Osmium-ruthenium nanoscale enzyme, and preparation method and application thereof

Abstract

This invention relates to the field of food safety technology, specifically to an osmium-ruthenium nanozyme, its preparation method, and its applications. The preparation method includes: dissolving soluble osmium salt, soluble ruthenium salt, glycine, and polyvinylpyrrolidone together in water to obtain a mixture; mixing the mixture with citric acid solution and carrying out a reduction reaction, followed by cooling to room temperature and purification by dialysis to obtain the osmium-ruthenium nanozyme. This invention prepares osmium-ruthenium nanozyme through a one-step hydrothermal reaction, exhibiting excellent peroxidase-like activity. The catechin dual detection platform constructed based on the osmium-ruthenium nanozyme of this invention, including a solution platform and a paper platform, has the advantages of fast response speed, simple operation, and relatively low cost, overcoming the shortcomings of existing technologies.

Description

Technical Field

[0001] This invention relates to the field of food safety technology, specifically to an osmium-ruthenium nanozyme, its preparation method, and its application. Background Technology

[0002] Catechins are phenolic active substances extracted from natural plants. They are commonly found in tea and are the main bioactive components of tea. Catechins have been reported to possess various biological activities, including anti-inflammatory, antibacterial, anticancer, cardiovascular protective, blood sugar lowering, and free radical scavenging effects, and are therefore widely used in the medical and food fields. However, once the catechin content exceeds the safe threshold, it can harm human health, causing symptoms such as nausea and dizziness. Therefore, establishing an efficient, sensitive, and reliable method for detecting catechins is of great significance 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 generally suffer from limitations such as complex and time-consuming operation, high cost, and inability to achieve visual detection, making it difficult to meet the current demand for efficient and accurate determination of catechins. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an osmium-ruthenium nanozyme, its preparation method, and its applications. Using soluble osmium salt, soluble ruthenium salt, glycine, and polyvinylpyrrolidone as raw materials, this invention prepares an osmium-ruthenium nanozyme with excellent peroxidase-like activity through a one-step hydrothermal reduction reaction. Based on this osmium-ruthenium nanozyme, a dual-detection platform for catechins is constructed. This dual-detection platform utilizes the osmium-ruthenium nanozyme to catalyze the generation of hydroxyl radicals (HO·) from H₂O₂. HO· oxidizes 3,3',5,5'-tetramethylbenzidine solution (TMB) for color development. Subsequently, catechins scavenge HO·, inhibiting the color development, thus achieving rapid and sensitive colorimetric detection of catechins. It has the advantages of fast response (8-12 minutes), simple operation (no complex instruments required), and low cost (easy-to-obtain raw materials), overcoming the shortcomings of existing detection methods.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The first objective of this invention is to provide a method for preparing the aforementioned osmium-ruthenium nanozyme, comprising the following steps:

[0007] S1. Dissolve soluble osmium salt, soluble ruthenium salt, glycine, and polyvinylpyrrolidone together 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 nanozymes, such as catalytic activity, optical performance, and electrochemical performance.

[0008] S2. Mix the mixture with a citric acid solution and carry out a reduction reaction. During the reduction reaction, the citric acid in the citric acid solution decomposes into aconitic acid and reducing fragments. 4+ and Ru 3+ Glycine is reduced to its atomic state and forms an alloy core. During this process, glycine exhibits amphoteric molecular properties, maintaining the pH stability of the reaction system. In the reduction reaction, glycine decomposes to produce reducing substances (such as ammonia and aldehydes), which assist in the reduction of Os. 4+ and Ru 3+ The amino and carboxyl groups on the glycine molecule are related to Os... 4+ Ru 3+ Forming coordinate bonds effectively slows down Os 4+ and Ru 3+ The reduction rate is controlled to avoid explosive nucleation, which is conducive to the formation of particles with uniform size. At the same time, free osmium and ruthenium atoms are deposited on the alloy core, and the osmium and ruthenium atoms diffuse into each other to form a homogeneous alloy. Polyvinylpyrrolidone is adsorbed on the surface of osmium and ruthenium atoms or the surface of the alloy core through the carbonyl oxygen on its pyrrole ring. Its long-chain molecular structure forms a steric hindrance layer, which effectively prevents the aggregation of nanoparticles. Subsequently, 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, which prevents O2 corrosion and particle aggregation. After dialysis purification, osmium-ruthenium nanozyme is obtained.

[0009] Preferably, the mass ratio of osmium salt solution, ruthenium salt solution, glycine and polyvinylpyrrolidone is 0.5~1.5:0.1~0.5:4~10:10~20.

[0010] Preferably, the dissolution conditions are: stirring at 300r / min to 500r / min for 5min to 30min at room temperature.

[0011] Preferably, the concentration of citric acid is 0.2 mmol / L to 0.5 mmol / L, and the total molar ratio of osmium ions and ruthenium ions to the molar ratio of citric acid is 1:3 to 5.

[0012] Preferably, the reduction reaction conditions are: stirring at 1200r / min to 1800r / min for 30min to 60min at 80℃ to 120℃. Citric acid has a relatively mild reducing ability at room temperature. Under heating conditions, citric acid molecules will undergo partial decarboxylation or dehydration reactions to generate more reducing intermediate products, such as aconitine. These thermal decomposition products have stronger reducing ability and can improve the reduction rate and accelerate the chemical reaction rate.

[0013] Preferably, the dialysis purification procedure is as follows: the mixture is dialyzed in deionized water using a 500 Da dialysis bag for 12 h to 36 h.

[0014] The second objective of this invention is to provide an osmium-ruthenium nanozyme, prepared using the above-described method.

[0015] The third objective of this invention is to provide a dual catechin detection platform, which is based on the osmium-ruthenium nanozyme described above, and the dual catechin detection platform is a solution platform or a paper platform.

[0016] The solution platform consisted of mixing osmium-ruthenium nanozyme with a solution of 3,3',5,5'-tetramethylbenzidine and H2O2 to obtain a blue oxidized form of 3,3',5,5'-tetramethylbenzidine diimide. Catechins were added to the above reaction system, and the catechins inhibited the oxidation of 3,3',5,5'-tetramethylbenzidine by the osmium-ruthenium nanozyme, causing the blue color to lighten.

[0017] The paper platform was prepared by loading osmium-ruthenium nanozymes onto paper, allowing it to air dry, then first applying a catechin solution, and after air drying, adding a 3,3',5,5'-tetramethylbenzidine solution and observing the color.

[0018] The detection principle is as follows: Os 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 state change of the metal center (Os or Ru atom), ultimately reducing H2O2 to HO·, which has 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.

[0019] Preferably, the detection conditions for the solution platform are as follows: dilute the osmium-ruthenium nanozyme solution (100 μg / mL) 200 to 300 times, and the concentration of 3,3',5,5'-tetramethylbenzidine is 3.5 mmol / L to 4.0 mmol / L. React the solution at 20°C to 25°C and pH 4 to 5.5 for 8 to 12 minutes.

[0020] Preferably, the detection conditions for the paper platform are as follows: diluting the osmium-ruthenium nanozyme solution with a mass concentration of 100 ug / mL by 60 to 70 times, setting the concentration of 3,3',5,5'-tetramethylbenzidine to 3.5 mmol / L to 4.0 mmol / L, setting the pH of the reaction system to 4.0 to 5.0, and setting the reaction time to 8 min to 12 min.

[0021] Preferably, the detection limit for the solution platform is 2.84 μmol / L, the detection limit for the paper platform is 56.25 μmol / L for naked eye detection, and the detection limit for grayscale analysis is 9.68 μmol / L.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. This invention provides a method for preparing osmium-ruthenium nanozymes, comprising dissolving soluble osmium salt, soluble ruthenium salt, glycine, and polyvinylpyrrolidone together in water to obtain a mixture; mixing the mixture with a citric acid solution and carrying out a reduction reaction, wherein during the reduction reaction, the citric acid in the citric acid solution decomposes into aconitic acid and reducing fragments, Os 4+ and Ru 3+ Glycine is reduced to its atomic state and forms an alloy core. During this process, glycine exhibits amphoteric molecular properties, maintaining the pH stability of the reaction system. In the reduction reaction, glycine decomposes to produce reducing substances (such as ammonia and aldehydes), which assist in the reduction of Os. 4+ and Ru 3+ The amino and carboxyl groups on the glycine molecule are related to Os... 4+ Ru 3+ Forming coordinate bonds effectively slows down Os 4+ and Ru 3+ The reduction rate is optimized to avoid explosive nucleation, which is beneficial for forming particles of uniform size. Simultaneously, free osmium and ruthenium atoms deposit onto the alloy core, and interdiffusion between osmium and ruthenium atoms forms a homogeneous alloy. Polyvinylpyrrolidone (PVP) adsorbs onto the surface of osmium and ruthenium atoms or the alloy core through the carbonyl oxygen on its pyrrole ring. Its long-chain molecular structure forms a steric hindrance layer, effectively preventing the aggregation of nanoparticles. Subsequently, cooling to room temperature allows citric acid to re-adsorb onto the homogeneous alloy surface during the cooling process, forming a protective layer that prevents O2 corrosion and particle aggregation. After dialysis purification, osmium-ruthenium nanozyme is obtained. This invention prepares osmium-ruthenium nanozyme through a one-step hydrothermal reaction, exhibiting excellent peroxidase-like activity. Compared to existing technologies, the catechin dual detection platform constructed based on the osmium-ruthenium nanozyme of this invention has the advantages of fast response speed, simple operation, and relatively low cost, overcoming the shortcomings of existing technologies.

[0024] 2. This invention constructs a dual-detection platform for catechins, comprising a solution platform and a paper platform. Osmium-ruthenium nanozyme, through its peroxidase-mimicking activity, effectively catalyzes the generation of hydroxyl radicals (HO∙) from H₂O₂, oxidizing 3,3',5,5'-tetramethylbenzidine (hereinafter referred to as TMB) to produce the blue product oxidized 3,3',5,5'-tetramethylbenzidine diimine (hereinafter referred to as oxTMB), thus turning both the solution and paper platforms blue. Catechins can scavenge the HO∙ generated by the osmium-ruthenium nanozyme catalyzing H₂O₂, 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 by observing the color changes of the solution and paper platforms.

[0025] Furthermore, the dual-platform direct colorimetric detection of catechins of this invention exhibits excellent sensitivity, with a detection limit of 2.84 μmol / L for the solution platform, a visual detection limit of 56.25 μmol / L for the paper platform, and a grayscale detection limit of 9.68 μmol / L.

[0026] 3. The dual catechin 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 dual catechin detection platform of the present invention and provided more ideas for designing and developing colorimetric sensing methods based on nanozymes for rapid detection of antioxidants. Attached Figure Description

[0027] Figure 1 The images show the characterization of osmium-ruthenium nanozymes, where a is a TEM image, b is the size distribution, c is the EDS spectrum, d is the XRD pattern, e is the zeta potential, and f is the UV-vis spectrum.

[0028] Figure 2 This is a graph showing the peroxidase-like activity of osmium-ruthenium nanozymes.

[0029] Figure 3 The figure shows the steady-state kinetics of osmium-ruthenium nanozymes, where a represents TMB and b represents H2O2.

[0030] Figure 4 The image shows the characterization of osmium-ruthenium nanozymes after storage at room temperature for 90 days. In the image, a represents TEM, b represents size distribution, and c represents peroxidase-like activity.

[0031] Figure 5 Figure 1 shows the feasibility test results for the catechin solution platform and the paper platform. In figure 1, a represents the solution platform; in the insets a1, a2 represents TMB + H2O2 + osmium-ruthenium nanozyme; a3 represents TMB + H2O2 + catechin (450 μmol / L) + osmium-ruthenium nanozyme; and b represents the paper platform; in the insets b1, b2 represents TMB + H2O2 + osmium-ruthenium nanozyme; and b3 represents TMB + H2O2.

[0032] Figure 6 The images show the fluorescence and ESR spectra measured at an excitation wavelength of 315 nm, where a is the fluorescence spectrum and b is the ESR spectrum.

[0033] Figure 7 The figure shows the optimization results of the detection conditions for the solution platform based on osmium-ruthenium nanozymes. In the figure, a is the concentration of osmium-ruthenium nanozymes, b is the reaction time, c is the pH value, and d is the reaction temperature.

[0034] Figure 8 The figure shows the optimization results of the detection conditions of the paper platform 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.

[0035] Figure 9 The graph shows the selectivity of osmium-ruthenium nanozyme for detecting catechins. In the graph, a represents the color and absorbance at 650 nm of the system with added catechins on the solution platform; b represents the color and absorbance at 650 nm of the system with added catechins and interfering substances on the solution platform; c represents the color and grayscale value of the system with added catechins on the paper platform; and d represents the color and grayscale value of the system with added catechins and interfering substances on the paper platform.

[0036] Figure 10 The graphs show the performance of the dual-colorimetric platform for detecting catechins. In the graphs, a represents 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 represents the color change and linear curve of the osmium-ruthenium nanozyme + TMB + H2O2 system in the paper platform under the action of different concentrations of catechins.

[0037] Figure 11 The figure shows the storage stability test results of the paper platform based on osmium-ruthenium nanozymes.

[0038] Figure 12 The graph shows the spiked detection results of actual samples using the dual-colorimetric detection platform and instrument detection method, where a represents 200 μmol / L and b represents 400 μmol / L. Detailed Implementation

[0039] The technical solution of the present invention will be clearly and completely described below with reference to the data in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0040] It should be noted that the technical terms used in this invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the following embodiments of this invention can be purchased on the market or prepared by existing methods.

[0041] In existing technologies, the detection of catechins mainly relies on instrumental analytical methods, such as gas chromatography, high-performance liquid chromatography, Raman spectroscopy, and electrochemical methods. However, these methods all require large instruments and generally suffer from limitations such as complex and time-consuming operation, high cost, and inability to achieve visual detection, making it difficult to meet the current demand for efficient and accurate determination of catechins.

[0042] To address the issues of equipment dependence, operational complexity, and inability to detect catechins with the naked eye in existing technologies, this invention utilizes a prepared osmium-ruthenium nanozyme to construct a dual-detection platform for catechins, replacing traditional instruments. Specifically, it leverages the high catalytic activity of osmium-ruthenium nanozyme to mimic peroxidase, converting catechin concentration into a visible color change through a colorimetric reaction (TMB-H2O2 system), eliminating the need for complex equipment. Furthermore, the dual-detection platform constructed in this invention achieves a high sensitivity of 2.84 μmol / L for solution platforms by optimizing pH, temperature, and the dilution factor of the osmium-ruthenium nanozyme. For paper platforms, combined with osmium-ruthenium nanozyme immobilization technology, it supports both naked-eye qualitative (56.25 μmol / L) and gray-scale quantitative (9.68 μmol / L) detection, meeting the needs of different scenarios.

[0043] 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:

[0044] Example 1

[0045] A method for preparing osmium-ruthenium nanozymes includes the following steps:

[0046] 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 sequence; after stirring in a vortex at 400 r / min for 10 min at room temperature, a mixture is obtained.

[0047] S2. Add the mixture to a three-necked flask containing 50 mL of 0.3 mmol / L citric acid solution (preheated to 100 °C), stir vigorously at 1800 r / min for 40 min, and allow it to cool naturally to room temperature. Then, dialyze it 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.

[0048] Example 2

[0049] An osmium-ruthenium nanozyme preparation method is the same as that 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, thus obtaining the osmium-ruthenium nanozyme.

[0050] Example 3

[0051] An osmium-ruthenium nanozyme preparation method is the same as that 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, thus obtaining the osmium-ruthenium nanozyme.

[0052] Example 4

[0053] An osmium-ruthenium nanozyme preparation method is the same as that in Example 1, except that the amount of citric acid solution in S2 is replaced from 50 mL to 30 mL to obtain the osmium-ruthenium nanozyme.

[0054] a. Experimental Section:

[0055] Study on the peroxidase-like activity of Os-Ru nanozymes:

[0056] First, the peroxidase-like activity of the Os-Ru nanozyme was evaluated to see if it could catalyze the oxidation of the chromogenic substrate TMB in the presence of H2O2. In short, 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 min. The UV-Vis absorption spectra (500 nm–800 nm) were then recorded using a microplate reader. The control groups (containing only TMB and H2O2) and the control group (containing only TMB and Os-Ru nanozyme) were used to observe the effect of H2O2 or Os-Ru nanozyme alone on the oxidation of TMB. The control group (containing neither H2O2 nor TMB) was used as the blank group, and all results were filtered out for blank values.

[0057] The catalytic properties of the Os-Ru nanozyme were further investigated using steady-state kinetic analysis. In short, 20 μL of Os-Ru nanozyme solution (diluted 256-fold) and 200 μL of substrate solution containing 10 mmol / L H₂O₂ and different concentrations of TMB (8.24 mmol / L, 4.12 mmol / L, 2.06 mmol / L, 1.03 mmol / L, 0.51 mmol / L, 0.26 mmol / L), or 2.0 mmol / L TMB and different 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, 0.15625 mmol / L) were added to a 96-well plate and reacted for 10 min at room temperature. The absorbance at 650 nm was then measured using a microplate reader. The group without added TMB and H2O2 was designated as the blank control group, and blank values ​​were removed from all results. The concentration of oxTMB produced by the above system was calculated using the following formula:

[0058] (1)

[0059] in, A This represents the system absorbance value measured at a wavelength of 650 nm. ξ The molar absorption coefficient of oxTMB is 3.9 × 10⁻⁶. 4 M -1 cm -1 ), b Represents the thickness of the absorption layer. c This represents the concentration of oxTMB.

[0060] Then, the concentration of oxTMB produced per unit time is calculated using the following formula ( V 0 ):

[0061] (2)

[0062] in, t The reaction time is 10 min in this invention.

[0063] Then, the reciprocal of the concentration of oxTMB produced per unit time (1 / V 0 The reciprocal of the concentration of the substrate TMB (or H2O2) and the concentration of the substrate TMB (or H2O2) (1 / [S] Plot the graph using the vertical and horizontal axes as the ordinates. Finally, calculate the Michaelis constant using the following formula ( ). K m ) and maximum reaction rate ( V max ):

[0064] (3)

[0065] Long-term stability and sustainability study of Os-Ru nanozymes:

[0066] The morphological and size changes of Os-Ru nanozymes after 90 days of storage at room temperature were monitored by TEM to investigate the long-term structural stability. Furthermore, the sustainability of the physicochemical properties of Os-Ru nanozymes was studied by monitoring their peroxidase-simulated activity after 90 days of storage at room temperature. In short, 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 for 10 min at room temperature, the absorbance of the system at 650 nm was measured using a microplate reader. The absorbance value measured on day 0 was set as 100% peroxidase-simulated activity.

[0067] Feasibility of direct colorimetric detection of catechins using an Os-Ru nanozyme-based dual detection platform:

[0068] The feasibility of direct detection of catechins using a dual-platform approach with Os-Ru nanozymes was explored by evaluating the inhibitory effect of high concentrations of catechins on the peroxidase-like activity of Os-Ru nanozymes on solution and paper platforms.

[0069] In short, on a solution platform, 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 substrate solution containing 2.0 mmol / L TMB and 10 mmol / L H2O2 were added to a 96-well plate. After reacting for 10 min at room temperature, the reaction was photographed using an iPhone 14 Pro, and the UV absorption spectrum of the reaction system in the wavelength range of 500 nm to 800 nm was obtained using a microplate reader. Furthermore, the group without catechin or without Os-Ru nanozyme served as the control group.

[0070] On a paper platform, 10 μL of Os-Ru nanozyme solution (64-fold dilution of the original solution) was first added to a clean test strip with a diameter of 8 mm, and allowed to air dry at room temperature to prepare a catechin test strip. Then, 10 μL of catechin solution (450 μmol / L) was added to the test strip, and allowed to air dry at room temperature. Next, 20 μL of reaction substrate solution (containing 3.75 mmol / L TMB and 10 mmol / L H2O2) was added to the test strip. After reacting for 10 min at room temperature, the color of the test strip was recorded using an iPhone 14 Pro, and the grayscale (G) value of the test strip was obtained using ImageJ software. Additionally, the group without catechin and Os-Ru nanozyme was designated as the control group, and the group containing catechin and Os-Ru nanozyme was designated as the blank group. △G is the difference between the G value of the blank group and the G value of the experimental or control group.

[0071] Os-Ru nanozyme-based colorimetric detection mechanism for catechins:

[0072] The colorimetric detection mechanism of catechins based on Os-Ru nanozymes was investigated by monitoring the generation of hydroxyl radicals (HO·) in the reaction system. Terephthalic acid (TA) and 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) were selected as HO· scavengers. The systems (TA (100 μL, 5 mmol / L) + H₂O₂ (800 μL, 10 mmol / L) + Os-Ru nanozyme (50 μL, 64-fold dilution of the original solution)) and (TA (100 μL, 5 mmol / L) + H₂O₂ (800 μL, 10 mmol / L) + Os-Ru nanozyme (50 μL, 64-fold dilution of the original solution) + catechins (900 μmol / L)) were reacted at 45°C for 30 min, and the fluorescence spectra were recorded using a fluorescence spectrophotometer with an excitation wavelength of 315 nm. Furthermore, the system... The electron spin resonance (ESR) spectra of the systems (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 reacting at pH 6 for 5 min.

[0073] Optimization of detection conditions for the dual-detection platform for catechins:

[0074] To achieve optimal sensing performance for catechin detection, the sensing conditions for both the solution platform and the paper platform were optimized.

[0075] For the solution platform, the optimized conditions include the concentration of Os-Ru nanozyme (expressed as dilution factor of the original solution: 0, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048), the enzyme-catalyzed reaction time (0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min), the reaction temperature (20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃), and the 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). In short, 200 μL of substrate solution (containing 2.0 mmol / L TMB and 10 mmol / L H2O2) and 20 μL of Os-Ru nanozyme solution diluted by a certain factor were first added to a 96-well plate. The system was then reacted at specific pH and temperature conditions for a certain period of time. Next, the absorbance of the system at 650 nm was measured using a microplate reader. The group without added substrate solution served as a blank control group. The final absorbance values ​​for all groups were the absorbance values ​​after removing the absorbance values ​​of the blank control group. Finally, a graph was plotted with absorbance values ​​on the ordinate and Os-Ru nanozyme concentration, reaction temperature, reaction time, and system pH values ​​on the x-axis.

[0076] For the paper platform, optimized conditions included Os-Ru nanozyme concentration (expressed as dilution factors of the original solution: 0, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048), enzyme 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 optimized 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 reacting for a certain time at room temperature and specific pH conditions, the color of the test strips was recorded by taking photos with an iPhone 14 Pro, and the grayscale (G) value of the test strips was obtained using ImageJ software. Test strips without added substrate solution were designated as the blank group, and the ΔG value of the test strips was the difference between the G values ​​of the blank group and the experimental group. Finally, a graph was plotted with the grayscale value ΔG as the ordinate and the Os-Ru nanozyme concentration, TMB solution concentration, reaction time, and system pH value as the abscissa.

[0077] The anti-interference capability of the catechin dual detection platform:

[0078] The inhibitory effect of common food-related interfering substances (such as ions, sugars, and amino acids) on the reaction system (osmium-ruthenium + TMB + H2O2) was investigated, and the anti-interference ability of the Os-Ru nanozyme-based dual-platform detection of catechins was evaluated. The concentration of each interfering substance was 900 μmol / L on both the solution and paper platforms.

[0079] On a solution colorimetric platform, 20 μL of Os-Ru nanozyme (256-fold dilution of the original solution), 10 μL of catechin (450 μmol / L) and / or interfering substance (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 min at room temperature and pH 4.5, the absorbance of the system at 650 nm was measured using a microplate reader. The group containing only Os-Ru nanozyme + TMB + H2O2 without the addition of catechin and interfering substance was designated as the positive control group (blank group).

[0080] On a paper colorimetric platform, 10 μL of Os-Ru nanozyme solution (a 64-fold dilution of the original solution) was first added to a clean test strip with a diameter of 8 mm and allowed to air dry to obtain a catechin test strip. Then, 10 μL of catechin (450 μmol / L) and / or interfering substance (900 μmol / L) was added to the test strip and allowed to air dry at room temperature. Next, 20 μL of reaction substrate solution (containing 3.75 mmol / L TMB and 10 mmol / L H2O2) was added to the test strip. The reaction was carried out at room temperature and pH 4.0 for 10 min. A photo was then taken with an iPhone 14 Pro, and the image was processed using ImageJ software to obtain the grayscale value (G) of the test strip. △G is the difference between the G value of the control group (without catechin, interfering substance, and reaction substrate) and the G value of the experimental group. The group without added catechin and interfering substance was designated as the blank group.

[0081] A two-colorimetric platform is used for catechin detection:

[0082] In summary, on a solution colorimetric platform, 20 μL of Os-Ru nanozyme (a 256-fold dilution of the original solution), 10 μL of catechin solutions at different concentrations (0 μmol / L–450 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 min at room temperature and pH 4.5, the absorbance of the system at 650 nm was measured using a microplate reader. The group without the reaction substrate solution served as a blank control. The final absorbance values ​​for all groups were the absorbance values ​​after removing the absorbance values ​​of the blank control group. Finally, a standard curve was plotted with absorbance values ​​on the ordinate and catechin concentration on the abscissa.

[0083] The limit of detection (LOD) for the solution platform is defined as the concentration of catechins corresponding to a signal-to-noise ratio (S / N) of 3.

[0084] (4)

[0085] Where SD is the standard deviation of the signal measured multiple times in the blank control group, and m is the slope of the standard curve.

[0086] On a paper colorimetric platform, 10 μL of catechin solutions of different concentrations (0 μmol / L to 450 μmol / L) were added to the test paper. After air-drying at room temperature, 20 μL of the reaction substrate solution (containing 3.75 mmol / L TMB and 10 mmol / L H2O2) was added. The test paper was then reacted for 10 min at room temperature and pH 4.0. Next, an iPhone 14 Pro was used to take a picture, and the grayscale value (G) of the test paper was obtained using ImageJ software. A standard curve was plotted with the grayscale value (G) on the ordinate and the catechin concentration on the abscissa. The subsequent steps were the same as those for the solution colorimetric platform described above.

[0087] Room temperature storage stability of paper colorimetric platforms:

[0088] To facilitate future storage, we investigated the stability of the Os-Ru nanozyme colorimetric strip at room temperature by measuring its catalytic activity on days 0, 10, 20, 30, 40, 50, and 60. In short, 20 μL of the substrate solution (containing 3.75 mmol / L TMB and 10 mmol / L H₂O₂) was added to the colorimetric strip at different storage days. After reacting at room temperature for 10 min, the color of the strip was recorded by photographing it with an iPhone 14 Pro, and the color was converted to a grayscale (G) value using ImageJ software. The G value on day 0 was set as 100% relative activity.

[0089] Spiked recovery analysis of catechins in real samples:

[0090] To verify the feasibility of the dual-colorimetric detection platform developed based on Os-Ru nanozymes in actual sample detection, three green tea beverages—Master Kong, Vita, and Yulu—were selected as actual samples for catechin spiked recovery analysis. First, the three green tea beverages were centrifuged at 25°C and 12000 rpm for 10 min, and the supernatant was collected. Then, the supernatant was diluted 100 times with distilled water and filtered through a 0.22 μm filter membrane. Next, different amounts of catechins were weighed and added to the diluted solutions of the three beverages to obtain catechin spiked recovery solutions with spike concentrations of 300 μmol / L, 150 μmol / L, and 0 μmol / L, respectively. Finally, the above catechin spiked recovery solutions were detected using the solution colorimetric platform and paper colorimetric platform constructed in this invention, as well as high-performance liquid chromatography-mass spectrometry.

[0091] b. Results and Discussion:

[0092] Characterization of Os-Ru nanozymes:

[0093] To verify the successful synthesis of Os-Ru nanozymes, they were characterized by TEM, EDS, XRD, Zeta potential, and UV-vis spectroscopy. Figure 1 Figures a and b show that the Os-Ru nanozymes are dispersed elliptical particles with an average particle size of approximately 19.48 nm. Figure 1 The results in Figure c show that the Os-Ru nanozyme contains Os and Ru elements, and two peaks appear at 1.91 keV and 2.56 keV, corresponding to the EDS peaks of Os and Ru elements, respectively. Furthermore, from... Figure 1 The d-plot revealed five diffraction peaks for the Os-Ru nanozyme at 38.0°, 38.4°, 41.8°, 43.6°, and 44.0°, corresponding to (100), (002), and (101) peaks of fcc Os (JCPDS06-0662) and (100) and (101) peaks of fcc Ru (JCPDS06-0663), respectively. Furthermore, Figure 1 Figures e and f show that the zeta potential of the Os-Ru nanozyme is approximately -18.79 ± 0.87 mV, with a maximum absorption peak near 526 nm. Figure 1 The Os-Ru nanozyme of this invention was successfully prepared.

[0094] Os-Ru nanozymes exhibit peroxidase-like activity:

[0095] To verify whether Os-Ru nanozymes possess peroxidase-like activity, TMB was selected as the chromogenic substrate to investigate the catalytic oxidation ability of Os-Ru nanozymes. Figure 2As shown, Os-Ru nanozymes cannot directly catalyze the oxidation of TMB, but in the presence of H2O2, TMB can be catalyzed to be oxidized into a blue product with the strongest absorption peak near 650 nm, indicating that Os-Ru nanozymes have peroxidase-like activity.

[0096] To further evaluate the peroxidase-like activity of Os-Ru nanozymes, steady-state kinetic studies were conducted. Michaelis constant ( K m The initial velocity (%) reflects the enzyme's affinity for the corresponding substrate; the smaller the value, the stronger the affinity. V max The value represents the maximum rate of the catalytic reaction; the larger the value, the faster the reaction rate. Figure 3 Figures a and b in the diagram are typical Michaelis-Menten curves established using TMB and H2O2 as substrates, respectively. After final calculations, the Os-Ru nanozyme exhibits [a] responsiveness to TMB. K m and V max The concentrations were 0.36 mmol / L and 6.75 × 10⁻⁶, respectively. -6 Ms -1 , for H2O2 K m and V max The concentrations were 2.67 mmol / L and 1.25 × 10⁻⁶, respectively. -6 Ms -1 .

[0097] Table 1. K content of Os-Ru nanozymes and other catalysts m and V max Test Result Table

[0098]

[0099] Note: "-" indicates that the project was not tested.

[0100] Table 1 lists HRP and other reported nanozymes with peroxidase-like activity. K m and V max The results showed that the Os-Ru nanozyme had a higher affinity for TMB and H2O2 and a higher maximum reaction rate than HRP and other nanozymes. This indicates that the Os-Ru nanozyme obtained in this invention has excellent peroxidase-like activity and is expected to become a candidate material for nano-peroxidases.

[0101] Long-term stability and sustainability of Os-Ru nanozymes:

[0102] To further verify the practical application potential of Os-Ru nanozymes, the long-term storage stability of their structure and the sustainability of their peroxidase-like activity were investigated. Figure 4 The results showed that the Os-Ru nanozyme retained its original morphology (dispersed elliptical particles, such as...) after being stored at room temperature for 90 days. Figure 4 (Figure a in the image), size (approximately 19.32 nm), Figure 4 (Figure b in the figure) and almost unchanged peroxidase-like activity ( Figure 4 (See Figure c in the table). These results demonstrate that Os-Ru nanozymes possess good long-term structural stability and sustained peroxidase-like activity.

[0103] Feasibility of colorimetric detection of catechins using a dual detection platform based on Os-Ru nanozymes:

[0104] To evaluate the feasibility of dual-platform colorimetric detection of catechins based on Os-Ru nanozymes, the effects of catechins on the absorbance and color of the system (Os-Ru nanozyme + TMB + H2O2) were investigated on both a solution platform and a paper platform. Figure 5 Figure a shows that after adding a high concentration (450 μmol / L) of catechin to the system, the color of the solution (test tube b) changed from blue to colorless, and the corresponding absorption peak at 650 nm also decreased significantly. This indicates that catechin can effectively inhibit the oxidation of TMB in the system. Therefore, Os-Ru nanozymes can be used to design a colorimetric detection solution platform for catechin.

[0105] Similarly, from Figure 5 As shown in Figure b, when the substrate solution (TMB + H₂O₂) is added to the paper (containing Os-Ru nanozyme), the paper turns blue (paper a), corresponding to a high ΔG value (gray value of the blank group minus gray value of the sample group). However, when a certain amount of high-concentration catechin (450 μmol / L) is added before adding the substrate solution, the test paper does not change color (test paper b). This indicates that catechin can also effectively inhibit the oxidation of TMB on the paper platform. Therefore, Os-Ru nanozyme can be used to design novel paper platforms for colorimetric detection of catechin.

[0106] In summary, Os-Ru nanozymes can be used to develop a dual detection platform (solution platform and paper platform) for colorimetric detection of catechins.

[0107] Os-Ru nanozyme-based colorimetric detection mechanism for catechins:

[0108] The essence of the peroxidase-like activity of nanozymes lies in the catalytic decomposition of H2O2 by nanozymes to generate hydroxyl radicals (HO·), which further oxidize 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 HO· generated from the decomposition of H2O2 by Os-Ru nanozymes. To verify this speculation, this invention used terephthalic acid (TA) and 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) as HO· scavengers to investigate the amount of HO· generated by Os-Ru nanozymes catalyzing H2O2 in the presence or absence of catechins.

[0109] Figure 6 Figure a shows that the system (TA + H₂O₂ + Os-Ru nanozyme) exhibits strong fluorescence emission near 435 nm. This is because the Os-Ru nanozyme catalyzes the production of a large amount of HO· from H₂O₂, which is captured by TA, resulting in fluorescent TA-OH. However, when catechin (450 μmol / L) is added, the fluorescence intensity of the system significantly decreases, indicating that catechin can remove the HO· produced by the Os-Ru nanozyme catalyzing H₂O₂. Furthermore, from... Figure 6 Figure b shows that the (DMPO+Os-Ru nanozyme+H2O2) system can effectively generate a high signal with a ratio of 1:2:2:1, corresponding to the ESR signal of HO·. This further indicates that catechins have the ability to scavenge HO· produced by Os-Ru nanozyme catalyzing H2O2.

[0110] Optimization of detection conditions for the dual-detection platform for catechins:

[0111] To optimize the sensing performance of the Os-Ru nanozyme-based dual-colorimetric platform in catechin detection, the detection conditions for both the solution platform and the paper platform were optimized. The results are as follows: Figure 7 and Figure 8 As shown.

[0112] The optimized conditions for the solution platform included the concentration of Os-Ru nanozyme (expressed as dilution factor), reaction time, pH value, and reaction temperature. The results are expressed as the absorbance value of the reaction system at 650 nm. Here, we selected the conditions corresponding to the system absorbance values ​​of 0.8–1.2 as the optimal detection conditions.

[0113] Figure 7Figure a shows that as the dilution factor increases (the concentration of Os-Ru nanozyme decreases), the absorbance of the system gradually increases and then decreases. The absorbance reaches its maximum value (approximately 2.18) when the Os-Ru nanozyme solution is diluted 64-fold. 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 caused by an excess of Os-Ru nanozyme in the system. When the dilution factor is greater than 64, the color of the reaction system gradually lightens from blue, corresponding to the gradual decrease in absorbance. When the Os-Ru nanozyme solution is diluted 256-fold, the system is blue, and the absorbance is within the optimal range (0.8~1.2). Therefore, 256 is chosen as the optimal dilution factor.

[0114] like Figure 7 As shown in Figure b, the absorbance of the reaction system gradually increases with the extension of reaction time, and the blue color of the system gradually deepens. When the reaction time is 10 minutes, the absorbance of the system is approximately 1.0; therefore, 10 minutes is chosen as the optimal reaction time.

[0115] from Figure 7 As shown in Figure c, the absorbance of the reaction system first increases and then decreases with increasing pH, and the color of the system changes from bright green to pale blue to blue to blue-green to yellow-green to colorless. When the pH is 4.5, the color of the system is blue, and the absorbance reaches its maximum value (approximately 1.12) at a wavelength of 650 nm. Therefore, pH 4.5 is chosen as the optimal reaction condition.

[0116] Figure 7 The d-plot 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, which can better catalyze the oxidation of TMB, making the blue color of the system deeper and the absorbance value increase. When the temperature is too high, most of the TMB in the system will undergo secondary oxidation, resulting in the system color turning green and the absorbance decreasing. When the reaction temperature is 20℃, the absorbance value of the system is around 1.0, and it is blue. Therefore, 20℃ is selected as the optimal reaction temperature.

[0117] In summary, the optimal detection conditions for the solution platform are: a dilution factor of 256 for Os-Ru nanozyme, a reaction time of 10 min, a pH of 4.5, and a reaction temperature of 20℃.

[0118] For the paper platform, optimization conditions included the concentration of Os-Ru nanozyme (expressed as dilution factor), reaction time, pH value, and TMB concentration. The results were expressed as ΔG value (gray value of blank paper - gray value of sample paper).

[0119] Figure 8 Figure a shows that as the dilution factor increases (the concentration of Os-Ru nanozyme decreases), the ΔG value of the test paper first gradually increases, then tends to stabilize, and the color of the test paper gradually changes from yellow-green to blue, and then to light blue. When the dilution factor is 64, the ΔG value of the paper reaches its maximum (54.30), accompanied by the most pronounced blue color. 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 the excessive oxidation of TMB caused by the excess Os-Ru nanozyme in the system. When the dilution factor is greater than 64, the ΔG value decreases significantly, and the paper color turns light blue or even colorless. This is because the catalytic activity of the Os-Ru nanozyme is weakened by the low concentration. Therefore, 64 is chosen as the optimal dilution factor.

[0120] Figure 8 Figure b shows that as the reaction time increases, the ∆G value of the paper first increases and then decreases, and the paper color gradually changes from light blue to dark blue, then to yellowish-green, and finally to almost colorless. When the reaction time is 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 paper color changes from dark blue to yellowish-green, and finally to almost colorless, while the ∆G value also decreases. This is due to the excessive reaction time leading to TMB peroxidation and oxTMB decomposition. Therefore, 10 minutes is chosen as the optimal reaction time.

[0121] from Figure 8 As shown in Figure c, pH value has a significant impact on the activity of Os-Ru nanozymes. With increasing pH, the ΔG value of the paper first gradually increases and then decreases. The highest ΔG value (approximately 68.64) is found at pH 4.0, at which point the paper is blue. Therefore, pH 4.0 is considered the optimal pH for the reaction.

[0122] from Figure 8 The d-plot shows that the concentration of TMB solution has a significant impact on the color and grayscale value of the paper. As the concentration of TMB solution decreases, the ΔG value of the paper first increases and then decreases, and the paper color changes from bluish-yellow to blue, and then to light blue. When the concentration of TMB solution is 3.75 mmol / L, the ΔG value of the paper is the highest and the color is the bluest; therefore, 3.75 mmol / L is selected as the optimal TMB solution concentration.

[0123] In summary, the optimal detection conditions for the paper platform are: a dilution factor of 64 for Os-Ru nanozyme, a reaction time of 10 min, a pH of 4.0, and a TMB concentration of 3.75 mmol / L.

[0124] The anti-interference capability of the catechin dual detection platform:

[0125] To evaluate the anti-interference ability of Os-Ru nanozyme in detecting catechins, the inhibitory effects of common food interfering substances (such as ions, sugars and amino acids) on the reaction system (Os-Ru nanozyme + TMB + H2O2) were investigated on both solution and paper platforms.

[0126] like Figure 9 As shown in Figure a, under the same conditions, the sample wells with added catechins were colorless, with very low absorbance at 650 nm. In contrast, the blank control wells and the sample wells with only the interfering substance were both blue, with similar absorbance values ​​(650 nm). Furthermore, Figure 9 Figure b shows that, in the presence of both catechins and interfering substances, all wells remained colorless, and the absorbance values ​​at 650 nm were very low. These results indicate that the solution platform exhibits good anti-interference capability in the detection of catechins.

[0127] Similarly, from Figure 9 As shown in Figure c, under the same conditions, only the sample paper with added catechins showed no color development (corresponding to a very low ΔG value). The blank control paper and the sample paper with only the interfering substance showed a blue-green color, and the ΔG value of the sample paper with the interfering substance was similar to that of the blank control paper. Furthermore, from... Figure 9 The d-plot in the image shows that all papers remained colorless and exhibited low ΔG values ​​in the presence of both catechins and interfering substances. These results indicate that the paper platform also demonstrates good selectivity for catechin detection. Therefore, the Os-Ru nanozyme-based dual-colorimetric platform exhibits excellent selectivity for catechin detection.

[0128] Catechin detection using a two-colorimetric platform:

[0129] Table 2 Comparison of catechin detection results using different detection methods

[0130]

[0131] Note: "-" indicates that the project was not tested.

[0132] Based on the ability of catechins to scavenge HO·, this invention first constructs a colorimetric detection solution platform for catechins using Os-Ru nanozyme as a recognizer and TMB as a signal generator. For example... Figure 10As shown in Figure a, with increasing catechin concentration, the solution color of the Os-Ru nanozyme + TMB + H2O2 + catechin system gradually lightens from blue to colorless, corresponding to a gradual decrease in absorbance at 650 nm. This is because with increasing catechin concentration, the amount of HO· produced by Os-Ru nanozyme catalyzing H2O2 decreases, thus preventing TMB from being oxidized into the blue oxTMB product with a specific absorption peak at 650 nm. Simultaneously, within the concentration range of 0 μmol / L to 450 μmol / L, the inhibitory effect of catechin on the oxidation of TMB catalyzed by Os-Rh nanozyme shows a good linear relationship, with a linear correlation coefficient (R0). 2 The limit of detection (LOD) for catechins calculated to be 0.997. Compared to other catechin detection methods (Table 2), this solution platform exhibits a lower LOD, indicating good sensitivity.

[0133] Based on the high sensitivity of the solution platform for detecting catechins, a colorimetric paper detection platform was established, making the detection more portable and convenient. The results are as follows: Figure 10 As shown in Figure b. From Figure 10 As clearly seen in Figure b, the blue color of the paper platform gradually fades until it disappears with increasing catechin concentration. Particularly when the concentration exceeds 56.25 μmol / L, the paper color is significantly different from the blank control paper; therefore, this concentration can be considered the visual detection limit of catechin on the paper platform. The paper color was converted to corresponding grayscale values ​​using ImageJ software, yielding a graph showing the relationship between catechin concentration and paper grayscale values. The results indicate a good linear relationship between the two within the concentration range of 0 μmol / L to 450 μmol / L, with R0... 2 0.990 ( Figure 10 (See Figure b in the original text). Calculations show that the detection limit for catechins using this colorimetric paper platform is 9.68 μmol / L, lower than many other studies. Therefore, this colorimetric paper platform provides a convenient and sensitive detection platform for catechins.

[0134] Furthermore, this invention also evaluated the stability of the comparative colored paper platform stored at room temperature for 60 days. For example... Figure 11 As shown, the activity of the colorimetric paper platform did not decrease significantly within 60 days, exhibiting good room temperature storage stability. These results demonstrate that the colorimetric paper platform developed in this invention is simple to store and easy to apply in practical catechin detection.

[0135] Spiked recovery analysis of catechins in real samples:

[0136] To evaluate the feasibility and reliability of the Os-Ru nanozyme-based dual-colorimetric detection platform for detecting catechins in practical applications, three green tea beverages containing catechins were selected as actual samples for a spiked recovery test. Figure 12 It can be seen that the catechin spike recovery results obtained by the Os-Ru nanozyme-based dual-colorimetric detection platform are very close to those obtained by the HPLC-MS method. Furthermore, Table 3 shows that the spike recoveries of catechins by the Os-Ru nanozyme-based colorimetric solution platform and colorimetric paper platform are 93.27%–103.01% and 91.78%–100.76%, respectively, with relative standard deviations (RSDs) of 2.17%–6.74% and 1.62%–4.57%, respectively. These are not significantly different from the catechin spike recoveries (94.36%–102.83%) and RSDs (1.69%–2.97%) detected by the HPLC-MS method. The results indicate that the established Os-Ru nanozyme-based dual-colorimetric detection platform has good accuracy and reliability in practical applications and has considerable practical application potential in the field of rapid catechin detection.

[0137] Table 3. Recovery rate and relative standard deviation of added catechins in actual samples.

[0138]

[0139] In summary, using Os-Ru nanozymes as probes, solution and paper platforms for catechin detection were successfully developed. Firstly, an Os-Ru nanozyme with excellent peroxidase-like activity was successfully synthesized via a one-step hydrothermal reaction, exhibiting activity against TMB and H2O2. K m The 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, a highly sensitive dual-platform (solution platform and paper platform) for direct colorimetric detection of catechins was established after optimizing the detection conditions. The linear range for catechin detection on both the solution platform and the paper platform was 0 μmol / L to 450 μmol / L. The detection limit was 2.84 μmol / L for the solution platform and 56.25 μmol / L (naked eye) and 9.68 μmol / L (grayscale value) for the paper platform. Moreover, the dual-detection platform demonstrated satisfactory accuracy and reliability in practical sample applications, with spiked recoveries of 91.78%–103.01% and RSDs of 1.62%–6.74%, showing 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.

[0140] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range, as well as any value between the two endpoints, can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

Claims

1. A method for preparing osmium-ruthenium nanozymes, characterized in that, Includes the following steps: Soluble osmium salt, soluble ruthenium salt, glycine and polyvinylpyrrolidone are dissolved together in water to obtain a mixture; The mixture is combined with a citric acid solution and subjected to a 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+ The osmium atoms are reduced to their atomic state and form an alloy core. At the same time, free osmium and ruthenium atoms are deposited on the alloy core and diffuse into each other to form a homogeneous alloy. Subsequently, the alloy is cooled to room temperature. During the cooling process, citric acid is re-adsorbed onto the surface of the homogeneous alloy to form a protective layer, preventing O2 erosion and particle aggregation. After dialysis purification, osmium-ruthenium nanozyme is obtained. The mass ratio of soluble osmium salt, soluble ruthenium salt, glycine, and polyvinylpyrrolidone is 0.5~1.5:0.1~0.5:4~10:10~20; The concentration of the citric acid solution is 0.2 mmol / L to 0.5 mmol / L, and the total molar ratio of osmium ions and ruthenium ions to the molar ratio of citric acid is 1:3 to 5. The reduction reaction conditions are: stirring at 80℃~120℃ for 30min~60min.

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

3. An osmium-ruthenium nanozyme, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 2.

4. A dual detection platform for catechins, characterized in that, Based on the osmium-ruthenium nanozyme constructed according to claim 3, 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, causing the blue color to lighten; The paper platform was prepared by loading osmium-ruthenium nanozymes onto paper, allowing it to air dry, then first applying a catechin solution, allowing it to air dry again, and then adding a 3,3',5,5'-tetramethylbenzidine solution and observing the color.

5. The catechin dual detection platform according to claim 4, characterized in that, The detection conditions for the solution platform were as follows: osmium-ruthenium nanozyme with a mass concentration of 100 μg / mL was diluted 200 to 300 times, 3,3',5,5'-tetramethylbenzidine was added at a concentration of 3.5 mmol / L to 4.0 mmol / L, and the reaction was carried out at 20℃ to 25℃ and pH 4 to 5.5 for 8 to 12 minutes after the addition of catechins.

6. The catechin dual detection platform according to claim 4, characterized in that, The detection conditions for the paper platform were as follows: osmium-ruthenium nanozyme solution with a mass concentration of 100 ug / mL was diluted 60 to 70 times, 3,3',5,5'-tetramethylbenzidine concentration was 3.5 mmol / L to 4.0 mmol / L, pH value was 4.0 to 5.0, and catechin solution was added dropwise and reacted for 8 to 12 minutes.

7. The catechin dual detection platform according to claim 4, characterized in that, The detection limit for the solution platform was 2.84 μmol / L, the detection limit for the paper platform was 56.25 μmol / L (naked eye detection), and the detection limit for the paper platform was 9.68 μmol / L (grayscale analysis detection).

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