Osmium-ruthenium nano-enzyme as well as preparation method and application thereof

Abstract

The invention relates to the technical field of food safety, in particular to osmium-ruthenium nano-enzyme as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving soluble osmium salt, soluble ruthenium salt, glycine and polyvinylpyrrolidone in water together to obtain a mixture; and mixing the mixture with a citric acid solution, carrying out a reduction reaction, then cooling to room temperature, and carrying out dialysis purification treatment to obtain the osmium-ruthenium nano-enzyme. The osmium-ruthenium nano-enzyme is prepared through one-step hydrothermal reaction, and the osmium-ruthenium nano-enzyme has excellent peroxidase-like activity. A catechin double-detection platform constructed based on the osmium-ruthenium nano-enzyme comprises a solution platform and a paper platform, has the advantages of high response speed, simplicity in operation and relatively low cost, and overcomes the defects in the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety, and in particular to an osmium-ruthenium nanozyme and a preparation method and application thereof. Background Art

[0002] Catechins are phenolic compounds extracted from natural plants. Commonly found in tea leaves, they are the primary biologically active ingredient in tea. Catechins are reported to exhibit a variety of biological activities, including anti-inflammatory, antibacterial, anti-cancer, cardiovascular protection, hypoglycemic, and free radical scavenging properties. Consequently, they are widely used in medical and food applications. However, exceeding safety thresholds can cause adverse health effects, such as nausea and dizziness. Therefore, developing an efficient, sensitive, and reliable method for catechin detection is crucial for ensuring food safety.

[0003] Currently, gas chromatography, high-performance liquid chromatography, Raman spectroscopy, and electrochemical methods are the main methods for determining catechins. However, these methods all require large instruments and are generally limited by complex and time-consuming operations, high costs, and the inability to detect with the naked eye. These methods are unable to meet the current demand for efficient and accurate catechin determination. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention provides an osmium-ruthenium nanozyme, its preparation method, and application. Using a soluble osmium salt, a soluble ruthenium salt, glycine, and polyvinylpyrrolidone as raw materials, the present invention prepares the osmium-ruthenium nanozyme with excellent peroxidase-like activity through a one-step hydrothermal reduction reaction. A dual-detection platform for catechins is constructed based on this osmium-ruthenium nanozyme. This dual-detection platform for catechins achieves rapid and sensitive colorimetric detection of catechins by catalyzing H₂O₂ to produce hydroxyl radicals (HO·). HO· oxidizes 3,3',5,5'-tetramethylbenzidine (TMB) solution to develop color. Subsequently, catechins scavenge HO· to ​​inhibit color development. This platform offers the advantages of a fast response time (8-12 minutes), simple operation (no complex instrumentation required), and low cost (raw materials are readily available), overcoming the shortcomings of existing detection methods.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: The first object of the present invention is to provide a method for preparing the above-mentioned osmium-ruthenium nanozyme, comprising the following steps: S1. Dissolve soluble osmium salt, soluble ruthenium salt, glycine and polyvinylpyrrolidone in water to obtain a mixture; the purpose of mixing is to ensure that the chemical environment of all reaction sites is consistent. Insufficient mixing will lead to uneven distribution of elements, which will seriously affect the performance of osmium-ruthenium nanozyme, such as catalytic activity, optical properties, and electrochemical properties.

[0006] S2, mixing the mixture with citric acid solution and performing reduction reaction. During the reduction reaction, the citric acid in the citric acid solution decomposes into aconitic acid and reducing fragments, Os 4+ and Ru 3+ is reduced to an atomic state and forms an alloy nucleus; in this process, glycine has amphiphilic molecular properties and can maintain the pH stability of the reaction system. During the reduction reaction, glycine decomposes to produce reducing substances (such as ammonia and aldehydes) to assist in the reduction of Os 4+ and Ru 3+ ; The amino and carboxyl groups on the glycine molecule interact with Os 4+ 、Ru 3+ Forming coordination bonds, effectively slowing down the Os 4+ and Ru 3+ The reduction rate is reduced, explosive nucleation is avoided, and it is conducive to the formation of particles of uniform size; at the same time, free osmium atoms and ruthenium atoms are deposited on the alloy core, and the osmium atoms and ruthenium atoms diffuse with each other to form a homogeneous alloy; polyvinylpyrrolidone is adsorbed on the surface of osmium atoms and ruthenium atoms or the surface of the alloy core through the carbonyl oxygen on its pyrrole ring, and its long-chain molecular structure forms a spatial steric hindrance layer, which effectively prevents the agglomeration of nanoparticles; it is then cooled to room temperature. During the cooling process, citric acid is re-adsorbed on the surface of the homogeneous alloy to form a protective layer, which prevents O2 erosion and particle aggregation. After dialysis purification, osmium-ruthenium nanozyme is obtained.

[0007] Preferably, the mass ratio of the osmium salt solution, the ruthenium salt solution, the glycine and the polyvinyl pyrrolidone is 0.5-1.5:0.1-0.5:4-10:10-20.

[0008] Preferably, the dissolution conditions are: stirring at 300 rpm to 500 rpm for 5 to 30 minutes at room temperature.

[0009] Preferably, the concentration of citric acid is 0.2 mmol / L to 0.5 mmol / L, and the molar ratio of the total molar amount of osmium ions and ruthenium ions to the molar amount of citric acid is 1:3 to 5.

[0010] Preferably, the reduction reaction conditions are: stirring at 1200 r / min to 1800 r / min for 30 min to 60 min at 80° C. to 120° C., wherein the reducing ability of citric acid at room temperature is relatively mild. Under heating conditions, the citric acid molecules will undergo partial decarboxylation or dehydration reactions to generate intermediates with stronger reducing activity, such as aconitic acid. These thermal decomposition products have stronger reducing ability, which can increase the reduction rate and accelerate the chemical reaction rate.

[0011] Preferably, the dialysis purification operation is: dialyzing the mixture in deionized water using a 500Da dialysis bag for 12 hours to 36 hours.

[0012] The second object of the present invention is to provide an osmium-ruthenium nanozyme prepared by the above preparation method.

[0013] The third object of the present invention is to provide a catechin dual detection platform, which is constructed based on the above-mentioned osmium-ruthenium nanozyme, and the catechin dual detection platform is a solution platform or a paper platform.

[0014] Among them, the solution platform is: mixing osmium-ruthenium nanozyme with 3,3',5,5'-tetramethylbenzidine solution and H2O2 to obtain blue oxidized 3,3',5,5'-tetramethylbenzidine diimine; adding catechin to the above reaction system, catechin inhibits the oxidation of 3,3',5,5'-tetramethylbenzidine by osmium-ruthenium nanozyme, and the blue color becomes lighter.

[0015] The paper platform is as follows: osmium-ruthenium nanozyme is loaded on the paper, and after it is naturally dried, catechin solution is first applied. After it is naturally dried, 3,3',5,5'-tetramethylbenzidine solution is added and the color is observed.

[0016] The detection principle is as follows: the Os atoms or Ru atoms on the surface of the osmium-ruthenium nanozyme provide active sites, adsorb and activate H2O2 molecules, and then transfer electrons through the valence change of the metal center (Os atom or Ru atom), ultimately reducing H2O2 to HO· with strong oxidizing properties. HO· oxidizes 3,3',5,5'-tetramethylbenzidine to generate blue oxidized 3,3',5,5'-tetramethylbenzidine diimine; catechins inhibit the oxidation of 3,3',5,5'-tetramethylbenzidine by scavenging HO·, making the solution or paper lighter in color, thereby achieving quantitative detection of catechins.

[0017] Preferably, the detection conditions of the solution platform are as follows: dilute the osmium-ruthenium nanozyme solution with a mass concentration of 100 ug / mL by 200 to 300 times, add 3,3',5,5'-tetramethylbenzidine at a concentration of 3.5 mmol / L to 4.0 mmol / L, and react at 20°C to 25°C and a pH of 4 to 5.5 for 8 min to 12 min.

[0018] Preferably, the detection conditions of the paper platform are: diluting the osmium-ruthenium nanozyme solution with a mass concentration of 100ug / mL by 60 to 70 times, the concentration of 3,3',5,5'-tetramethylbenzidine is 3.5mmol / L to 4.0mmol / L, the pH value of the reaction system is 4.0 to 5.0, and the reaction time is 8min to 12min.

[0019] Preferably, the detection limit of the solution platform is 2.84 μmol / L, the detection limit of the paper platform with the naked eye is 56.25 μmol / L, and the detection limit of the grayscale analysis is 9.68 μmol / L.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a method for preparing osmium-ruthenium nanozyme, comprising dissolving a soluble osmium salt, a soluble ruthenium salt, glycine and polyvinylpyrrolidone in water to obtain a mixture; mixing the mixture with a citric acid solution and performing a reduction reaction. During the reduction reaction, the citric acid in the citric acid solution decomposes into aconitic acid and reducing fragments, and Os 4+ and Ru 3+ is reduced to an atomic state and forms an alloy nucleus; in this process, glycine has amphiphilic molecular properties and can maintain the pH stability of the reaction system. During the reduction reaction, glycine decomposes to produce reducing substances (such as ammonia and aldehydes) to assist in the reduction of Os 4+ and Ru 3+ ; The amino and carboxyl groups on the glycine molecule interact with Os 4+ 、Ru 3+ Forming coordination bonds, effectively slowing down the Os 4+ and Ru 3+ The reduction rate is reduced to avoid explosive nucleation, which is conducive to the formation of particles of uniform size; at the same time, free osmium atoms and ruthenium atoms are deposited on the alloy core, and the osmium atoms and ruthenium atoms diffuse with each other to form a homogeneous alloy; polyvinyl pyrrolidone is adsorbed on the surface of osmium atoms and ruthenium atoms or the surface of the alloy core through the carbonyl oxygen on its pyrrole ring, and its long-chain molecular structure forms a steric hindrance layer, which effectively prevents the agglomeration of nanoparticles; it is then cooled to room temperature, and during the cooling process, citric acid is re-adsorbed on the surface of the homogeneous alloy to form a protective layer, preventing O2 erosion and particle aggregation, and dialysis purification treatment is performed to obtain osmium-ruthenium nanozyme. The present invention prepares osmium-ruthenium nanozyme through a one-step hydrothermal reaction, which has excellent peroxidase-like activity. Compared with the existing technology, the catechin dual detection platform constructed based on the osmium-ruthenium nanozyme of the present invention has the advantages of fast response speed, simple operation and relatively low cost, which overcomes the defects of the existing technology.

[0021] 2. This invention constructs a dual catechin detection platform, comprising a solution platform and a paper platform. The osmium-ruthenium nanozyme, through its peroxidase-mimicking activity, effectively catalyzes H₂O₂ to produce hydroxyl radicals (HO₀), which oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to form the blue product, oxidized 3,3',5,5'-tetramethylbenzidine diimide (oxTMB), causing the solution and paper platforms to turn blue. Catechins, on the other hand, scavenge the HO₀ produced by the osmium-ruthenium nanozyme, thereby preventing the oxidation of TMB and causing the solution and paper platforms to lighten in color. Based on this, direct colorimetric detection of catechins can be easily achieved based on the color changes of the solution and paper platforms.

[0022] In addition, the dual platform for direct colorimetric detection of catechins in the present invention has good sensitivity, with a detection limit of 2.84 μmol / L for the solution platform, a naked eye detection limit of 56.25 μmol / L for the paper platform, and a grayscale detection limit of 9.68 μmol / L.

[0023] 3. The catechin dual detection platform of the present invention was applied to the detection of green tea beverages. The spiked recovery rate was 91.78%~103.01%, and the relative standard deviation (RSD) was less than 6.74%, which further confirmed the reliability and applicability of the catechin dual detection platform of the present invention, and provided more ideas for the design and development of nanozyme-based colorimetric sensing methods for rapid detection of antioxidants. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Characterization diagram of osmium-ruthenium nanozyme, where a is the TEM image, b is the size distribution, c is the EDS spectrum, d is the XRD diagram, e is the zeta potential diagram, and f is the UV-vis spectrum.

[0025] Figure 2 This is a diagram showing the peroxidase-like activity effect of osmium-ruthenium nanozyme.

[0026] Figure 3 This is the steady-state kinetic detection result of osmium-ruthenium nanozyme, where a is TMB and b is H2O2.

[0027] Figure 4 This is the characterization diagram of osmium-ruthenium nanozyme after storage at room temperature for 90 days, where a is TEM, b is size distribution, and c is peroxidase-like activity.

[0028] Figure 5 The figures show the feasibility test results of the catechin solution platform and the paper platform, where a is the solution platform, the illustration a1 in a is TMB+H2O2+osmium-ruthenium nanozyme, a2 is TMB+H2O2+catechin (450μmol / L)+osmium-ruthenium nanozyme, and a3 is TMB+H2O2); b is the paper platform, the illustration b1 in b is TMB+H2O2+osmium-ruthenium nanozyme; b2: TMB+H2O2+catechin (450μmol / L)+osmium-ruthenium nanozyme; b3 is TMB+H2O2.

[0029] Figure 6 Figure 2 is the fluorescence spectrum and ESR spectrum measured at an excitation wavelength of 315 nm, where a is the fluorescence spectrum and b is the ESR spectrum.

[0030] Figure 7 This is a diagram showing the optimization results of the solution platform detection conditions based on osmium-ruthenium nanozyme, where a is the concentration of osmium-ruthenium nanozyme, b is the reaction time, c is the pH value, and d is the reaction temperature.

[0031] Figure 8 This is a graph showing the optimization results of the paper platform detection conditions based on osmium-ruthenium nanozyme, where a is the concentration of osmium-ruthenium nanozyme, b is the reaction time, c is the pH value, and d is the TMB concentration.

[0032] Figure 9 This is the selectivity diagram of osmium-ruthenium nanozyme for detecting catechin, where a is the color of the system with catechin added and the absorbance value at 650nm on the solution platform, b is the color of the system with catechin and interfering substances added and the absorbance value at 650nm on the solution platform, c is the color and grayscale value of the system with catechin added on the paper platform, and d is the color and grayscale value of the system with catechin and interfering substances added on the paper platform.

[0033] Figure 10 Figure 3 is the performance diagram of the dual colorimetric platform for detecting catechins, where a is the color change and linear curve of the osmium-ruthenium nanozyme + TMB + H2O2 system in the solution platform under the action of different concentrations of catechins, and b is the color change and linear curve of the osmium-ruthenium nanozyme + TMB + H2O2 system on the paper platform under the action of different concentrations of catechins.

[0034] Figure 11 This figure shows the storage stability test results of the paper platform based on osmium-ruthenium nanozyme.

[0035] Figure 12 This is the result of the dual-colorimetric detection platform and instrument detection method for actual sample spike detection, where a is 200 μmol / L and b is 400 μmol / L. DETAILED DESCRIPTION

[0036] The following will clearly and completely describe the technical solution of the present invention in conjunction with the data in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0037] It should be noted that the professional terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the following embodiments of the present invention can be purchased from the market or prepared by existing methods.

[0038] Existing technologies primarily rely on instrumental analysis methods, such as gas chromatography, high-performance liquid chromatography, Raman spectroscopy, and electrochemical methods. However, these methods require large instruments and are often complex, time-consuming, and expensive to perform, often hindering visual detection. These methods, however, are unable to meet the current demand for efficient and accurate catechin determination.

[0039] In response to the problems of equipment dependence, complex operation and inability to detect with the naked eye in the above-mentioned existing technologies, the present invention uses the prepared osmium-ruthenium nanozyme to construct a catechin dual detection platform to replace traditional instruments, that is, using its high catalytic activity to simulate peroxidase, and converting the catechin concentration into a visual color change through a color development reaction (TMB-H2O2 system), without the need for complex equipment; in addition, the catechin dual detection platform constructed by the present invention, for the solution platform, achieves a high-sensitivity detection of 2.84μmol / L by optimizing the pH, temperature and dilution ratio of the osmium-ruthenium nanozyme; for the paper platform, combined with the osmium-ruthenium nanozyme immobilization technology, supports naked-eye qualitative (56.25μmol / L) and grayscale quantitative (9.68μmol / L) detection, meeting the needs of different scenarios.

[0040] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments: Example 1 A method for preparing an osmium-ruthenium nanozyme comprises the following steps: S1. Add 900 μL of deionized water to a 1.5 mL test tube, then add 50 μL of 40 mmol / L K2OsCl6 solution, 50 μL of 20 mmol / L RuCl3∙3H2O solution, 6.0 mg of glycine, and 15 mg of PVP in that order; vortex and stir at 400 rpm for 10 min at room temperature to obtain a mixed solution.

[0041] S2. The mixed solution was added into a three-necked flask containing 50 mL of 0.3 mmol / L citric acid solution (preheated to 100°C), stirred vigorously at 1800 r / min for 40 min, and naturally cooled to room temperature. The mixture was dialyzed in deionized water for 24 h using a 500 Da dialysis bag to obtain osmium-ruthenium nanozyme, namely Os-Ru nanozyme, abbreviated as Os-Ru.

[0042] Example 2 A method for preparing an osmium-ruthenium nanozyme is the same as the preparation method in Example 1, except that the amount of K2OsCl6 solution in S1 is replaced from 50 μL to 150 μL, the amount of RuCl3∙3H2O solution is replaced from 50 μL to 83.33 μL, the amount of glycine is replaced from 6.0 mg to 10 mg, and the amount of PVP is replaced from 15.0 mg to 20 mg, to obtain an osmium-ruthenium nanozyme.

[0043] Example 3 A method for preparing an osmium-ruthenium nanozyme is the same as the preparation method in Example 1, except that the amount of RuCl3∙3H2O solution in S1 is replaced from 50 μL to 16.7 μL, the amount of glycine is replaced from 6.0 mg to 4.0 mg, and the amount of PVP is replaced from 15.0 mg to 10 mg, to obtain an osmium-ruthenium nanozyme.

[0044] Example 4 A method for preparing an osmium-ruthenium nanozyme is the same as the preparation method in Example 1, except that the amount of citric acid solution in S2 is replaced from 50 mL to 30 mL to obtain an osmium-ruthenium nanozyme.

[0045] a. Experimental part: Study on the peroxidase-like activity of Os-Ru nanozymes: First, the peroxidase-like activity of the Os-Ru nanozyme was evaluated to determine whether it could catalyze the oxidation of the chromogenic substrate TMB in the presence of H2O2. Briefly, 20 μL of Os-Ru nanozyme (a 256-fold dilution of the original solution) and 200 μL of the reaction substrate solution (containing 2.0 mmol / L TMB and 10 mmol / L H2O2) were added to a 96-well plate and reacted at room temperature for 10 minutes. UV-visible absorption spectra (500 nm–800 nm) were then recorded using a microplate reader. A control group (TMB + H2O2 alone) and a control group (TMB + Os-Ru nanozyme alone) were used to investigate the effect of H2O2 or Os-Ru nanozyme alone on TMB oxidation. The group without H2O2 or TMB was designated as a blank group, and all results were removed from the blank value.

[0046] The catalytic properties of the Os-Ru nanozyme were further explored through steady-state kinetic analysis. Briefly, 20 μL of the Os-Ru nanozyme solution (256-fold dilution of the original solution) and 200 μL of a substrate solution containing 10 mmol / L H₂O₂ and various concentrations of TMB (8.24 mmol / L, 4.12 mmol / L, 2.06 mmol / L, 1.03 mmol / L, 0.51 mmol / L, and 0.26 mmol / L), or 2.0 mmol / L of TMB and various concentrations of H₂O₂ (10 mmol / L, 5 mmol / L, 2.5 mmol / L, 1.25 mmol / L, 0.625 mmol / L, 0.3125 mmol / L, and 0.15625 mmol / L) were added to a 96-well plate and reacted for 10 minutes at room temperature. The absorbance of the system was then measured at a wavelength of 650 nm using a microplate reader. The group without TMB and H2O2 was set as the blank control group, and all results were subtracted from the blank value. The oxTMB concentration generated by the above system was calculated as follows:

[0047] (1) in, A Represents the system absorbance value measured at a wavelength of 650nm, ξ represents the molar absorption coefficient of oxTMB (3.9×10 4 M -1 cm -1 ), b represents the thickness of the absorption layer, c represents the concentration of oxTMB.

[0048] Then the oxTMB concentration produced per unit time was calculated according to the following formula ( V 0 ): (2) in, t is the reaction time, which is 10 min in the present invention.

[0049] Then, the reciprocal of the oxTMB concentration produced per unit time (1 / V 0 ) and the reciprocal of the substrate TMB (or H2O2) concentration (1 / [S] ) as the vertical and horizontal axes. Finally, the Michaelis constant is calculated according to the following formula ( K m ) and maximum reaction speed ( V max ):

[0050] (3) Study on the long-term stability and sustainability of Os-Ru nanozymes: The morphological and dimensional changes of the Os-Ru nanozyme after 90 days of storage at room temperature were monitored by TEM to investigate its long-term structural stability. Furthermore, the sustainability of the physicochemical properties of the Os-Ru nanozyme was investigated by monitoring its peroxidase-mimicking activity after 90 days of storage at room temperature. Briefly, 20 μL of Os-Ru nanozyme (a 256-fold dilution of the original solution) and 200 μL of reaction substrate solution (containing 2.0 mmol / L TMB and 10 mmol / L H2O2) were added to a 96-well plate. After reacting at room temperature for 10 minutes, the absorbance of the system at a wavelength of 650 nm was measured using a microplate reader. The absorbance value measured on day 0 was set as 100% peroxidase-mimicking activity.

[0051] Feasibility of direct colorimetric detection of catechins based on Os-Ru nanozyme dual detection platform: By evaluating the inhibitory ability of high-concentration catechins on the peroxidase-like activity of Os-Ru nanozymes on both the solution platform and the paper platform, the feasibility of directly detecting catechins using the Os-Ru nanozyme dual platform was explored.

[0052] Briefly, 20 μL of Os-Ru nanozyme (a 256-fold dilution of the original solution), 10 μL of catechin solution (450 μmol / L), and 200 μL of a reaction substrate solution containing 2.0 mmol / L TMB and 10 mmol / L H₂O₂ were added to a 96-well plate on a solution platform. After reacting for 10 minutes at room temperature, photos were taken using an iPhone 14 Pro, and the UV absorption spectrum of the reaction system was acquired using a microplate reader within a wavelength range of 500 nm to 800 nm. In addition, groups without catechin or Os-Ru nanozyme were set as controls.

[0053] On a paper platform, 10 μL of an Os-Ru nanozyme solution (a 64-fold dilution of the original solution) was first added to a clean 8 mm diameter test paper and allowed to air dry at room temperature to prepare a catechin test strip. Then, 10 μL of a catechin solution (450 μmol / L) was added to the test paper and allowed to air dry at room temperature. Next, 20 μL of a reaction substrate solution (containing 3.75 mmol / L TMB and 10 mmol / L H₂O₂) was added to the test paper. After reacting for 10 minutes at room temperature, the color of the test paper was recorded using an iPhone 14 Pro, and the grayscale (G) value of the test paper was obtained using Image J software. A control group without catechin and Os-Ru nanozyme was designated as the control group, while a blank group with catechin and Os-Ru nanozyme was designated as the blank group. ΔG is the difference between the G value of the blank group and the G value of the test or control group.

[0054] Colorimetric detection mechanism of catechins based on Os-Ru nanozymes: The colorimetric detection mechanism of catechin based on Os-Ru nanozyme was studied by monitoring the generation of hydroxyl radicals (HO·) in the reaction system. Terephthalic acid (TA) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were selected as HO· scavengers. After the system (TA (100μL, 5mmol / L) + H2O2 (800μL, 10mmol / L) + Os-Ru nanozyme (50μL, 64-fold dilution of the original solution)) and the system ((TA (100μL, 5mmol / L) + H2O2 (800μL, 10mmol / L) + Os-Ru nanozyme (50μL, 64-fold dilution of the original solution) + catechin (900μmol / L)) reacted at 45°C for 30min, the fluorescence spectra of the system were recorded using a fluorescence spectrophotometer with an excitation wavelength of 315nm. In addition, the system Electron spin resonance (ESR) spectra of (DMPO (100 μL, 50 mmol / L) + Os-Ru nanozyme (50 μL, 64-fold dilution of the original solution) + H2O2 (800 μL, 10 mmol / L)) and (DMPO (100 μL, 50 mmol / L) + Os-Ru nanozyme (50 μL, 64-fold dilution of the original solution) + H2O2 (800 μL, 10 mmol / L) + catechin (900 μmol / L)) were monitored using a Bruker A300 spectrometer after reaction at pH 6 for 5 min.

[0055] Optimization of detection conditions for the catechin dual detection platform: To achieve the best sensing performance for catechin detection, the sensing conditions of the solution platform and paper platform were optimized, respectively.

[0056] For the solution platform, the optimized conditions included Os-Ru nanozyme concentration (expressed as the dilution multiple of the original solution: 0, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048), enzymatic reaction time (0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min), reaction temperature (20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C) and pH value of the reaction system (2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9). Briefly, 200 μL of reaction substrate solution (containing 2.0 mmol / L TMB and 10 mmol / L H2O2) and 20 μL of a diluted Os-Ru nanozyme solution were first added to a 96-well plate. The above system was then reacted for a certain period of time under specific pH and temperature conditions. Next, the absorbance of the system at a wavelength of 650 nm was measured using a microplate reader. The group without the addition of reaction substrate solution was set as the blank control group, and the final absorbance value of all groups was the absorbance value after subtracting the absorbance value of the blank control group. Finally, a plot was drawn with the absorbance value as the vertical axis and the Os-Ru nanozyme concentration, reaction temperature, reaction time, and system pH as the horizontal axis.

[0057] For the paper platform, optimized conditions included Os-Ru nanozyme concentration (expressed as the dilution factor of the original solution: 0, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048), enzymatic reaction time (2 min, 5 min, 10 min, 15 min, 20 min, 25 min), TMB concentration (4.58 mmol / L, 3.75 mmol / L, 2.91 mmol / L, 2.08 mmol / L, 1.25 mmol / L, 0.41 mmol / L), and system pH (2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9). The optimization procedure was as follows: 10 μL of diluted Os-Ru nanozyme solution and 20 μL of reaction substrate solution containing a certain concentration of TMB were added to the test paper. After a certain reaction time at room temperature and a specific pH, the test strips were photographed with an iPhone 14 Pro to record their color, and their grayscale (G) values ​​were calculated using Image J software. A test strip without the addition of the reaction substrate solution was designated a blank group, and the ΔG value of the test strips was the difference between the G values ​​of the blank and experimental groups. Finally, a plot was plotted with the grayscale ΔG value as the ordinate and the Os-Ru nanozyme concentration, TMB solution concentration, reaction time, and system pH as the abscissa.

[0058] Anti-interference ability of catechin dual detection platform: The ability of the Os-Ru nanozyme-based dual-platform to detect catechins was evaluated by investigating the inhibitory effects of common interfering substances in food (such as ions, sugars, and amino acids) on the reaction system (osmium-ruthenium + TMB + H2O2). The test concentration of each interfering substance was 900 μmol / L in both the solution and paper platforms.

[0059] On a solution colorimetric platform, 20 μL of Os-Ru nanozyme (a 256-fold dilution of the original solution), 10 μL of catechin (450 μmol / L) or / and interfering substances (900 μmol / L), and 200 μL of reaction substrate solution (containing 2.0 mmol / L TMB and 10 mmol / L H2O2) were mixed in a 96-well plate. After reacting for 10 minutes at room temperature and a pH of 4.5, the absorbance of the system was measured at 650 nm using a microplate reader. A group containing only Os-Ru nanozyme, TMB, and H2O2, without catechin or interfering substances, was designated as the positive control (blank group).

[0060] On a paper colorimetric platform, 10 μL of the Os-Ru nanozyme solution (a 64-fold dilution of the original solution) was first dripped onto a clean 8 mm diameter test paper and allowed to air dry to produce a catechin test strip. Subsequently, 10 μL of catechin (450 μmol / L) or / and an interfering substance (900 μmol / L) was dripped onto the test strip and allowed to air dry at room temperature. Next, 20 μL of a reaction substrate solution (containing 3.75 mmol / L TMB and 10 mmol / L H₂O₂) was dripped onto the test strip. The reaction was allowed to proceed at room temperature and pH 4.0 for 10 minutes. The test strip was then photographed using an iPhone 14 Pro and processed using Image J software to determine the grayscale value (G). ΔG is the difference between the G value of the control group (without catechin, interfering substances, or reaction substrate) and the G value of the test group. The group without catechin and interfering substances was designated as the blank group.

[0061] Dual colorimetric platform for catechin detection: Briefly, on a solution colorimetric platform, 20 μL of Os-Ru nanozyme (a 256-fold dilution of the original solution), 10 μL of catechin solution at varying concentrations (0 μmol / L to 450 μmol / L), and 200 μL of substrate solution (containing 2.0 mmol / L TMB and 10 mmol / L H₂O₂) were mixed in a 96-well plate. After 10 minutes of reaction at room temperature and pH 4.5, the absorbance of the system was measured at 650 nm using a microplate reader. A group without substrate solution was designated as a blank control, and the final absorbance values ​​for all groups were calculated by subtracting the absorbance of the blank control. Finally, a standard curve was plotted with absorbance as the y-axis and catechin concentration as the x-axis.

[0062] The catechin concentration corresponding to a signal-to-noise ratio (S / N) of 3 was defined as the limit of detection (LOD) of the solution platform.

[0063] (4) Wherein, SD is the standard deviation of the multiple measurement signals of the blank control group, and m is the slope of the standard curve.

[0064] On the paper colorimetric platform, 10 μL of catechin solution of varying concentrations (0 μmol / L to 450 μmol / L) was dripped onto the test paper. After air-drying at room temperature, 20 μL of reaction substrate solution (containing 3.75 mmol / L TMB and 10 mmol / L H2O2) was added. The test paper was then reacted for 10 minutes at room temperature and a pH of 4.0. Next, an iPhone 14 Pro was used to take a photo, and the grayscale value (G) of the test paper was obtained using Image J software. A standard curve was drawn with the grayscale value (G) as the ordinate and the catechin concentration as the abscissa. The subsequent steps were consistent with the solution colorimetric platform described above.

[0065] Room temperature storage stability of the paper colorimetric platform: Considering the convenience of future storage, we explored the stability of Os-Ru nanozyme colorimetric test strips at room temperature by measuring their catalytic reaction activity on days 0, 10, 20, 30, 40, 50, and 60. Briefly, 20 μL of reaction substrate solution (containing 3.75 mmol / L TMB and 10 mmol / L H2O2) was added dropwise to the colorimetric test strips stored for different days. After reacting at room temperature for 10 minutes, the color of the test strips was recorded using an iPhone 14 Pro. The color of the test strips was then converted into grayscale (G) values ​​using Image J software. The G value on day 0 was set as 100% relative activity.

[0066] Recovery analysis of spiked catechins in real samples: To verify the feasibility of the dual colorimetric detection platform developed based on Os-Ru nanozymes in actual sample testing, three green tea beverages—Master Kong, Vita, and Gyokuro—were selected as actual samples for catechin spike recovery analysis. First, the three green tea beverages were centrifuged at 25°C and 12,000 rpm for 10 minutes, and the supernatants were collected. The supernatants were then diluted 100-fold with distilled water and filtered through a 0.22 μm filter membrane. Subsequently, different amounts of catechins were weighed and added to the dilutions of the three beverages to obtain spiked catechin recovery solutions at concentrations of 300 μmol / L, 150 μmol / L, and 0 μmol / L, respectively. Finally, the catechin-spiked recovery solutions were analyzed using the solution and paper colorimetric platforms constructed in this invention, as well as high-performance liquid chromatography-mass spectrometry.

[0067] b. Results and Discussion Os-Ru nanozyme characterization: To verify the successful synthesis of Os-Ru nanozyme, it was characterized by TEM, EDS, XRD, Zeta potential and UV-vis spectroscopy. Figure 1 The results in Figures a and b show that Os-Ru nanozymes are dispersed elliptical particles with an average particle size of approximately 19.48 nm. Figure 1 The results of Figure c in Figure 3 show that Os-Ru nanozyme contains Os and Ru elements, and two peaks appear at 1.91keV and 2.56keV, corresponding to the EDS peaks of Os and Ru elements respectively. Figure 1 Figure d shows that Os-Ru nanozyme has five diffraction peaks at 38.0°, 38.4°, 41.8°, 43.6° and 44.0°, which can correspond to (100), (002) and (101) of fcc Os (JCPDS06-0662) and (100) and (101) of fcc Ru (JCPDS06-0663). In addition, Figure 1 Figures e and f in Figure 3 show that the zeta potential of Os-Ru nanozyme is about -18.79±0.87mV, with a maximum absorption peak near 526nm. Figure 1 It was concluded that the Os-Ru nanozyme of the present invention was successfully prepared.

[0068] Peroxidase-like activity of Os-Ru nanozymes: In order to verify whether Os-Ru nanozyme has peroxidase-like activity, TMB was selected as the chromogenic substrate to explore the catalytic oxidation ability of Os-Ru nanozyme. Figure 2 As shown, Os-Ru nanozyme cannot directly catalyze the oxidation of TMB, but in the presence of H2O2, TMB can be catalytically oxidized into a blue product with the strongest absorption peak near 650nm, indicating that Os-Ru nanozyme has peroxidase-like activity.

[0069] To further evaluate the peroxidase-like activity of Os-Ru nanozyme, steady-state kinetic studies were performed. The Michaelis constant ( K m ) value reflects the affinity of the enzyme to the corresponding substrate. The smaller the value, the stronger the affinity. V max ) represents the maximum speed of the catalytic reaction. The larger the value, the faster the reaction speed. Figure 3 Figures a and b in the figure are typical Michaelis-Menten curves established with TMB and H2O2 as substrates, respectively. Km and V max 0.36mmol / L and 6.75×10 -6 Ms -1 , for H2O2 K m and V max 2.67mmol / L and 1.25×10 -6 Ms -1 .

[0070] Table 1 K of Os-Ru nanozymes and other catalysts m and V max Test results table Note: “-” means the item was not tested.

[0071] Table 1 lists the HRP and other reported nanozymes with peroxidase-like activity. K m and V max The results showed that the affinity and maximum reaction rate of Os-Ru nanozyme for TMB and H2O2 were higher than those of HRP and other nanozymes, indicating that the Os-Ru nanozyme obtained in the present invention has excellent peroxidase-like activity and is expected to become a candidate material for nanoperoxidase.

[0072] Long-term stability and sustainability of Os-Ru nanozymes: To further verify the practical application potential of Os-Ru nanozyme, the long-term storage stability of its structure and the sustainability of its peroxidase-like activity were explored. Figure 4 The results showed that Os-Ru nanozymes could maintain their original morphology (dispersed elliptical particles, such as Figure 4 Figure a), size (about 19.32nm, Figure 4 b) and almost unchanged peroxidase-like activity ( Figure 4 These results indicate that the Os-Ru nanozyme possesses good long-term structural stability and sustained peroxidase-like activity.

[0073] Feasibility of colorimetric detection of catechins based on Os-Ru nanozyme dual detection platform: In order to evaluate the feasibility of dual-platform colorimetric detection of catechins based on Os-Ru nanozyme, the effects of catechins on the absorbance and color of the system (Os-Ru nanozyme + TMB + H2O2) were investigated on the solution platform and paper platform, respectively. Figure 5Panel a shows that after adding a high concentration of catechin (450 μmol / L) to the system, the color of the solution (test tube b) changes from blue to colorless, and the corresponding absorption peak at 650 nm also decreases significantly. This indicates that catechin effectively inhibits the oxidation of TMB in the system. Therefore, Os-Ru nanozymes can be used to design a colorimetric detection solution platform for catechin.

[0074] Likewise, from Figure 5 As shown in Figure b, when the substrate solution (TMB + H₂O₂) is added to the paper (containing the Os-Ru nanozyme), the paper turns blue (paper a), corresponding to a high ΔG value (grayscale value of the blank group minus grayscale value of the sample group). However, when a high concentration of catechin (450 μmol / L) is added before the substrate solution, the paper does not change color (paper b). This indicates that catechin can also effectively inhibit the oxidation of TMB on the paper platform. Therefore, the Os-Ru nanozyme can be used to design a novel paper platform for colorimetric detection of catechins.

[0075] In summary, Os-Ru nanozymes can be used to develop a dual detection platform (solution platform and paper platform) for colorimetric detection of catechins.

[0076] Colorimetric detection mechanism of catechins based on Os-Ru nanozymes: The essence of nanozyme peroxidase-like activity is the production of hydroxyl radicals (HO·) by the nanozyme-catalyzed decomposition of H2O2, which then oxidizes chromogenic substrates such as TMB and ABTS. Therefore, it is reasonable to speculate that catechins can inhibit the oxidation of TMB in the reaction system by scavenging the HO· produced by the Os-Ru nanozyme-catalyzed decomposition of H2O2. To verify this hypothesis, the present invention used terephthalic acid (TA) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as HO· scavengers to investigate the amount of HO· produced by Os-Ru nanozymes in the presence or absence of catechins.

[0077] Figure 6 The results in Figure a show that the system (TA+H2O2+Os-Ru nanozyme) has a strong fluorescence emission near 435nm. This is because Os-Ru nanozyme catalyzes H2O2 to produce a large amount of HO·, which is captured by TA, thereby producing fluorescent TA-OH. However, when catechin (450μmol / L) is added, the fluorescence intensity of the system decreases significantly, indicating that catechin can scavenge the HO· produced by Os-Ru nanozyme catalyzing H2O2. In addition, Figure 6Figure b shows that the (DMPO+Os-Ru nanozyme+H2O2) system effectively generates a high signal with a ratio of 1:2:2:1, corresponding to the ESR signal of HO·. This further demonstrates that catechins have the ability to scavenge HO· produced by Os-Ru nanozyme-catalyzed H2O2.

[0078] Optimization of detection conditions for the catechin dual detection platform: In order to achieve the best sensing performance of the dual colorimetric platform based on Os-Ru nanozyme in catechin detection, the detection conditions of the solution platform and paper platform were optimized respectively. Figure 7 and Figure 8 shown.

[0079] The optimized conditions for the solution platform include the concentration of the Os-Ru nanozyme (expressed as the dilution factor), reaction time, pH, and reaction temperature. Results are expressed as the absorbance of the reaction system at 650 nm. Here, we selected the conditions corresponding to an absorbance of 0.8 to 1.2 as the optimal detection conditions.

[0080] Figure 7 Panel a shows that as the dilution factor increases (and the Os-Ru nanozyme concentration decreases), the absorbance gradually increases and then decreases. When the Os-Ru nanozyme solution is diluted 64-fold, the absorbance reaches its maximum value (approximately 2.18). When the dilution factor is less than 64, the color of the reaction system turns yellow-green, brown, or even black, and the absorbance decreases. This phenomenon is due to excessive oxidation of TMB by the excess Os-Ru nanozyme in the system. When the dilution factor exceeds 64, the color of the reaction system gradually fades from blue to lighter, corresponding to the gradual decrease in absorbance. When the Os-Ru nanozyme solution is diluted 256-fold, the color of the system is blue, and the absorbance is within the optimal range (0.8–1.2), so 256 was selected as the optimal dilution factor.

[0081] like Figure 7 As shown in Figure b, as the reaction time increases, the absorbance of the reaction system gradually increases, and the blue color of the system gradually deepens. At a reaction time of 10 minutes, the absorbance of the system is approximately 1.0, so 10 minutes is selected as the optimal reaction time.

[0082] from Figure 7 As shown in Figure c, as the pH value increases, the absorbance of the reaction system first increases and then decreases, and the color of the system changes from bright green to light blue to blue to blue-green to yellow-green to colorless. At a pH of 4.5, the color of the system turns blue, and the absorbance at a wavelength of 650nm reaches a maximum of approximately 1.12. Therefore, a pH of 4.5 was selected as the optimal reaction condition.

[0083] Figure 7 Figure d shows that as the reaction temperature increases, the absorbance of the reaction system first increases and then decreases, and the color of the system changes from blue to dark blue to blue-green to green to yellow. This is because as the reaction temperature increases, the activity of the Os-Ru nanozyme increases, better catalyzing the oxidation of TMB, causing the blue color of the system to deepen and the absorbance to increase. When the temperature is too high, most of the TMB in the system undergoes secondary oxidation, causing the color of the system to turn green and the absorbance to decrease. At a reaction temperature of 20°C, the absorbance of the system is around 1.0, indicating a blue color, making 20°C the optimal reaction temperature.

[0084] In summary, the optimal detection conditions of the solution platform are: dilution factor of Os-Ru nanozyme is 256, reaction time is 10 min, pH value is 4.5, and reaction temperature is 20 °C.

[0085] For the paper platform, the optimized conditions included the concentration of Os-Ru nanozyme (expressed as dilution multiple), reaction time, pH value and TMB concentration, and the results were expressed as △G value (gray value of blank paper - gray value of sample paper).

[0086] Figure 8 Figure (a) shows that as the dilution ratio increases (and the Os-Ru nanozyme concentration decreases), the ΔG value of the test paper first gradually increases before leveling off, and the color of the test paper gradually changes from yellow-green to blue and then to light blue. At a dilution ratio of 64, the ΔG value of the paper reaches its maximum (54.30), accompanied by a distinct blue hue. When the dilution ratio is less than 64, the paper color turns yellow or even yellow-green, and the ΔG value decreases. This is due to excessive oxidation of TMB by the excess Os-Ru nanozyme in the system. When the dilution ratio exceeds 64, the ΔG value decreases significantly, and the paper color turns light blue or even colorless. This is due to the weakening of the catalytic activity of the Os-Ru nanozyme by low concentration. Therefore, 64 is selected as the optimal dilution ratio.

[0087] Figure 8 Figure b shows that as the reaction time increases, the ∆G value of the paper first increases and then decreases, and the color of the paper gradually changes from light blue to dark blue, then to yellow-green, and finally to nearly colorless. At a reaction time of 10 minutes, the ∆G value reaches its maximum (approximately 58.20), at which point the blue color of the paper is most pronounced. When the reaction time exceeds 10 minutes, the color of the paper changes from dark blue to yellow-green, and finally to almost colorless, and the ∆G value of the paper decreases accordingly. This is due to the overoxidation of TMB and the decomposition of oxTMB caused by prolonged reaction time. Therefore, 10 minutes is selected as the optimal reaction time.

[0088] from Figure 8As shown in Figure c, pH also significantly affects the activity of the Os-Ru nanozyme. As pH increases, the ∆G value of the paper gradually increases and then decreases. At pH 4.0, the ∆G value of the paper is the highest (approximately 68.64), and the paper appears blue. Therefore, pH 4.0 is selected as the optimal reaction pH.

[0089] from Figure 8 Figure d shows that the concentration of the TMB solution also significantly affects the color and grayscale values ​​of the paper. As the TMB concentration decreases, the ∆G value of the paper first increases and then decreases, and the color of the paper changes from bluish-yellow to blue and then to light blue. When the TMB concentration is 3.75 mmol / L, the ∆G value is the highest and the color is the bluest, making 3.75 mmol / L the optimal TMB concentration.

[0090] In summary, the optimal detection conditions of the paper platform are: dilution factor of Os-Ru nanozyme is 64, reaction time is 10 min, pH value is 4.0, and TMB concentration is 3.75 mmol / L.

[0091] Anti-interference ability of catechin dual detection platform: In order to evaluate the anti-interference ability of Os-Ru nanozyme in detecting catechins, the inhibitory effects of common interfering substances in food (such as ions, sugars and amino acids) on the reaction system (Os-Ru nanozyme + TMB + H2O2) were investigated on the solution platform and paper platform respectively.

[0092] like Figure 9 As shown in Figure a, under the same conditions, the sample wells with catechin added are colorless and have a very low absorbance at 650nm. In contrast, the blank control wells and the sample wells with only interfering substances added are both blue and have similar absorbance (650nm). Figure 9 Panel b shows that in the presence of both catechins and interfering substances, all wells appear colorless, with minimal absorbance at 650 nm. These results demonstrate that this solution platform has excellent anti-interference capabilities for catechin detection.

[0093] Likewise, from Figure 9 As shown in Figure c, under the same conditions, only the sample paper with catechin added showed no color (the corresponding △G value was very low), while the blank control paper and the sample paper with only the interfering substance added showed blue-green color, and the △G value of the sample paper with the interfering substance added was close to that of the blank control paper. Figure 9Figure d shows that in the presence of both catechins and interfering substances, all papers showed no color development and exhibited low ΔG values. These results demonstrate that the paper platform also exhibits good selectivity for the detection of catechins. Therefore, the dual colorimetric platform based on the Os-Ru nanozyme exhibits excellent selectivity for the detection of catechins.

[0094] Dual colorimetric platform for catechin detection: Table 2 Comparison of catechin detection results using different detection methods Note: “-” means the item was not tested.

[0095] Based on the ability of catechins to scavenge HO·, the present invention first uses Os-Ru nanozymes as identifiers and TMB as signal generators to construct a catechin colorimetric detection solution platform. Figure 10 As shown in Figure a, with the increase of catechin concentration, the solution color of the Os-Ru nanozyme + TMB + H2O2 + catechin system gradually changes from the original blue to colorless, which corresponds to the gradual decrease of its absorbance value at a wavelength of 650nm. This is because as the concentration of catechin in the system increases, the amount of HO· produced by H2O2 catalyzed by Os-Ru nanozyme decreases, thereby preventing TMB in the system from being oxidized into a blue oxTMB product with a specific absorption peak at 650nm; at the same time, in the concentration range of 0μmol / L~450μmol / L, the inhibitory effect of catechin on the oxidation of TMB catalyzed by Os-Rh nanozyme shows a good linear relationship, and the linear correlation coefficient (R 2 ) was 0.997. The calculated limit of detection (LOD) for catechins using this solution platform was 2.84 μmol / L. Compared with other catechin detection methods (Table 2), this solution platform has a lower LOD, demonstrating the good sensitivity of this method.

[0096] Based on the high sensitivity of solution platform for detecting catechins, a colorimetric paper detection platform was established to make the detection more portable and convenient. Figure 10 As shown in Figure b. Figure 10 It can be clearly seen in Figure b that as the catechin concentration increases, the blue color of the paper platform gradually becomes lighter until it disappears. In particular, when the concentration exceeds 56.25μmol / L, the color of the paper is significantly different from that of the blank control paper. Therefore, this concentration can be regarded as the naked eye detection limit of the paper platform for catechins. Image J software was used to convert the color of the paper into the corresponding grayscale value, and a relationship diagram between the catechin concentration and the grayscale value of the paper was obtained. The results showed that within the concentration range of 0μmol / L~450μmol / L, the two showed a good linear relationship, R 2 is 0.990( Figure 10(Figure b in the figure). The calculated detection limit for catechins using this colorimetric paper platform is 9.68 μmol / L, which is lower than that of many other studies. Therefore, this colorimetric paper platform provides a convenient and sensitive platform for the detection of catechins.

[0097] In addition, the present invention also evaluated the stability of the color paper platform stored at room temperature for 60 days. Figure 11 As shown, the activity of the colorimetric paper platform did not decrease significantly within 60 days, and it has good room temperature storage stability. The above results show that the colorimetric paper platform developed by the present invention is easy to store and is easy to promote and apply in actual catechin detection.

[0098] Recovery analysis of spiked catechins in real samples: In order to evaluate the feasibility and reliability of the dual colorimetric detection platform based on Os-Ru nanozymes for the detection of catechins in practical applications, three green tea beverages containing catechins were selected as actual samples for spike recovery tests. Figure 12 As can be seen, the spiked catechin recoveries measured by the Os-Ru nanozyme-based dual colorimetric detection platform are very close to those determined by the HPLC-MS method. Furthermore, as shown in Table 3, the spiked catechin recoveries for the Os-Ru nanozyme-based colorimetric solution platform and the colorimetric paper platform ranged from 93.27% to 103.01% and 91.78% to 100.76%, respectively, with relative standard deviations (RSDs) of 2.17% to 6.74% and 1.62% to 4.57%, respectively. These recoveries are comparable to the spiked catechin recoveries (94.36% to 102.83%) and RSDs (1.69% to 2.97%) determined by the HPLC-MS method. These results demonstrate that the established Os-Ru nanozyme-based dual colorimetric detection platform exhibits excellent accuracy and reliability in practical applications and has considerable potential for rapid catechin detection.

[0099] Table 3 Recovery rate and relative standard deviation of catechins added to actual samples In summary, Os-Ru nanozyme was used as a probe to successfully develop a solution platform and a paper platform for catechin detection. First, Os-Ru nanozyme with excellent peroxidase-like activity was successfully synthesized by a one-step hydrothermal reaction. K mThe values ​​were 0.36 mmol / L and 2.67 mmol / L, respectively. Furthermore, catechins were found to effectively scavenge HO· generated by the system (Os-Ru nanozyme + H2O2). Based on this, after optimizing the detection conditions, a highly sensitive dual platform (solution platform and paper platform) was established for the direct colorimetric detection of catechins. The linear range for both the solution and paper platforms was 0 μmol / L–450 μmol / L. The detection limits for the solution platform were 2.84 μmol / L, while those for the paper platform were 56.25 μmol / L (naked eye) and 9.68 μmol / L (grayscale value). Furthermore, the dual detection platform demonstrated satisfactory accuracy and reliability in real-world sample applications, with spike recoveries ranging from 91.78% to 103.01% and RSDs from 1.62% to 6.74%, demonstrating considerable potential for practical application. In summary, this study can provide more ideas for the rational design and development of colorimetric sensing methods for rapid detection of antioxidants based on nanozymes, thereby broadening the application of nanozymes in practical, rapid and on-site detection of antioxidants.

[0100] It should be noted that when the present invention involves numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes preferred embodiments. Although preferred embodiments of the present invention have been described, those skilled in the art will be able to make additional changes and modifications to these embodiments once they understand the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

1. A method for preparing an osmium-ruthenium nanozyme, characterized in that: The steps include: Dissolving a soluble osmium salt, a soluble ruthenium salt, glycine and polyvinyl pyrrolidone in water to obtain a mixture; The mixture is mixed with citric acid solution and subjected to reduction reaction. During the reduction reaction, the citric acid in the citric acid solution decomposes into aconitic acid and reducing fragments, and Os 4+ and Ru 3+ It is reduced to an atomic state and forms an alloy core; at the same time, free osmium atoms and ruthenium atoms are deposited on the alloy core, and the osmium atoms and ruthenium atoms diffuse with each other to form a homogeneous alloy; then it is cooled to room temperature. During the cooling process, citric acid is re-adsorbed on the surface of the homogeneous alloy to form a protective layer, preventing O2 erosion and particle aggregation. After purification by dialysis, osmium-ruthenium nanozyme is obtained.

2. The method for preparing an osmium-ruthenium nanozyme according to claim 1, wherein: The mass ratio of soluble osmium salt, soluble ruthenium salt, glycine and polyvinyl pyrrolidone is 0.5-1.5:0.1-0.5:4-10:10-20.

3. The method for preparing an osmium-ruthenium nanozyme according to claim 1, wherein: The concentration of the citric acid solution is 0.2 mmol / L to 0.5 mmol / L, and the molar ratio of the total molar amount of osmium ions and ruthenium ions to the molar amount of citric acid is 1:3 to 5.

4. The method for preparing an osmium-ruthenium nanozyme according to claim 1, wherein: The reduction reaction conditions are: stirring the reaction at 80°C~120°C for 30min~60min.

5. The method for preparing an osmium-ruthenium nanozyme according to claim 1, wherein: The dialysis purification operation is as follows: the homogeneous alloy is dialyzed in deionized water using a 500Da dialysis bag for 12h~36h.

6. An osmium-ruthenium nanozyme, characterized in that The method is prepared according to any one of claims 1 to 5.

7. A catechin dual detection platform, characterized in that: Based on the osmium-ruthenium nanozyme constructed according to claim 6, the catechin dual detection platform is a solution platform or a paper platform; The solution platform is as follows: osmium-ruthenium nanozyme is mixed with 3,3',5,5'-tetramethylbenzidine solution and H2O2 to obtain blue oxidized 3,3',5,5'-tetramethylbenzidine diimine; catechin is added to the above reaction system, and catechin inhibits the oxidation of 3,3',5,5'-tetramethylbenzidine by osmium-ruthenium nanozyme, and the blue color becomes lighter; The paper platform is as follows: osmium-ruthenium nanozyme is loaded on the paper, and after it is naturally dried, catechin solution is first applied. After it is naturally dried, 3,3',5,5'-tetramethylbenzidine solution is added and the color is observed.

8. The catechin dual detection platform according to claim 7, characterized in that: The detection conditions of the solution platform are as follows: dilute the osmium-ruthenium nanozyme with a mass concentration of 100ug / mL by 200-300 times, the concentration of 3,3',5,5'-tetramethylbenzidine by 3.5mmol / L-4.0mmol / L, and react for 8min-12min after adding catechin at 20℃-25℃ and pH 4-5.

5.

9. The catechin dual detection platform according to claim 7, characterized in that: The detection conditions of the paper platform are as follows: dilute the osmium-ruthenium nanozyme solution with a mass concentration of 100ug / mL by 60 to 70 times, add the catechin solution at a concentration of 3.5mmol / L to 4.0mmol / L, and let it react for 8min to 12min.

10. The catechin dual detection platform according to claim 7, characterized in that: The detection limit of the solution platform was 2.84 μmol / L, the detection limit of the paper platform with naked eyes was 56.25 μmol / L, and the detection limit of the paper platform with grayscale analysis was 9.68 μmol / L.

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