30 minutes. At this point, the Au concentration in the solution will increase. 3+ It will interact with the Cu nanocube surface Cu 0 Electrodisplacement occurs, generating atomically dispersed Au single atoms on the surface of the nanocube, yielding Au1Cu single-atom nanozymes.

[0039] The post-processing steps include: c) cooling the solution to room temperature, washing the Au1Cu single-atom nanozyme sample with a mixed solvent of n-hexane and ethanol, repeating the process three times, and dispersing the sample in hexane or toluene to form a uniform dispersion.

[0040] Specifically, the reaction temperature in step a is 250-270℃, for example, 250℃, 255℃, 260℃, 265℃, or 270℃, preferably 270℃, which directly affects the size of the nanocubes. The reaction temperature in step b is 200-220℃, for example, 200℃, 205℃, 210℃, 215℃, or 220℃, preferably 220℃. The higher the reaction temperature in step a, the larger the nanoparticle size; the higher the reaction temperature in step b, the higher the loading of Au single atoms. Therefore, temperature directly affects the loading of Au single atoms.

[0041] More preferably, in one more specific embodiment, the method for synthesizing Au1Cu single-atom nanozymes includes:

[0042] a. Dissolve 70 mg of cuprous bromide and 500 mg of tri-zinc-based phosphorus oxide in 7 mL of oleylamine. Under an argon atmosphere, heat the solution to 80 °C and hold for 15 minutes. Then, continue heating the solution to 270 °C at a heating rate of 10 °C / min and hold for 1 hour. At this time, Cu nanocube structures with a side length of about 76 ± 8 nm are generated in the solution.

[0043] b. Cool the solution to 220°C. Dissolve 40 mg of tetrachloroauric acid trihydrate in 10 mL of oleylamine and stir for 10 minutes to form a gold precursor solution. Take 1 mL of the gold precursor solution and inject it into the reaction solution using a syringe. Maintain the solution at 220°C for 30 minutes. At this point, the Au content in the precursor solution will be... 3+ It will interact with the Cu nanocube surface. 0 Electrodisplacement occurs, generating atomically dispersed Au single atoms on the surface of the nanocube, yielding Au1Cu single-atom nanozymes.

[0044] Post-processing steps: c. Cool the solution to room temperature, add 20 mL of n-hexane and 15 mL of ethanol, sonicate for 2 minutes, centrifuge, remove the supernatant, and repeat three times. Then disperse the sample in toluene to form a 1 mg / mL dispersion.

[0045] Furthermore, the assembly method for nanozyme assembly membrane materials includes:

[0046] At room temperature, Au1Cu single-atom nanozyme dispersion is added dropwise to the surface of the base membrane one by one. After the dispersion has completely evaporated, the next drop is added. The total number of drops is 10-25 to obtain the nanozyme assembled membrane material.

[0047] Specifically, adding the material dropwise can avoid the problem of a messy assembly structure and defects on the membrane caused by adding too much material at once.

[0048] According to an embodiment of the present invention, the pore size of the base membrane is 50 nm, and nylon filter membranes with a pore size of 50 nm and polyethersulfone filter membranes with a pore size of 50 nm can be used.

[0049] According to embodiments of the present invention, the smaller the nanocubes, the larger the specific surface area and the better the catalytic activity. The pore size of the base film should be as small as possible smaller than the nanocube size, so a base film with a pore size of 50 nm is selected to support the nanocubes.

[0050] More preferably, in one more specific embodiment, the assembly method of the nanozyme assembly membrane material includes:

[0051] At room temperature, a nylon filter membrane with a diameter of 50 mm and a pore size of 50 nm was fixed in a vacuum filtration device with a ground glass joint of size 24#. Au1Cu single-atom nanozyme dispersion was then added dropwise to the surface of the base membrane in increments of 200 μL. The dispersion was allowed to evaporate completely before the next addition was made, with a total of 3 mL of dispersion added to obtain the nanozyme assembled membrane material. The nanozyme assembled membrane material, loaded onto the nylon base membrane, could be removed from the vacuum filtration device along with the base membrane.

[0052] Furthermore, the volume of the added dispersion affects the thickness of the nanozyme assembly membrane material. When 3 mL of dispersion is added, the thickness of the formed nanozyme assembly membrane material is approximately 3 μm.

[0053] In an embodiment of the present invention, the method for plotting the glucose concentration standard curve in the blood glucose detection method includes: preparing a 5 mM 3,3′,5,5′-tetramethylbenzidine (TMB) ethanol solution and a 0.2 M acetate-sodium acetate buffer solution with pH=5.2, and preparing a series of glucose solutions with concentration gradients by adding an appropriate amount of glucose to the buffer solution. 50 μL of TMB ethanol solution and 50 μL of glucose solution are added to 1900 μL of buffer solution to obtain 2 mL of reaction solution (final glucose concentrations are 0 μM, 0.2 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 3 μM, 5 μM, 7 μM, 10 μM, 15 μM, 20 μM, 30 μM, 50 μM, and 100 μM). Using the "flow-through method" of the present invention, the nanozyme assembly membrane material loaded on the nylon-based membrane is fixed in a vacuum filter with a ground glass joint size of 24#, the vacuum pump is turned on, and 2 mL of reaction solution is added. After the reaction solution is filtered and passed through a nanozyme-assembled membrane material, the glucose in it is oxidized to gluconic acid and hydrogen peroxide. The generated hydrogen peroxide is then decomposed to generate hydroxyl radicals, which cause TMB to change color. By adding a series of glucose solutions with different concentration gradients and measuring the absorbance of the product solution at 652 nm, a standard curve of glucose concentration versus product solution absorbance was plotted.

[0054] In the embodiments of this invention, the "flow-through method" exhibits superior mass transfer kinetics. Under pressure differential, the substrate undergoes convective transport through the membrane channels, significantly reducing the diffusion boundary layer thickness and mitigating concentration polarization, making the reaction more readily approach the intrinsic catalytic rate. Simultaneously, the confined space provided by the membrane channels allows for local substrate enrichment and increases the effective collision frequency, thereby contributing to improved apparent reaction rates and shorter response times. This "convective-confined coupling" mass transfer mode is difficult to achieve in dispersed systems where pure diffusion is dominant. Regarding stability, nanozymes in dispersed systems are prone to aggregation, sedimentation, or loss with the system during actual detection, leading to activity fluctuations and decreased batch repeatability. In contrast, nanozyme particles in membrane structures are stably loaded on the base membrane surface, significantly improving the stability of the detection system. Furthermore, the "flow-through method" demonstrates stronger resistance to complex matrices. The membrane material itself possesses certain size sieving properties, which can, to a certain extent, prevent large molecules or interfering substances from complex matrices from entering the catalytic region, thereby improving the sensitivity of the detection system.

[0055] Furthermore, in the dispersed system, nanozymes are essentially disposable and difficult to recover, while the membrane material composed of nanozymes flowing through the system can be reused. Moreover, the nanozymes and substrates coexist continuously throughout the detection process, with the catalytic reaction proceeding continuously, resulting in a significant time-dependent and continuously evolving colorimetric signal. In contrast, in the flowing system, the reaction solution rapidly separates from the immobilized nanozymes upon passing through the membrane catalytic interface, effectively terminating the catalytic process. This ensures that the obtained colorimetric signal remains stable after detection and no longer changes over time. This signal-locking effect resulting from the "reaction-separation" coupling helps improve the repeatability of readings and the comparability of results.

[0056] Furthermore, rapid measurement methods for detecting blood glucose concentration include:

[0057] A blood glucose sample solution was prepared by adding an appropriate amount of glucose to an artificial blood sample. The sample solution was diluted to the detection range of the standard curve. 50 μL of TMB ethanol solution and 50 μL of the diluted sample solution were added to 1900 μL of buffer solution to obtain the reaction solution. The nylon-based membrane-supported nanozyme assembly was fixed in a vacuum filter with a 24# ground glass joint. The vacuum pump was turned on, and 2 mL of the reaction solution was added. The absorbance of the product solution at 652 nm was measured, and the glucose concentration in the sample solution was calculated by substituting this absorbance into the standard curve.

[0058] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0059] Example 1

[0060] Synthesis of Au1Cu single-atom nanozymes:

[0061] 70 mg of cuprous bromide and 500 mg of tri-zinc-based phosphorus oxide were dissolved in 7 mL of oleylamine. Under an argon atmosphere, the solution was heated to 80 °C and held for 15 minutes. Then, the solution was further heated to 270 °C at a heating rate of 10 °C / min and held for 1 hour. At this point, Cu nanocubes with a side length of 76 ± 8 nm were formed in the solution. The results are as follows: Figure 1 As shown in Figure a.

[0062] Cool the solution to 220°C. Dissolve 40 mg of tetrachloroauric acid trihydrate in 10 mL of oleylamine and stir for 10 minutes to form a gold precursor solution. Take 1 mL of the gold precursor solution and inject it into the reaction solution using a syringe, then maintain the solution at 220°C for 30 minutes. At this point, the Au concentration in the solution will be... 3+ It will interact with the Cu nanocube surface. 0 Electrodisplacement occurs, generating atomically dispersed gold single atoms (e.g.) on the surface of the nanocubes. Figure 1 (As shown in b), this is the Au1Cu single-atom nanozyme. The Au mass fraction of this Au1Cu single-atom nanozyme is 2.7 wt%.

[0063] Cool the solution to room temperature, add 20 mL of n-hexane and 15 mL of ethanol, sonicate for 2 minutes, centrifuge, remove the supernatant, and repeat three times. Then disperse the sample in toluene to form a 1 mg / mL dispersion.

[0064] according to Figure 1 As can be seen, its cubic structure can be observed, and the atomic brightness of the outer layer of the nanocube is not uniform, with the brighter areas being Au atoms.

[0065] Example 2

[0066] Synthesis of Au1Cu single-atom nanozymes:

[0067] 70 mg of cuprous bromide and 500 mg of tri-zinc-based phosphorus oxide were dissolved in 7 mL of oleylamine. Under an argon atmosphere, the solution was heated to 80 °C and held for 15 minutes. Then, the solution was further heated to 250 °C at a heating rate of 10 °C / min and held for 1 hour. At this point, Cu nanocubic structures with a side length of 42 ± 6 nm were formed in the solution. Figure 8 As shown.

[0068] The solution was cooled to 200°C. 40 mg of tetrachloroauric acid trihydrate was dissolved in 10 mL of oleylamine and stirred for 10 minutes to form a gold precursor solution. 1 mL of the gold precursor solution was injected into the reaction solution using a syringe and kept at 200°C for 30 minutes to obtain Au1Cu single-atom nanozyme. The Au mass fraction of this Au1Cu single-atom nanozyme was 2.1 wt%.

[0069] Cool the solution to room temperature, add 20 mL of n-hexane and 15 mL of ethanol, sonicate for 2 minutes, centrifuge, remove the supernatant, and repeat three times. Then disperse the sample in hexane to form a 1 mg / mL dispersion.

[0070] Example 3

[0071] Preparation of nanozyme assembly membrane materials:

[0072] At room temperature, a nylon filter membrane with a diameter of 50 mm and a pore size of 50 nm was fixed in a vacuum filtration device with a ground joint specification of 24#. Then, the Au1Cu single-atom nanozyme dispersion prepared in Example 1 was added dropwise to the surface of the base membrane in batches of 200 μL each time. After the dispersion was completely evaporated, the next addition was carried out. A total of 3 mL of dispersion was added to obtain the nanozyme assembled membrane material (e.g., Figure 2 As shown in Figure a). The thickness of the nanozyme assembly membrane material is 3 μm (as shown in Figure a). Figure 2 (As shown in b).

[0073] Example 4

[0074] Preparation of nanozyme assembly membrane materials:

[0075] At room temperature, a nylon filter membrane with a diameter of 50 mm and a pore size of 50 nm was fixed in a vacuum filtration device with a ground joint specification of 24#. Then, the Au1Cu single-atom nanozyme dispersion prepared in Example 1 was added dropwise to the surface of the base membrane in batches of 200 μL each time. After the dispersion was completely evaporated, the next addition was carried out. A total of 4.5 mL of dispersion was added to obtain the nanozyme assembled membrane material. The thickness of the nanozyme assembled membrane material was 5 μm.

[0076] Example 5

[0077] Preparation of nanozyme assembly membrane materials:

[0078] At room temperature, a polyethersulfone filter membrane with a diameter of 25 mm and a pore size of 50 nm was fixed in a vacuum filtration device with a ground joint specification of 24#. Then, the Au1Cu single-atom nanozyme dispersion prepared in Example 2 was added dropwise to the surface of the base membrane in batches of 200 μL each time. The addition was repeated only after the dispersion had completely evaporated, for a total of 3 mL of dispersion was added, resulting in a nanozyme assembled membrane material. This nanozyme assembled membrane material has a thickness of 3 μm. Figure 9As shown.

[0079] Example 6

[0080] Plotting the glucose concentration standard curve:

[0081] Prepare a 5 mM TMB ethanol solution and a 0.2 M acetate-sodium acetate buffer solution at pH 5.2. Prepare a series of glucose solutions with varying concentrations by adding appropriate amounts of glucose to the buffer solutions. Add 50 μL of TMB ethanol solution and 50 μL of glucose solution to 1900 μL of buffer solution to obtain reaction solutions (final glucose concentrations of 0 μM, 0.2 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 3 μM, 5 μM, 7 μM, 10 μM, 15 μM, 20 μM, 30 μM, 50 μM, and 100 μM). Figure 3 As shown, the nanozyme-assembled membrane material from Example 3 was fixed in a vacuum filtration device with a ground glass joint of size 24#. The vacuum pump was turned on, creating negative pressure inside the receiving bottle, and 2 mL of reaction solution was added. After the reaction solution passed through the nanozyme-assembled membrane material during the vacuum filtration process, the glucose in it was oxidized to gluconic acid and hydrogen peroxide. The generated hydrogen peroxide was decomposed to generate hydroxyl radicals, which caused TMB to change color. By adding a series of glucose solutions with varying concentrations, the absorbance of the product solution at 652 nm was measured (e.g., ...). Figure 4 As shown in Figure a), a standard curve of glucose concentration versus absorbance of the product solution can be plotted (e.g., Figure 4 As shown in Figure b, y = 0.046x + 0.035), its detection limit is 0.11 μM.

[0082] Further, by preparing a 2mM TMB ethanol solution and performing the above steps, the corresponding standard curve was obtained as: y = 0.023x + 0.029; or by using the nanozyme assembly membrane material assembled in Example 4 and performing the above steps, the corresponding standard curve was obtained as: y = 0.069x + 0.040 (e.g., ...). Figure 4 (As shown in b).

[0083] Different standard curves can then be obtained, proving that the reaction process can be adjusted by regulating the film thickness and reactant concentration.

[0084] Example 7

[0085] Rapid measurement of blood glucose concentration:

[0086] A 5 mM glucose sample solution was prepared by adding glucose to artificial blood. Using a volumetric flask, 10 mL of the sample solution was diluted to 500 mL with buffer solution to completely dilute it to the detection range of the standard curve. 50 μL of TMB ethanol solution and 50 μL of the completely diluted sample solution were added to 1900 μL of buffer solution to obtain the reaction solution. The nanozyme-assembled membrane material from Example 3 was fixed in a vacuum filter with a ground glass joint of 24#. The vacuum pump was turned on, and 2 mL of the reaction solution was added. The absorbance of the product solution at 652 nm was measured, and the glucose concentration in the sample solution was calculated by substituting it into the standard curve (y = 0.046x + 0.035). Three independent parallel tests were performed using three nanozyme-assembled membrane materials from Example 3 (numbered 1, 2, and 3), with each test lasting approximately 3 minutes. The results are as follows: Figure 5 As shown.

[0087] according to Figure 5 It can be seen that the test results of the three membrane materials are close to the standard concentration, and the standard deviation is small.

[0088] Comparative Example 1

[0089] (1) Control group (Au1Cu single-atom nanozyme): 100 mL of the Au1Cu single-atom nanozyme dispersion prepared in Example 1, 10 mg of polyvinylpyrrolidone and 10 mg of cetyltrimethylammonium bromide were added to 200 mL of deionized water, sonicated for 1 h and then allowed to stand overnight. The upper layer of foam was removed to obtain an aqueous dispersion of Au1Cu single-atom nanozyme. The dispersion was washed by centrifugation with deionized water, repeated three times, and then redispersed in water to form a 2 mg / mL aqueous dispersion.

[0090] A glucose solution was prepared by adding an appropriate amount of glucose to a buffer solution. 50 μL of TMB ethanol solution and 50 μL of glucose solution were added to 1900 μL of buffer solution to obtain the reaction solution (final glucose concentration 100 μM).

[0091] 100 μL of Au1Cu single-atom nanozyme aqueous dispersion was added to the reaction solution and incubated at room temperature for 1 h. The absorbance of the product solution at 652 nm was measured.

[0092] (2) Experimental group (nanozyme assembly membrane material): The nanozyme assembly membrane material from Example 3 was fixed in a vacuum filter with a ground joint size of 24#. The vacuum pump was turned on and 2 mL of reaction solution was added. The absorbance of the product solution at 652 nm was measured.

[0093] according to Figure 6It is known that Au1Cu single-atom nanozymes do not exhibit a glucose signal response during traditional incubation catalysis. However, the "flow-through" catalysis process of the nanozyme assembly membrane material in this invention exhibits a good glucose signal response, demonstrating that the assembly strategy greatly improves the catalytic performance of the nanozyme. Furthermore, the "flow-through" method of this invention can complete the detection in only 1-3 minutes, significantly shortening the detection time compared to the 1 hour required by traditional methods.

[0094] Comparative Example 2

[0095] The corresponding 100 μM solutions of the substances to be detected were prepared by adding glucose, ascorbic acid, uric acid, dopamine, glutathione, fructose, and lactic acid to the buffer solution.

[0096] 50 μL of TMB ethanol solution and 50 μL of the analyte solution were added to 1900 μL of buffer solution to obtain the reaction solution (final concentration 100 μM). The nanozyme assembly membrane material from Example 3 was fixed in a vacuum filter with a ground glass joint of size 24#. The vacuum pump was turned on, and 2 mL of the reaction solution was added. The absorbance of the product solution at 652 nm was measured.

[0097] according to Figure 7 It is known that the nanozyme assembled membrane material in this invention only shows a signal response to glucose, while showing almost no signal response to other biomolecules, which proves the good selectivity of the nanozyme assembled membrane material in blood glucose detection. This is due to the selectivity of the Au active site in the nanozyme assembled membrane material for glucose oxidation and the size selectivity of the channel in the membrane material.

[0098] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nanoenzyme assembly membrane material, characterized in that: The nanoenzyme assembly membrane material includes a base membrane and Au1Cu single-atom nanoenzymes, wherein the Au1Cu single-atom nanoenzymes are dispersed on the surface of the base membrane. The Au1Cu single-atom nanozyme has a side length of 36-84 nm for the Cu nanocubes, and the surface of the Cu nanocubes has atomically dispersed Au single atoms with a mass fraction of 2%-3% for Au. The thickness of the nanozyme assembly membrane material is 2-5 μm.

2. The nanoenzyme assembly membrane material according to claim 1, characterized in that: The pore size of the base film is 50 nm.

3. A method for preparing a nanozyme assembly membrane material as described in claim 1 or 2, characterized in that: Au1Cu single-atom nanozyme dispersion was added dropwise to the surface of the base membrane one by one. After the dispersion evaporated, the next dropwise addition was performed. The total number of drops was 10-25 to obtain the Au1Cu nanozyme assembled membrane material.

4. The preparation method according to claim 3, characterized in that: The concentration of the dispersion is 0.5-1 mg / mL.

5. The preparation method according to claim 3, characterized in that: The single drop volume of the dispersion is 200 μL.

6. A method for detecting blood glucose, characterized in that: The nanozyme assembly membrane material according to claim 1 or 2 was used for detection.

7. The method according to claim 6, characterized in that: The reaction solution is passed through the nanozyme assembled membrane material, and the absorbance of the product solution at 652 nm is detected. The glucose concentration in the test solution can be obtained by substituting the absorbance value into the standard curve. The reaction solution is a mixture of the test solution and an ethanol solution of 3,3',5,5'-tetramethylbenzidine.

8. A method for preparing the Au1Cu single-atom nanozyme as defined in claim 1, characterized in that: a. Dissolve cuprous bromide and tri-zinc-based phosphorus oxide in oleylamine, heat to 70-90℃ under inert gas protection and hold for 10-20 minutes, then continue heating the solution to 250-270℃ at a heating rate of 10-15℃ / min and hold for 0.5-1 hour to generate Cu nanocubes; b. Cool the reaction solution in step a to 200-220°C, then inject an oleylamine solution of chloroauric acid into the reaction solution and maintain it at 200-220°C for 15-30 minutes to obtain the Au1Cu single-atom nanozyme.

9. The use of the Au1Cu single-atom nanozyme as defined in claim 1 in the preparation of a blood glucose detection kit.

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