A reagent for analyzing multiple markers in blood based on cascade enzyme catalytic reaction, and a preparation method and application thereof

Abstract

The application discloses a kind of based on cascade enzyme catalytic reaction in blood multiple marker analysis reagent and its preparation method and application.The analysis reagent includes reagent F and includes reagent A, reagent B, reagent C1, reagent C2, reagent D, reagent E one or several;Wherein, reagent F is the nanoenzyme of NF@Co-ZIF-L with peroxidase-like activity, and the multiple markers include at least one of cholesterol, lactic acid, uric acid, glucose.The multiple marker analysis reagent based on cascade enzyme catalytic reaction prepared by the application can quickly realize the detection of multiple markers such as glucose, lactic acid, cholesterol and uric acid in blood, and meets the actual demand of personalized diagnosis.Through the synergistic effect of cascade enzyme catalytic reaction and NF@Co-ZIF-L with peroxidase-like activity, the biological markers to be measured are converted into fluorescent signal, and the sensitivity of detection is improved.

Description

Technical Field

[0001] This invention belongs to the field of serum analysis technology, specifically relating to a fluorescent analytical reagent for multiple biomarkers in blood based on a cascade enzyme catalytic reaction, its preparation method, and its application. Background Technology

[0002] For some metabolic diseases, such as diabetes, hypertension, and dyslipidemia, especially in a home environment, frequent monitoring of relevant biomarkers is necessary to rationally control diet and lifestyle, and adjust treatment strategies. For example, serum cholesterol (Chol) levels affect the occurrence of cerebral thrombosis and arteriosclerosis; for every 1% decrease in serum cholesterol levels, the risk of these diseases decreases by approximately 2%. Uric acid (UA) monitoring is of great significance for assessing the treatment effect of gout patients and preventing gout recurrence. Serum lactate (LA) is closely related to a variety of serious diseases such as ischemia, infection, and traumatic shock. Importantly, simultaneous detection of multiple biomarkers better meets the practical needs of personalized diagnosis. For example, many metabolic diseases require combined measurement of cholesterol and glucose. Therefore, developing detection reagents with high sensitivity for multiple biomarkers in the blood is of great significance.

[0003] In the comparison of the kinetic properties of peroxidase-like active materials, natural horseradish peroxidase (HRP), although possessing classic catalytic activity, has limited affinity for substrates (K_m for OPD is 7.3 mM); Zhao et al. (Zhao, C., Xiong, C., Liu, X., Qiao, M., Li, Z., Yuan, T., Wang, J., Qu, Y., Wang, X., Zhou, F., Xu, Q., Wang, S., Chen, M., Wang, W., Li, Y., Yao, T., Wu, Y., & Li, Y. (2019). Unraveling the enzyme-like activity of heterogeneous singleatom catalyst. Chemical Communications(55, 2285–2288.) developed single-atom iron catalysts (FeSAEs) to mimic the catalytic function of natural heme-containing enzymes, achieving excellent enzyme-like activity (K_m of 0.572 mM for OPD); Kulandaivel et al. (Kulandaivel, S., Lin, C.-H., & Yeh, Y.-C. (2022). The bi-metallic MOF-919 (Fe-Cu) nanozyme capable of bifunctional enzyme-mimicking catalytic activity. Chemical Communications (He, N., Zhu, X., Liu, F., Yu, R., Xue, Z., & Liu, X. (2022). Rational design of FeS2-encapsulated covalentorganic frameworks as stable and reusable nanozyme for dual-signal detection glutathione in cell lysates. Chemical Engineering Journal, 445, 136543.) developed MIL-100(Fe) nanozyme to mimic peroxidase activity, achieving catalytic oxidation of organic substrates (Km of 1.06 mM for OPD). In contrast, the NF@Co-ZIF-L nanozyme developed in this invention, used for the immediate detection of four metabolic markers in serum—cholesterol, lactic acid, uric acid, and glucose—achieves superior results due to its lowest Michaelis constant (Km = 0.12 mM) for OPD and its high maximum reaction rate (Vmax = 0.829 × 10⁻⁶). -7 M·s -1 It exhibits superior substrate affinity compared to the aforementioned control materials and natural enzymes. Furthermore, by combining a smartphone app with artificial neural network algorithms to collaboratively analyze the RGB and HSV parameters of fluorescence images, it achieves accurate offline quantitative detection of multiple targets without the need for large instruments and professional operators, demonstrating significant comprehensive advantages in terms of convenience, detection throughput, and intelligent data processing. Summary of the Invention

[0004] To address the issues of low sensitivity and difficulty in detecting multiple targets in existing methods, this invention targets four substances: cholesterol, lactic acid, uric acid, and glucose. Quantitative analysis of these targets relies on the in-situ synthesis of a cobalt-based foliated zeolite imidazole acid framework (Co-ZIF-L) with peroxidase-like activity on nickel foam, using the corresponding oxidases. In this process, the oxidase recognizes specific target molecules and catalyzes the oxidation reaction to generate H₂O₂. Then, the peroxidase-like Co-ZIF-L catalyzes the oxidation of o-phenylenediamine (OPD) by H₂O₂, generating a highly fluorescent substrate, 2,3-diaminopiperazine (OPDox). Based on this, the biomarker to be tested can be converted into a fluorescent signal of OPDox.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows.

[0006] This invention provides a reagent for analyzing multiple biomarkers in blood based on a cascade enzyme catalytic reaction. The reagent includes reagent F and one or more of reagents A, B, C1, C2, D, and E.

[0007] Specifically, Reagent A is an ultrapure aqueous solution of glucose oxidase with a concentration of 1.0 ± 0.1 mg / mL. -1 ; Reagent B is an ultrapure aqueous solution of lactate oxidase with a concentration of 1.0 ± 0.1 mg / mL. -1 ; Reagent C1 is an ultrapure aqueous solution of cholesterol oxidase with a concentration of 1.0 ± 0.1 mg / mL. -1 ; Reagent C2 is an ultrapure aqueous solution of cholesterol lipase with a concentration of 1.0 ± 0.1 mg / mL. -1 ; Reagent D is an ultrapure aqueous solution of uricase with a concentration of 1.0 ± 0.1 mg / mL. -1 ; Reagent E is an ultrapure aqueous solution of o-phenylenediamine with a concentration of 10.0 ± 1.0 mM; Reagent F is an NF@Co-ZIF-L nanozyme with peroxidase-like activity.

[0008] The NF@Co-ZIF-L nanozyme was synthesized in water from nickel foam and ZIF-L precursor, and the preparation method is as follows: First, a certain area of ​​nickel foam was washed successively with 3M hydrochloric acid, ethanol, and deionized water, and then dried for later use. Next, 1.1817-1.4443 g of 2-methylimidazole (C4H6N2) and 0.524-0.640 g of cobalt nitrate hexahydrate (Co(NO3)2•6H2O) were added to 49.0-51.0 mL of deionized water and stirred until dissolved. Then, the two components were mixed and stirred for 30 seconds to form a purple solution. The nickel foam (NF) substrate was then rapidly immersed in the purple solution at room temperature for 60 minutes. Finally, the synthesized NF@Co-ZIF-L was washed with ethanol and deionized water, dried, and cut into 1.0 cm × 1.0 cm cubes for later use.

[0009] The mass ratio of 2-methylimidazole to cobalt nitrate hexahydrate is 2.2 ± 0.1 : 1.0. More preferably, it is 2.2:1.

[0010] The present invention also provides an application of the above-mentioned blood biomarker analysis reagent based on cascade enzyme catalysis reaction, for rapid detection of multiple target substances in blood, including but not limited to cholesterol, lactic acid, uric acid, glucose, etc.

[0011] 1. Glucose detection: The method for establishing the standard curve is as follows: First, 100 μL of glucose solutions of different concentrations (0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 mM) were mixed with 10 μL of reagent A and incubated at 37°C for 30 minutes. Then, 100 μL of OPD reagent E and 790 μL of phosphate-buffered saline (PBS, 0.02 M, pH 7.4) were added to the above solution. Next, reagent F was added to the solution. Finally, after reacting in the dark at room temperature for 30 minutes, the above solution was transferred to a quartz tube, and fluorescence was measured under 360 nm excitation to establish the standard curve.

[0012] When testing blood samples, take 100 μL of serum and follow the above procedure to measure fluorescence intensity and calculate the glucose concentration in serum based on the standard curve.

[0013] 2. Lactic acid, cholesterol, and uric acid detection: First, incubate 100 μL of different concentrations of lactic acid, cholesterol, and uric acid with 10 μL of reagent B, reagent C1, and reagent D, respectively, at 37°C for 30 minutes. Then, perform other procedures according to the glucose detection method described above, i.e., establish a standard curve based on the fluorescence signal, and then calculate the specific sample concentration by measuring the sample fluorescence signal.

[0014] This invention provides a blood biomarker analysis reagent based on a cascade enzyme catalytic reaction. By utilizing a metabolic biomarker cascade reaction, an NF@Co-ZIF-L with peroxidase-like activity is synthesized. The H2O2 oxidizes OPD (o-phenylenediamine) catalyzed by this substance to produce a highly fluorescent substrate OPDox, thereby achieving highly sensitive determination of the target biomarker.

[0015] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: (1) The blood multi-marker analysis reagent based on cascade enzyme catalysis of the present invention can quickly detect multiple markers such as glucose, lactic acid, cholesterol and uric acid in blood (in vitro detection), which meets the actual needs of personalized diagnosis.

[0016] (2) Through the synergistic effect of cascade enzyme catalysis and NF@Co-ZIF-L with peroxidase-like activity, the biomarker to be tested is converted into a fluorescent signal, which improves the detection sensitivity. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the metabolic biomarker cascade reaction catalyzed by the corresponding oxidase and NF@Co-ZIF-L.

[0018] Figure 2 Low- and high-magnification scanning electron microscope images of NF@Co-ZIF-L.

[0019] Figure 3 This is a photograph of cholesterol at different concentrations reacting with reagents based on cascade enzyme catalysis under 365nm UV light irradiation.

[0020] Figure 4 This is a photograph of lactic acid at different concentrations reacting with reagents based on cascade enzyme catalysis under 365nm UV light irradiation.

[0021] Figure 5 This is a photograph of uric acid at different concentrations reacting with reagents based on cascade enzyme catalysis under 365nm UV light irradiation.

[0022] Figure 6 This is a photograph of glucose at different concentrations reacting with reagents based on cascade enzyme catalysis under 365nm UV light irradiation.

[0023] Figure 7 To obtain working curves for determining different concentrations of biomarkers using reagents based on cascade enzyme catalysis.

[0024] Figure 8 The image shows the fluorescence of serum diluted 10-fold with a cascade enzyme-based catalytic reaction assay reagent. Detailed Implementation

[0025] The method of the present invention will be described below through specific embodiments, but the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0026] To further understand the present invention, preferred embodiments of the present invention will be described below in conjunction with specific implementation methods.

[0027] Example 1 (1) Preparation of reagent F: First, a certain area of ​​nickel foam was washed successively with 3M hydrochloric acid, ethanol, and deionized water, and then dried for later use. Next, 1.313 g of 2-methylimidazole (C4H6N2) and 0.582 g of cobalt nitrate hexahydrate (Co(NO3)2•6H2O) were added to 50 mL of deionized water and stirred until dissolved. Then, the two components were mixed and stirred for 30 seconds to form a purple solution. The nickel foam (NF) substrate was then rapidly immersed in the purple solution at room temperature for 60 minutes. Finally, the synthesized NF@Co-ZIF-L was washed with ethanol and deionized water, dried, and cut into 1.0 cm × 1.0 cm cubes for later use.

[0028] (2) Calibration of the standard curve First, 100 μL of cholesterol at different concentrations (0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 mM) was mixed with 10 μL of reagent C1 and incubated at 37°C for 30 minutes. Then, 100 μL of OPD reagent E and 790 μL of phosphate-buffered saline (PBS, 0.02 M, pH 7.4) were added to the above solution. Next, reagent F was added to the solution. Finally, after reacting in the dark at room temperature for 30 minutes, the solution was transferred to a quartz tube, and fluorescence was measured under 360 nm excitation to establish a standard curve.

[0029] When detecting cholesterol, 100 μL of diluted sample was pre-treated with reagent C2 to hydrolyze cholesterol esters into free cholesterol, and then incubated with 10 μL of reagent C1 at 37°C for 30 minutes.

[0030] (3) Add 100 μL of OPD reagent E and 790 μL of phosphate-buffered saline (PBS, 0.02 M, pH 7.4) to the above solution. Then, add reagent F to the solution. Finally, after reacting in the dark at room temperature for 30 minutes, transfer the above solution to a quartz tube and measure the fluorescence under 360 nm excitation to establish a standard curve. When testing blood samples, take 100 μL of serum and follow the above procedure to measure the fluorescence intensity. Calculate the cholesterol concentration in the serum based on the standard curve.

[0031] Example 2 (1) Preparation of reagent F: First, a certain area of ​​nickel foam was washed successively with 3M hydrochloric acid, ethanol, and deionized water, and then dried for later use. Next, 1.313 g of 2-methylimidazole (C4H6N2) and 0.582 g of cobalt nitrate hexahydrate (Co(NO3)2•6H2O) were added to 50 mL of deionized water and stirred until dissolved. Then, the two components were mixed and stirred for 30 seconds to form a purple solution. The nickel foam (NF) substrate was then rapidly immersed in the purple solution at room temperature for 60 minutes. Finally, the synthesized NF@Co-ZIF-L was washed with ethanol and deionized water, dried, and cut into 1.0 cm × 1.0 cm cubes for later use.

[0032] (2) Calibration of the standard curve First, 100 μL of glucose solutions of different concentrations (0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 mM) were mixed with 10 μL of reagent A and incubated at 37°C for 30 minutes. Then, 100 μL of OPD reagent E and 790 μL of phosphate-buffered saline (PBS, 0.02 M, pH 7.4) were added to the above solution. Next, reagent F was added to the solution. Finally, after reacting in the dark at room temperature for 30 minutes, the solution was transferred to a quartz tube, and fluorescence was measured under 360 nm excitation to establish a standard curve.

[0033] When detecting glucose, incubate with 10 μL of reagent A at 37°C for 30 minutes.

[0034] (3) Add 100 μL of OPD reagent E and 790 μL of phosphate-buffered saline (PBS, 0.02 M, pH 7.4) to the above solution. Then, add reagent F to the solution. Finally, after reacting in the dark at room temperature for 30 minutes, transfer the above solution to a quartz tube and measure the fluorescence under 360 nm excitation to establish a standard curve. When testing blood samples, take 100 μL of serum and follow the above procedure to measure the fluorescence intensity. Calculate the glucose concentration in the serum based on the standard curve.

[0035] Example 3 A method for preparing analytical reagents for multiple biomarkers in blood based on cascade enzyme catalysis includes the following steps: (1) Preparation of reagent F: First, a certain area of ​​nickel foam was washed successively with 3M hydrochloric acid, ethanol, and deionized water, and then dried for later use. Next, 1.313 g of 2-methylimidazole (C4H6N2) and 0.582 g of cobalt nitrate hexahydrate (Co(NO3)2•6H2O) were added to 50 mL of deionized water and stirred until dissolved. Then, the two components were mixed and stirred for 30 seconds to form a purple solution. The nickel foam (NF) substrate was then rapidly immersed in the purple solution at room temperature for 60 minutes. Finally, the synthesized NF@Co-ZIF-L was washed with ethanol and deionized water, dried, and cut into 1.0 cm × 1.0 cm cubes for later use.

[0036] (2) Calibration of the standard curve First, 100 μL of lactic acid at different concentrations (0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 mM) was mixed with 10 μL of reagent B and incubated at 37°C for 30 minutes. Then, 100 μL of OPD reagent E and 790 μL of phosphate-buffered saline (PBS, 0.02 M, pH 7.4) were added to the above solution. Next, reagent F was added to the solution. Finally, after reacting in the dark at room temperature for 30 minutes, the above solution was transferred to a quartz tube, and fluorescence was measured under 360 nm excitation to establish a standard curve.

[0037] When detecting lactic acid, incubate with 10 μL of reagent B at 37°C for 30 minutes.

[0038] Add 100 μL of OPD reagent E and 790 μL of phosphate-buffered saline (PBS, 0.02 M, pH 7.4) to the above solution. Then, add reagent F to the solution. Finally, after reacting in the dark at room temperature for 30 minutes, transfer the above solution to a quartz tube and measure fluorescence under 360 nm excitation to establish a standard curve. When testing blood samples, take 100 μL of serum and follow the above procedure, measure the fluorescence intensity, and calculate the lactate concentration in the serum based on the standard curve.

[0039] Example 4 (1) Preparation of reagent F: First, a certain area of ​​nickel foam was washed successively with 3M hydrochloric acid, ethanol, and deionized water, and then dried for later use. Next, 1.313 g of 2-methylimidazole (C4H6N2) and 0.582 g of cobalt nitrate hexahydrate (Co(NO3)2•6H2O) were added to 50 mL of deionized water and stirred until dissolved. Then, the two components were mixed and stirred for 30 seconds to form a purple solution. The nickel foam (NF) substrate was then rapidly immersed in the purple solution at room temperature for 60 minutes. Finally, the synthesized NF@Co-ZIF-L was washed with ethanol and deionized water, dried, and cut into 1.0 cm × 1.0 cm cubes for later use.

[0040] (2) Calibration of the standard curve First, 100 μL of uric acid at different concentrations (0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 mM) was mixed with 10 μL of reagent D and incubated at 37°C for 30 minutes. Then, 100 μL of OPD reagent E and 790 μL of phosphate-buffered saline (PBS, 0.02 M, pH 7.4) were added to the above solution. Next, reagent F was added to the solution. Finally, after reacting in the dark at room temperature for 30 minutes, the above solution was transferred to a quartz tube, and fluorescence was measured under 360 nm excitation to establish a standard curve.

[0041] When testing for uric acid, incubate with 10 μL of reagent D at 37°C for 30 minutes.

[0042] (3) Add 100 μL of OPD reagent E and 790 μL of phosphate-buffered saline (PBS, 0.02 M, pH 7.4) to the above solution. Then, add reagent F to the solution. Finally, after reacting in the dark at room temperature for 30 minutes, transfer the above solution to a quartz tube and measure the fluorescence under 360 nm excitation to establish a standard curve. When testing blood samples, take 100 μL of serum and follow the above procedure to measure the fluorescence intensity. Calculate the uric acid concentration in the serum based on the standard curve.

[0043] like Figure 1 As shown in the schematic diagram, during the implementation process, the serum sample is first incubated with the corresponding specific oxidase solution (such as cholesterol oxidase, uricase, lactate oxidase, or glucose oxidase) at 37°C for 30 minutes. This allows the four metabolic markers (Chol, UA, LA, Glu) to be specifically catalyzed and oxidized by their respective oxidases, quantitatively generating the intermediate product hydrogen peroxide (H2O2). Subsequently, a pre-cut NF@Co-ZIF-L (1.0 cm) is added to the reaction system. 2 The target concentration is converted into a fluorescence signal by a cascade of conversion between the target concentration and the chromogenic substrate o-phenylenediamine (OPD, 10 mM). NF@Co-ZIF-L, with its peroxidase-like activity, catalyzes the decomposition of H2O2 generated in the first step to produce a highly oxidizing hydroxyl radical (·OH), which in turn oxidizes the colorless OPD substrate into 2,3-diaminophenazine (oxOPD) with strong yellow fluorescence.

[0044] like Figure 2As shown, the morphology, structure, and elemental composition of NF@Co-ZIF-L were systematically revealed using various characterization techniques, including scanning electron microscopy (SEM), transmission electron microscopy (TEM), high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), and energy dispersive spectroscopy (EDS). Low-magnification SEM images showed that Co-ZIF-L nanosheets grew uniformly and densely on the surface of nickel foam (NF). High-magnification SEM and TEM images further revealed a clear two-dimensional leaf-like morphology with an average size of approximately 1 μm and a thickness of approximately 194 nm. Selected area electron diffraction (SAED) patterns showed regular diffraction spots, confirming its single-crystal nature. HAADF-STEM and corresponding EDS elemental distribution images showed that Co, N, and C elements were uniformly distributed within the nanosheets, proving that Co was successfully coordinated and embedded in the ZIF-L framework. Furthermore, X-ray diffraction (XRD) patterns and X-ray photoelectron spectroscopy (XPS, including Co2p and N2p) were also observed. The 1s fine spectrum further verified the successful preparation of Co-ZIF-L and the chemical valence state and coordination environment of Co. Combined, these results confirm the successful synthesis of NF@Co-ZIF-L nanozymes with peroxidase-like activity.

[0045] like Figure 3 As shown in the figure, the fluorescence images of reaction solutions with different concentrations of cholesterol (Chol, 0, 5, 10, 20, 50, 100, 200, 500, 1000 µM) in a portable POCT device are displayed. It can be seen from the figure that as the cholesterol concentration gradually increases from 0 µM to 1000 µM, the yellow fluorescence intensity of the solution in the test tube shows a clear increasing trend from dark to bright, indicating that the oxOPD fluorescence signal generated by the cascade catalytic reaction mediated by NF@Co-ZIF-L nanozyme is positively correlated with the cholesterol concentration.

[0046] like Figure 4 As shown in the figure, this image displays the fluorescence images of reaction solutions with different concentrations of lactic acid (LA, 0, 5, 10, 20, 50, 100, 200, 500, 1000 µM) in a portable POCT device. As the lactic acid concentration gradually increases from 0 µM to 1000 µM, the intensity of the yellow fluorescence in the test tube shows a clear concentration-dependent increasing trend. This result indicates that the NF@Co-ZIF-L-mediated cascade catalytic reaction can effectively convert lactic acid concentration into a visually recognizable fluorescence signal.

[0047] like Figure 5As shown in the figure, this image displays the fluorescence images of reaction solutions corresponding to different concentrations of uric acid (UA, 0, 5, 10, 20, 50, 100, 200, 500, 1000 µM) in a portable POCT device. Similar to the lactic acid detection results, the intensity of the yellow fluorescence of the solution in the test tube also shows a regular increase from weak to bright as the uric acid concentration increases. This further verifies that the NF@Co-ZIF-L nanozyme has good cascade catalytic response capability for different targets (uric acid catalyzed by uricase to produce H2O2).

[0048] like Figure 6 As shown, this figure displays fluorescence images of reaction solutions with different concentrations of glucose (Glu, 0, 5, 10, 20, 50, 100, 200, 500, 1000 µM) in a portable POCT device. Consistent with the previous two targets, increasing glucose concentration also drives a gradual increase in fluorescence intensity, with a clear dynamic change from the non-fluorescent control tube to the bright yellow fluorescence at high concentrations.

[0049] like Figure 7 As shown, the fluorescence response values ​​(characterized by relative fluorescence intensity or other derived parameters) of four targets—cholesterol (Chol), lactate (LA), uric acid (UA), and glucose (Glu)—at different concentrations (0–700 µM) are summarized. The data trends show that as the concentration of the four targets gradually increases from 0 µM to 700 µM, their corresponding fluorescence response values ​​all exhibit varying degrees of increase. The response values ​​of cholesterol, uric acid, and glucose continuously increase throughout the concentration range, while the response value of lactate tends to plateau after reaching approximately 200 µM, showing a clear saturation trend.

[0050] like Figure 8 The image shows the fluorescence spectrum of serum diluted 10-fold and analyzed using a cascade enzyme-catalyzed reaction assay reagent. This figure illustrates the user interface and real-time analysis capabilities of the "CLUG" smartphone application. During use, the app captures fluorescence images of the reaction solution in the POCT device using the phone's camera. It then uses an automatic rectangular bounding box (1 cm × 0.8 cm) to precisely locate the bottom region of the test tube, automatically extracting the linear tristimulus values ​​(RGB, e.g., 203, 201, 193) and hue parameters (HSV, e.g., Hue=225.0, Saturation=1.0, Value=1.0) for each pixel within that region. The app then inputs this multidimensional data into an embedded artificial neural network (ANN) algorithm model for computation and directly outputs the predicted target concentration value (e.g., Concentration=34.1) on the interface. The four different test interfaces in the figure demonstrate the app's parallel processing capabilities and the consistency of its output results when analyzing different samples.

Claims

1. A reagent for analyzing multiple biomarkers in blood based on a cascade enzyme catalytic reaction, characterized in that, The analytical reagents include reagent F and one or more of reagents A, B, C1, C2, D, and E; wherein reagent F is an NF@Co-ZIF-L nanozyme with peroxidase-like activity, and the multiple markers include at least one of cholesterol, lactic acid, uric acid, and glucose.

2. The analytical reagent for multiple biomarkers in blood based on a cascade enzyme catalytic reaction according to claim 1, characterized in that, Reagent A is an ultrapure aqueous solution of glucose oxidase with a concentration of 1.0 ± 0.1 mg / mL. -1 .

3. The blood biomarker analysis reagent based on cascade enzyme catalysis according to claim 1, characterized in that, Reagent B is an ultrapure aqueous solution of lactate oxidase with a concentration of 1.0 ± 0.1 mg / mL. -1 .

4. The analytical reagent for multiple biomarkers in blood based on a cascade enzyme catalytic reaction according to claim 1, characterized in that, Reagent C1 is an ultrapure aqueous solution of cholesterol oxidase with a concentration of 1.0 ± 0.1 mg / mL. -1 .

5. The analytical reagent for multiple biomarkers in blood based on a cascade enzyme catalytic reaction according to claim 1, characterized in that, Reagent C2 is an ultrapure aqueous solution of cholesterol lipase with a concentration of 1.0 ± 0.1 mg / mL. -1 .

6. The analytical reagent for multiple biomarkers in blood based on a cascade enzyme catalytic reaction according to claim 1, characterized in that, Reagent D is an ultrapure aqueous solution of uricase with a concentration of 1.0 ± 0.1 mg / mL. -1 .

7. The analytical reagent for multiple biomarkers in blood based on a cascade enzyme catalytic reaction according to claim 1, characterized in that, Reagent E is an ultrapure aqueous solution of o-phenylenediamine with a concentration of 10.0 ± 1.0 mM.

8. The analytical reagent for multiple biomarkers in blood based on a cascade enzyme catalytic reaction according to claim 1, characterized in that, The preparation method of the NF@Co-ZIF-L nanozyme includes: firstly, washing the nickel foam with hydrochloric acid, ethanol, and deionized water in sequence, and drying it for later use; adding 2-methylimidazole and cobalt nitrate hexahydrate to deionized water and stirring until dissolved, then mixing the two components and stirring to form a purple solution; and quickly immersing the nickel foam into the purple solution at room temperature to obtain NF@Co-ZIF-L.

9. The analytical reagent for multiple biomarkers in blood based on a cascade enzyme catalytic reaction according to claim 1, characterized in that, The mass ratio of 2-methylimidazole to cobalt nitrate hexahydrate is 2.2 ± 0.1 : 1.

0.

10. The application of the blood biomarker analytical reagent based on cascade enzyme catalysis reaction as described in any one of claims 1-9, for the in vitro detection of cholesterol, lactic acid, uric acid, and glucose in blood.

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