A cascade catalytic nanocomposite and a preparation method thereof, and a cholesterol detection kit

Abstract

This invention relates to the field of functional nanomaterials and their biodetection technology, and discloses a cascade catalytic nanocomposite, its preparation method, and a cholesterol detection kit. The cascade catalytic nanocomposite comprises: a metal-organic framework, copper ions doped within the metal-organic framework, and cholesterol oxidase immobilized by the metal-organic framework. The preparation method includes: preparing a mixed solution containing a zinc source and a copper source; sequentially adding a cholesterol oxidase solution and an organic ligand solution to the mixed solution, reacting to form the nanocomposite. The cholesterol detection method includes: reacting the sample to be tested with the nanocomposite and a chromogenic substrate, monitoring the color or absorbance changes of the reaction system to determine the cholesterol content. This composite integrates cholesterol oxidase and peroxidase-like activities, exhibiting excellent thermal stability and cascade catalytic efficiency.

Description

Technical Field

[0001] This invention relates to the field of functional nanomaterials and their biodetection technology, and particularly to a cascade catalytic nanocomposite and its preparation method, as well as a cholesterol detection kit. Background Technology

[0002] Cholesterol is an essential lipid molecule for life activities, constituting the cell membranes of all tissues and organs in the body. It is also an important precursor for the synthesis of various steroid hormones that regulate growth, development, and reproductive functions, as well as bile acids. Elevated blood cholesterol levels are significantly associated with the risk of atherosclerotic cardiovascular disease. The prevalence of hypercholesterolemia in my country is as high as 4.9%, and it continues to rise, projected to lead to approximately 9.2 million new cardiovascular events between 2010 and 2030. This disease has become a major public health problem that seriously threatens the health of the Chinese public and hinders the achievement of the "Healthy China" goal. However, existing cholesterol detection and analysis methods generally suffer from drawbacks such as complex operation and long response times, resulting in low early diagnosis and treatment achievement rates for this disease. Therefore, developing a novel cholesterol detection method that is highly stable, low-cost, and convenient is of great significance for the early identification and effective intervention of cardiovascular diseases.

[0003] Currently, cholesterol detection methods mainly include chromatography, colorimetry, fluorescence, and electrochemical analysis. Among these, the enzyme-colorimetric method, based on cholesterol oxidase, is the most widely used. Its principle is to utilize cholesterol oxidase to catalyze the production of hydrogen peroxide from cholesterol, and cholesterol is indirectly determined by detecting the hydrogen peroxide content. Table 1 summarizes the basic principles and reaction times of different detection methods. Fluorescence detection is relatively convenient; for example, using gold nanoclusters modified with thiol-β-cyclodextrin as a fluorescent probe, high-sensitivity cholesterol detection can be achieved in about 8 minutes, with a detection limit of 16.07 μM. However, the signal from fluorescence methods is easily interfered with by various factors, affecting detection accuracy. While colorimetric detection is more accurate, the response time generally exceeds 30 minutes. This is mainly due to the strong hydrophobicity of cholesterol and the low catalytic activity of cholesterol oxidase at room temperature, resulting in a slow catalytic reaction rate and limiting the versatility and convenience of the detection.

[0004] Table 1. Principles and testing time of cholesterol content detection methods

[0005] To address the issues of low reaction efficiency and easy enzyme inactivation in cholesterol detection methods, researchers have proposed using carriers to immobilize natural enzymes to improve their stability. For example, using ZIF-8 as a carrier, cholesterol oxidase and platinum nanozymes were loaded to construct a cascaded nanoreactor for nanozyme-enzyme-linked immunosorbent assay (ELISA), achieving high-throughput and rapid evaluation of cancer biomarkers. However, the high cost of precious metals hinders large-scale clinical and home-use applications. Low-cost transition metal ions, such as copper ions, can trigger the colorimetric reaction of 3,3',5,5'-tetramethylbenzidine mediated by hydrogen peroxide, exhibiting peroxidase-like activity. These studies offer the possibility of constructing cascade catalytic reaction systems using metal ions and cholesterol oxidase to achieve convenient and rapid colorimetric cholesterol detection. However, how to effectively integrate these ions with cholesterol oxidase and construct a highly stable cascade catalytic system remains a pressing technical challenge in the field. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a cascade catalytic nanocomposite and its preparation method, as well as a cholesterol detection kit, in order to solve the problems of poor enzyme stability, long detection time, and reliance on professional instruments and personnel in existing cholesterol detection methods.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a cascade catalytic nanocomposite comprising: a metal-organic framework, copper ions doped in the metal-organic framework, and cholesterol oxidase immobilized by the metal-organic framework; The metal-organic framework is formed by the coordination of zinc ions with organic ligands; The molar ratio of copper ions to zinc ions is (3:25). (2:15); The mass ratio of cholesterol oxidase to zinc ions is 1:5.

[0008] Optionally, the organic ligand is 2-methylimidazole.

[0009] Optionally, the copper ions are provided in the form of a copper salt, which includes one or more of copper chloride, copper nitrate, and copper sulfate.

[0010] Optionally, the zinc ions are provided in the form of zinc salts, which include one or more of zinc nitrate, zinc acetate, and zinc sulfate.

[0011] A second aspect of the present invention provides a method for preparing a cascaded catalytic nanocomposite, the method comprising the following steps: Prepare a mixed solution containing a zinc source and a copper source; Cholesterol oxidase solution and organic ligand solution were added sequentially to the mixed solution at 20°C. The reaction was carried out at 30 °C, and a metal-organic framework immobilized with the cholesterol oxidase and doped with the copper ions was formed through in-situ self-assembly, thus obtaining the cascade catalytic nanocomposite.

[0012] Optionally, the reaction time is 0.5 seconds. 2 hours.

[0013] A third aspect of the present invention provides a cholesterol detection kit comprising the above-described cascade catalytic nanocomposite.

[0014] A fourth aspect of the present invention provides a method for detecting cholesterol, the method using the above-described cascade catalytic nanocomposite, comprising the following detection steps: The cholesterol sample to be tested is reacted with the cascaded catalytic nanocomposite and the chromogenic substrate; Monitor the color or absorbance changes of the reaction system, and determine the cholesterol content in the cholesterol sample to be tested based on the changes.

[0015] Optionally, the contact reaction is carried out at a temperature of 60°C for 5 minutes.

[0016] Optionally, the chromogenic substrate includes one or more of 3,3',5,5'-tetramethylbenzidine, o-phenylenediamine, and 3,3'-diaminobenzidine.

[0017] Optionally, the step of monitoring the color change of the reaction system specifically includes: acquiring an image of the reaction system, extracting the RGB color channel values ​​of the image, and determining the cholesterol content in the cholesterol sample to be tested based on a preset correspondence between the RGB color channel values ​​and cholesterol concentration.

[0018] The present invention has the following beneficial effects: This invention provides a cascaded catalytic nanocomposite, its preparation method, and a cholesterol detection kit. The invention constructs an integrated cascaded catalytic nanocomposite by in-situ immobilizing cholesterol oxidase within a metal-organic framework (MOF) formed by the coordination of zinc ions and organic ligands, while simultaneously doping this framework with copper ions during its synthesis. This MOF, through its spatial confinement effect, protects the internal cholesterol oxidase, effectively inhibiting the unfolding and inactivation of the enzyme protein structure at high temperatures, significantly improving the structural stability and catalytic activity of cholesterol oxidase under high-temperature conditions. Simultaneously, the uniformly doped copper ions within the framework exert a peroxidase-like catalytic function. During detection, the hydrogen peroxide produced by cholesterol oxidase is immediately catalyzed by neighboring copper ions, used to oxidize the chromogenic substrate and generate a color signal. This tightly integrated cascaded catalytic pathway significantly shortens the reaction time. Because the high-temperature stability of enzyme activity is guaranteed, the entire detection process can be carried out at higher temperatures, further accelerating the reaction kinetics. Therefore, this cascaded catalytic nanocomposite successfully solves the problems of poor enzyme thermal stability and slow reaction rates in traditional enzymatic detection methods, achieving rapid and stable cholesterol detection. In addition, this method can also quantify by monitoring changes in the color or absorbance of the reaction system. Combined with smartphone image analysis technology, it makes the detection operation simpler and faster, and the results are intuitive and visible, making it suitable for home and on-site rapid screening scenarios. Attached Figure Description

[0019] Figure 1 Transmission electron microscope morphology of the cascaded catalytic nanocomposite provided in an embodiment of the present invention.

[0020] Figure 2 The X-ray diffraction pattern of the cascaded catalytic nanocomposite provided in the embodiments of the present invention.

[0021] Figure 3 X-ray photoelectron spectroscopy of the cascaded catalytic nanocomposite provided in an embodiment of the present invention.

[0022] Figure 4 The image shows a circular dichroism chromatogram of free cholesterol oxidase and cascade catalytic nanocomposite.

[0023] Figure 5 This is a comparison of the thermal stability of free cholesterol oxidase and cholesterol oxidase immobilized in a cascade catalytic nanocomposite.

[0024] Figure 6 The graph shows the change in absorbance of the reaction system at a wavelength of 453.7 nm over time during the catalytic cholesterol reaction of the cascade catalytic nanocomposite.

[0025] Figure 7This is a comparison of the storage stability of solution-state free cholesterol oxidase and cascade catalytic nanocomposite.

[0026] Figure 8 This is a comparison of the enzyme activity and stability of powdered free cholesterol oxidase and cascade catalytic nanocomposite.

[0027] Figure 9 The image shows a circular dichroism chromatogram of powdered free cholesterol oxidase and cascade catalytic nanocomposite.

[0028] Figure 10 The figure shows the substrate selectivity test results for the cascaded catalytic nanocomposite.

[0029] Figure 11 This is a standard curve for cholesterol detection based on cascade catalytic nanocomposites.

[0030] Figure 12 This is an example diagram illustrating the application effect of the intelligent output system in the detection method of the present invention. Detailed Implementation

[0031] This invention provides a cascade catalytic nanocomposite and its preparation method, as well as a cholesterol detection kit. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0032] The cascaded catalytic nanocomposite provided by this invention can serve as a core detection element for rapid cholesterol screening in homes, communities, or resource-limited areas. Convenient, rapid, and low-cost detection can be achieved through simple colorimetric reactions and image analysis techniques.

[0033] This invention provides a cascade catalytic nanocomposite, which includes: a metal-organic framework, copper ions doped in the metal-organic framework, and cholesterol oxidase immobilized by the metal-organic framework; The metal-organic framework is formed by the coordination of zinc ions with organic ligands; The molar ratio of copper ions to zinc ions is (3:25). (2:15); The mass ratio of cholesterol oxidase to zinc ions is 1:5.

[0034] This design utilizes the spatial confinement effect of a metal-organic framework to provide physical protection for the internal cholesterol oxidase, effectively inhibiting the unfolding and inactivation of the enzyme protein structure at high temperatures. Simultaneously, copper ions uniformly doped within the framework function as peroxidase-like catalytic agents. During detection, the hydrogen peroxide generated from the cholesterol reaction catalyzed by cholesterol oxidase can be efficiently utilized by neighboring copper ions to oxidize the chromogenic substrate and generate a color signal, thus constructing an efficient and compact cascade catalytic pathway. This synergistic effect solves the problems of poor thermal stability of cholesterol oxidase and the difficulty in stable integration of free enzymes and enzyme-like catalysts, leading to low cascade reaction efficiency in previous technologies, enabling rapid and stable detection of cholesterol at high temperatures.

[0035] When the ratio of copper ions, zinc ions, and cholesterol oxidase is within a defined range, the material is uniform in size, the enzyme conformation is intact, and it exhibits excellent catalytic performance and stability.

[0036] In some embodiments, the organic ligand is preferably 2-methylimidazole.

[0037] When the organic ligand is 2-methylimidazole, it can rapidly self-assemble with zinc ions to form a ZIF-8 type metal-organic framework. This framework has good biocompatibility and is easy to synthesize. Its pore size is suitable for achieving in-situ efficient immobilization of cholesterol oxidase under mild conditions, while forming a stable crystal structure for copper ion doping.

[0038] In some embodiments, the copper ions are provided in the form of copper salts, which include one or more of copper chloride, copper nitrate, and copper sulfate.

[0039] In some embodiments, the zinc ions are provided in the form of zinc salts, which include one or more of zinc nitrate, zinc acetate, and zinc sulfate.

[0040] Using these soluble salts facilitates the formation of a homogeneous mixture of metal precursors in solution, ensuring that copper ions can be uniformly doped into the metal-organic framework during subsequent self-assembly reactions.

[0041] This invention provides a method for preparing a cascaded catalytic nanocomposite, the method comprising the following steps: Prepare a mixed solution containing a zinc source and a copper source; Cholesterol oxidase solution and organic ligand solution were added sequentially to the mixed solution at 20°C. The reaction was carried out at 30°C, and a metal-organic framework immobilized with the cholesterol oxidase and doped with the copper ions was formed through in-situ self-assembly, thus obtaining the cascade catalytic nanocomposite.

[0042] In some embodiments, the reaction time is 0.5 seconds. 2 hours.

[0043] Specifically, the preparation method of the cascaded catalytic nanocomposite includes the following steps: Step S1, preparing the mixed solution: Dissolve the zinc salt (as the zinc source) and the copper salt (as the copper source) in deionized water to prepare a mixed solution containing zinc ions and copper ions. The molar ratio of copper ions to zinc ions is (3:25). (2:15); Step S2, in-situ self-assembly reaction: A certain concentration of cholesterol oxidase solution is first added to the mixed solution obtained in step S1, followed by the rapid addition of an organic ligand solution (e.g., 2-methylimidazole solution). The mixture is placed in an environment of 20-30°C, and incubated at 200... The reaction mixture was stirred at 400 rpm for 0.5 seconds. Two hours. During this process, zinc and copper ions undergo coordination self-assembly with organic ligands, simultaneously immobilizing cholesterol oxidase in solution and forming a metal-organic framework, with copper ions also being incorporated into the framework simultaneously. This one-step synthesis process is simple and efficient, ensuring tight integration of the enzyme and catalytic site.

[0044] Step S3, Post-treatment: Centrifuge the mixture from step S2 at 4°C and 10,000 rpm for 10 minutes to collect the precipitate. Wash the precipitate with deionized water. The reaction was repeated three times to remove unreacted raw materials and free enzymes, and finally, the powdered cascade catalytic nanocomposite was obtained by freeze drying.

[0045] This invention provides a cholesterol detection kit, which comprises the above-mentioned cascade catalytic nanocomposite.

[0046] This invention provides a method for detecting cholesterol, which uses the aforementioned cascade catalytic nanocomposite and includes the following detection steps: The cholesterol sample to be tested is reacted with the cascaded catalytic nanocomposite and the chromogenic substrate; Monitor the color or absorbance changes of the reaction system, and determine the cholesterol content in the cholesterol sample to be tested based on the changes.

[0047] In some embodiments, the contact reaction is carried out at a temperature of 60°C for a duration of 5 minutes.

[0048] In some embodiments, the chromogenic substrate includes one or more of 3,3',5,5'-tetramethylbenzidine, o-phenylenediamine, and 3,3'-diaminobenzidine.

[0049] In some embodiments, the step of monitoring the color change of the reaction system specifically includes: acquiring an image of the reaction system, extracting the RGB color channel values ​​of the image, and determining the cholesterol content in the cholesterol sample to be tested based on a preset correspondence between the RGB color channel values ​​and cholesterol concentration.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0051] The following detailed description uses specific examples.

[0052] Example 1: Preparation and Characterization of Cascade Catalytic Nanocomposites Prepare a copper-zinc mixed solution by adding 2-MIM solution (308 mg / mL), ChOx solution (4 mg / mL), and deionized water to the copper-zinc mixed solution (zinc nitrate 12 mg / mL; copper chloride 1.808 mg / mL). The volume ratio of the copper-zinc mixed solution, 2-MIM solution, ChOx solution, and deionized water is 5:5:4:6. Stir the mixture at 300 rpm for 1 hour at room temperature. Centrifuge the resulting mixture at 10,000 rpm and 4℃ for 10 min, repeating this process three times. Collect the precipitate to obtain the cascade catalytic nanocomposite ChOx@CuZIF-8 (denoted as CCZ).

[0053] Comparative Example 1: Preparation of the composite without copper ions The preparation process was the same as in Example 1, except that an equimolar amount of zinc nitrate solution was used instead of the copper-zinc mixed solution to obtain a metal-organic framework complex ChOx@ZIF-8 (denoted as CZ) that only immobilizes cholesterol oxidase.

[0054] Figure 1 The prepared CCZ nanoparticles have a particle size of approximately 60 nm. Figure 2 This indicates that CCZ encapsulation of ChOx maintains the crystal structure of ZIF-8. Figure 3 The successful doping of Cu in CCZ has been confirmed. 2+ Ions. The protein secondary structure of CCZ was analyzed using circular dichroism spectroscopy, such as... Figure 4 As shown, typical α-helix and β-sheet signals appeared at 192 nm and 215 nm, respectively, which are consistent with the spectral characteristics of the free enzyme, indicating that the secondary structure of the enzyme remained intact during synthesis and no obvious conformational changes occurred.

[0055] Example 2: Temperature and reaction kinetics for CCZ cholesterol determination Prepare a series of cholesterol solutions of different concentrations (using PBS to prepare the detection solution, with concentrations of 0, 0.1, 0.2, 0.5, 1.0, and 1.5 mM). Take 1 mL of cholesterol solution (1 mM), add 10 μL of CCZ solution and 100 μL of TMB chromogenic substrate solution (10 mM), and react at room temperature (25℃) for 10 min.

[0056] In Comparative Example 1, each reaction solution was consistently supplemented with a mixture of ChOx solution (3.5 mg / mL), TMB solution (10 mM), and HRP solution (0.01 mg / mL), and heated to 25°C, 37°C, 50°C, and 65°C for 10 min. Using the same enzyme concentration as a baseline, the CCZ group was supplemented with (4 mg / mL), with all other conditions identical for comparison. Results are as follows: Figure 5 The optimal reaction temperature of CCZ is significantly increased to 60℃ compared to the free enzyme, and CCZ endows ChOx with better thermal stability.

[0057] The reaction kinetics of CCZ at 60℃ were further investigated, and the results are as follows: Figure 6 The absorbance at 453.7 nm was measured every 30 seconds, showing that the reaction time was shortened to 5 min, confirming that increasing the reaction temperature can significantly accelerate the catalytic rate of CCZ.

[0058] Based on Example 2, 60°C was selected as the reaction temperature and 5 min as the reaction time.

[0059] Example 3: Stability detection of free and immobilized enzymes 10 μL of free ChOx solution (3.5 mg / mL) was added to 1 mL of cholesterol solution with the same concentration as in Example 2. After reacting in a 60°C water bath for 5 min, the absorbance was measured at 453.7 nm. Under the same reaction system and conditions as in Example 2, the enzyme activity stability of the free enzyme, CZ, and CCZ groups was compared. CZ was used instead of free ChOx for detection to evaluate the Cu content in CCZ. 2+ The catalytic activity was observed. For the detection of CCZ samples, the cholesterol concentration and TMB addition were kept consistent, but no additional HRP enzyme was added.

[0060] After storing the free enzyme, CZ, and CCZ groups at room temperature for a certain period of time, enzyme activity was tested.

[0061] Based on Michaelis-Menten equation The reaction kinetics of the catalytic processes of free enzyme, CZ, and CCZ were analyzed, and the enzyme activity retention rates (%) of free enzyme, CZ, and CCZ were compared based on vmax. The results are as follows: Figure 7 (A), (B), (C).

[0062]

[0063] Under the same reaction conditions, CCZ consistently exhibited significantly higher catalytic activity than the free enzyme at the same time point. After a 6-day storage period, the activity of CCZ showed no significant change, while the free enzyme almost completely lost its catalytic activity. This comparison directly confirms that CCZ encapsulation can protect ChOx from inactivation caused by RT storage, significantly improving enzyme stability.

[0064] Further investigation was conducted into the free enzymes and CCZ activities in lyophilized powder form at different storage times. For example... Figure 8 As shown, free ChOx lyophilized powder lost its catalytic activity after being stored at room temperature for 6 days, while the catalytic activity of CCZ lyophilized powder after being stored at room temperature for 50 days was not significantly different from that in its initial state. From a structural biology perspective, the catalytic function of enzymes is highly dependent on the integrity and correctness of their spatial conformation: the secondary structure is the skeletal basis for maintaining the stability of the tertiary structure, and the correct folding of the tertiary structure is a prerequisite for the formation of a functional active site. Figure 9 The results showed that the free enzyme ChOx underwent significant changes in circular dichroism chromatograms after 6 days of storage at room temperature, indicating that its regular secondary structures, such as α-helices and β-sheets, had been disrupted, and the overall protein conformation tended towards disorder. In contrast, the secondary structure of the solidified CCZ protein remained unchanged after long-term storage (over 50 days) following lyophilization. The ZIF-8 in this invention may form multi-point contacts with the peptide backbone and side chains of the enzyme protein through its abundant surface interaction sites. These interactions provide conformational support for the peptide chain, effectively inhibiting unwinding or misfolding caused by thermal motion, thereby maintaining the stability of the secondary structure at the molecular level. This encapsulation effect also exhibits selective permeability, allowing the enzyme to contact and catalyze cholesterol molecules while stabilizing the overall enzyme conformation. This dynamic stabilization mechanism, while maintaining structural stability, ensures the exposure of the ChOx active site and the preservation of the catalytic microenvironment, ultimately achieving long-term maintenance of the functional integrity of ChOx.

[0065] In summary, in Example 3, CCZ significantly improved the stability of ChOx, and the preservation effect of freeze-dried storage on enzyme activity was far better than that of solution.

[0066] Example 4: Substrate selectivity of CCZ 1 mM solutions of interfering agents were prepared, including calcium chloride (CaCl2), magnesium chloride (MgCl2), sodium chloride (NaCl), D-glucose, glycine, urea, PBS solution, and cholesterol solution, with 1 mL of each solution. 100 μL of 10 mM TMB solution and 10 μL of CCZ (4 mg / mL) sample solution were added to each solution, mixed well, and reacted at 60 °C for 5 min. After the reaction, the absorbance of each reaction solution was measured at 453.7 nm. The absorbance of the cholesterol group was used as a reference to compare and analyze the response signals of other interfering agent groups, thereby evaluating the specificity and anti-interference ability of CCZ for cholesterol detection. Figure 10 This indicates that CCZ exhibits significant catalytic activity towards cholesterol, while showing no significant catalytic effect on common interfering substances in the samples, such as CaCl2, MgCl2, NaCl, D-glucose, glycine, and urea, demonstrating excellent substrate selectivity and specificity. This good selectivity lays the foundation for the application of CCZ in complex biological samples.

[0067] Example 5: Application of CCZ in rapid cholesterol detection Prepare a series of cholesterol solutions at different concentrations (using PBS to prepare the detection solution, with concentrations of 0, 0.1, 0.2, 0.25, 0.5, 1.0, 1.5, and 3.0 mM). Take 1 mL of each cholesterol solution at different concentrations and add 50 μL of CCZ sample (concentration 4 mg / mL). React at a constant temperature of 60℃. After 5 minutes of reaction, measure the absorbance at λ=453.7 nm, record the correlation between absorbance and cholesterol concentration, and plot a standard curve. Calculate the linear regression equation based on the standard curve, and determine the limit of detection (LOD) of the system by combining the three-times standard deviation (3σ) and slope (k) of the blank group signal, to evaluate the detection sensitivity. The results are as follows: Figure 11 The absorbance of the cholesterol reaction solution at λ=453.7 nm increased significantly with increasing cholesterol concentration, and the absorbance showed a good linear relationship with cholesterol concentration (Y = 0.5023*X + 0.05655, R). 2 =0.9926 (unit: mM). The cholesterol detection limit was extrapolated from the standard curve to be 0.0038 mM.

[0068] 1.5 mM standard cholesterol solution, dairy product samples, and mouse serum samples were tested. The results output by the ELISA reader (as shown in Table 2) prove that CCZ can accurately determine the cholesterol content in different samples.

[0069] Table 2 Analysis of Cholesterol Content Measurement Results by CCZ

[0070] Example 6: Image Signal Acquisition and Regression Model Construction To achieve rapid visualization and health assessment of CCZ detection data, this invention constructs an intelligent output system based on image recognition and multiple linear regression. The specific process is as follows: In the PyCharm platform, images of different cholesterol concentration groups after the reaction are processed, and the RGB color values ​​of the reaction solution are extracted. Based on this analysis result, a multiple linear regression model between RGB values ​​and absorbance is established in Matlab: (R) 2 =0.9975). This regression model was integrated into the PyCharm platform to develop a visual interface system that outputs detection results based on image input. An example of its operation is shown below. Figure 12 The results from the ELISA reader and the intelligent system were 2.761 mM and 2.748 mM, respectively, with a relative error of 0.47%. The test results for the remaining samples are shown in Table 2. The concentration calculated by the intelligent system is close to the concentration detected by the ELISA reader, indicating that the intelligent output system can be used to determine the cholesterol content in most samples. This system can quickly, conveniently, and cost-effectively achieve cholesterol detection and can be applied to home or primary screening scenarios.

[0071] In summary, this invention achieves cascade catalysis of cholesterol oxidation and colorimetric substrate oxidation. CCZ not only significantly improves the reaction efficiency and stability of the free enzyme, but also reduces detection costs by utilizing the catalytic activity of copper ions. By combining a multiple linear regression model of the RGB values ​​and absorbance of the reaction system, an intelligent detection system was developed, enabling rapid and accurate determination and visualization of cholesterol content, providing a new strategy for portable and rapid cholesterol detection.

[0072] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A cascaded catalytic nanocomposite, characterized in that, The cascaded catalytic nanocomposite comprises: a metal-organic framework, copper ions doped in the metal-organic framework, and cholesterol oxidase immobilized by the metal-organic framework; The metal-organic framework is formed by the coordination of zinc ions with organic ligands.

2. The cascaded catalytic nanocomposite according to claim 1, characterized in that, The molar ratio of copper ions to zinc ions is (3:25). (2:15); the mass ratio of the cholesterol oxidase to the zinc ions is 1:

5.

3. The cascaded catalytic nanocomposite according to claim 1, characterized in that, The organic ligand is 2-methylimidazole; the copper ions are provided in the form of copper salts, which include one or more of copper chloride, copper nitrate, and copper sulfate; the zinc ions are provided in the form of zinc salts, which include one or more of zinc nitrate, zinc acetate, and zinc sulfate.

4. A method for preparing the cascaded catalytic nanocomposite according to any one of claims 1-3, characterized in that, The preparation method includes the following steps: Prepare a mixed solution containing a zinc source and a copper source; Cholesterol oxidase solution and organic ligand solution are added sequentially to the mixed solution, and the reaction is carried out at 20-30 °C. The metal-organic framework with cholesterol oxidase immobilized and copper ions doped is formed through in-situ self-assembly, thus obtaining the cascade catalytic nanocomposite.

5. The method for preparing the cascaded catalytic nanocomposite according to claim 4, characterized in that, The reaction time is 0.5-2 hours.

6. A cholesterol detection kit, characterized in that, The cholesterol detection kit comprises the cascade catalytic nanocomposite as described in any one of claims 1-3.

7. A method for detecting cholesterol, characterized in that, The detection method uses the cascaded catalytic nanocomposite according to any one of claims 1-3, and includes the following detection steps: The cholesterol sample to be tested is reacted with the cascaded catalytic nanocomposite and the chromogenic substrate; Monitor the color or absorbance changes of the reaction system, and determine the cholesterol content in the cholesterol sample to be tested based on the changes.

8. The method for detecting cholesterol according to claim 7, characterized in that, The contact reaction was carried out at a temperature of 60°C for 5 minutes.

9. The method for detecting cholesterol according to claim 7, characterized in that, The chromogenic substrate includes one or more of 3,3',5,5'-tetramethylbenzidine, o-phenylenediamine, and 3,3'-diaminobenzidine.

10. The method for detecting cholesterol according to claim 7, characterized in that, The step of monitoring the color change of the reaction system specifically includes: acquiring an image of the reaction system, extracting the RGB color channel values ​​of the image, and determining the cholesterol content in the cholesterol sample to be tested based on a preset correspondence between the RGB color channel values ​​and cholesterol concentration.

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