PtRhRuCu quaternary alloy nanoszyme and preparation method and application thereof

Abstract

The application relates to a PtRhRuCu quaternary alloy nanoscale enzyme and a preparation method and application thereof, relates to the field of catalytic materials and analytical chemistry, and the quaternary alloy nanoscale enzyme is a nanoparticle composed of Pt, Rh, Ru and Cu and having an isolated island-shaped structure. The preparation method of the PtRhRuCu quaternary alloy nanoscale enzyme comprises the following steps: S1, mixing K2PtCl4 solution, K3RhCl6 solution, RuCl3 solution and CuCl2 solution to obtain a mixed solution I; S2, adding polyvinylpyrrolidone powder to the mixed solution I while stirring, heating to 150-220 DEG C, and constant-temperature reaction for 6-8 hours to obtain a mixed solution II; and S3, cooling the mixed solution II to room temperature, centrifuging, washing, separating out solid precipitates, and freeze-drying to obtain the PtRhRuCu quaternary alloy nanoscale enzyme. The application introduces the transition group metal element Cu into the three noble metals Pt, Rh and Ru, causes a multi-element effect, significantly improves the peroxidase-like activity, inhibits the oxidase-like activity, and achieves the effect of "directional regulation" of catalytic activity.

Description

Technical Field

[0001] This invention relates to the fields of catalytic materials and analytical chemistry, specifically to a PtRhRuCu quaternary alloy nanozyme, its preparation method, and its application. Background Technology

[0002] Enzymes are crucial biocatalysts in biological systems, exhibiting excellent catalytic activity and selectivity due to their unique protein scaffold, cofactors, and metal atom coordination. However, the practical application of natural enzymes is greatly limited by drawbacks such as high costs for product separation and purification, easy inactivation, difficulties in storage and recovery, and high environmental sensitivity. Nanozymes are novel functional nanomaterials with enzyme-like activity. Compared with natural enzymes, nanozymes offer advantages such as high stability, low cost, ease of preparation, adjustable activity, and low environmental requirements, making them widely applicable in fields such as biosensing, biomedicine, food safety, environmental monitoring, and agricultural production.

[0003] Metal nanozymes possess unique electronic structures and excellent catalytic activity. Noble metals (Au, Ag, Pt, Pd) and multivalent metals (Fe, Cu, Co, Ce, Mn) are often used as catalytic active centers to accelerate the enzymatic reaction process of chromogenic substrates. The output signal has high sensitivity and specificity, making it very suitable for application in the field of colorimetric sensing. The difficulty and challenge lies in continuously enhancing the catalytic activity, selectivity and stability of metal nanozymes.

[0004] Previous studies have shown that composition is an important factor in changing the electronic and geometric structure of nanomaterials, which can induce the modification of physical and chemical properties and help optimize the regulation of catalytic activity. To date, researchers have developed a series of binary and ternary metal nanozymes, which have shown satisfactory performance in colorimetric biodetection of various ions, small molecules and biomarkers. However, most of the existing binary and ternary alloy nanozymes have non-targeted enhancement multi-enzyme characteristics. This means that when the activity of one mimic enzyme is enhanced, the activity of other mimic enzymes is very likely to be enhanced at the same time ([1] Enhanced Multiple Enzymelike Activity of PtPdCu Trimetallic Nanostructures for Detection of Fe 2+and Evaluation of Antioxidant Capability. Yuanyang Mao, Fumin Jia, Tianyun Jing, Tingting Li, Huimin Jia, and Weiwei He. ACS Sustainable Chemistry & Engineering. 2021, 9(1), 569-579. [2] Metal nanozymes with multiple catalytic activities: regulating strategies and biological applications. Qi Yang, Yuanyang Mao, Quan Liu and Weiwei He. Rare Metals. 2023, 42, 2928-2948.). When faced with certain situations requiring "directional regulation" of catalytic activity to achieve biosensing functions in complex disease diagnostic technologies, there are significant shortcomings. Therefore, designing high-performance nanozymes with targeted enhancement activity is of great significance. Summary of the Invention

[0005] The present invention aims to provide a PtRhRuCu quaternary alloy nanozyme, its preparation method and application, so as to obtain an alloy nanozyme with targeted regulation effect.

[0006] To solve the above technical problems, the specific solution adopted in this invention is as follows: a PtRhRuCu quaternary alloy nanozyme, which is composed of nanoparticles with an isolated island-like structure composed of four elements: Pt, Rh, Ru, and Cu. The atomic percentage composition of the PtRhRuCu quaternary alloy nanozyme is Pt: 25%-35%, Rh: 20%-30%, Ru: 1%-6%, Cu: 35%-45%, with an average particle size of 25-30 nm.

[0007] A method for preparing a PtRhRuCu quaternary alloy nanozyme includes the following steps:

[0008] S1: Mix K2PtCl4 solution, K3RhCl6 solution, RuCl3 solution and CuCl2 solution to obtain mixed solution I;

[0009] S2: While stirring, add polyvinylpyrrolidone (PVP) powder to mixed solution I, heat to 150-220℃, and react at a constant temperature for 6-8 hours to obtain mixed solution II;

[0010] S3: Cool the mixed solution II to room temperature, centrifuge, wash, separate the solid precipitate, freeze dry, and the PtRhRuCu quaternary alloy nanozyme is obtained.

[0011] As a further optimization of the above technical solution, in step S1, equal volumes of K2PtCl4 solution, K3RhCl6 solution, RuCl3 solution and CuCl2 solution are mixed in deionized water to obtain mixed solution I. The volumes of K2PtCl4 solution, K3RhCl6 solution, RuCl3 solution and CuCl2 solution are all V1, and the volume of deionized water is V2, V1:V2 = 1:5-10.

[0012] As a further optimization of the above technical solution, the concentrations of K2PtCl4 solution, K3RhCl6 solution, RuCl3 solution and CuCl2 solution are all 20-30mM.

[0013] As a further optimization of the above technical solution, in the mixed solution I, V1 is 1 ml, V2 is 6-8 ml, and the amount of PVP powder added is 420-460 mg.

[0014] As a further optimization of the above technical solution, in step S3, the centrifugation speed is 12000-15000 rpm / min and the duration is 25-30 min.

[0015] Application of PtRhRuCu quaternary alloy nanozymes as peroxide (POD) mimicry enzymes.

[0016] The application of PtRhRuCu quaternary alloy nanozymes in glucose concentration detection utilizes the peroxidase-like activity of PtRhRuCu quaternary alloy nanozymes to detect hydrogen peroxide produced after glucose oxidase oxidizes glucose, thereby achieving glucose concentration detection.

[0017] The application of PtRhRuCu quaternary alloy nanozymes in glucose concentration detection includes the following specific steps:

[0018] 1) Plotting the working curve: Mix glucose oxidase with glucose solutions of different concentrations to obtain multiple mixture solutions with the same total volume; incubate the mixture solutions in a water bath at 37-40℃ for 80-100 min, then add 3,3',5,5'-tetramethylbenzidine (TMB) and PtRhRuCu quaternary alloy nanozyme solution respectively, mix thoroughly, and let stand at room temperature for 3-6 min; use a UV-Vis spectrophotometer to detect the absorbance value, and plot the detected absorbance value at 650 nm against the glucose concentration to plot the working curve;

[0019] 2) Glucose solution concentration detection: Mix glucose oxidase with the glucose solution to be tested to obtain a mixture solution with the same volume as in step 1). Incubate in a water bath at 37-40℃ for 80-100 min. Then add TMB and PtRhRuCu quaternary alloy nanoenzyme solution, mix well, and let stand at room temperature for 3-6 min. Use a UV-Vis spectrophotometer to detect the absorbance value. Substitute the detected absorbance value at 650 nm into the working curve obtained in step 1) to obtain the concentration of the glucose solution to be tested.

[0020] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention proposes a PtRhRuCu quaternary alloy nanozyme, its preparation method, and its application. By purposefully introducing the transition metal element Cu into three noble metals Pt, Rh, and Ru, a multi-element effect is induced, forming a unique geometric and electronic structure and modulated electronic states. This significantly enhances the peroxidase-like activity while inhibiting the oxidase-like (OXD) activity, achieving a "directed regulation" effect on catalytic activity. Compared with existing metal nanozymes, not only are further improvements in catalytic activity and stability achieved, but also the targeted regulation of enzyme-like activity is realized, providing a new strategy for optimizing enzyme-like catalytic activity.

[0021] This invention achieves a richer catalytic gain by increasing the number of principal components in the alloy nanozyme. The combination of the excellent catalytic activity of noble metals and the multivalent states of transition metals helps to promote charge separation and transfer, thereby improving catalytic efficiency. In addition, the unique geometric and electronic structure induced by the multi-components can effectively regulate the d-band center of the metal nanoparticles, affecting the interfacial adsorption / desorption behavior between the metal surface and the substrate, thus achieving selective regulation. Attached Figure Description

[0022] Figure 1 Transmission electron microscopy image of PtRhRuCu quaternary alloy nanozyme;

[0023] Figure 2 Comparison of OXD and POD activities of different alloy nanozymes with and without H2O2;

[0024] Figure 3 PtRhRuCu quaternary alloy nanoenzyme POD activity kinetic curves at different concentrations;

[0025] Figure 4 A comparison of the POD activity of PtRhRuCu quaternary alloy nanozymes under different pH conditions.

[0026] Figure 5 Comparison of the activities of OXD and POD nanoenzymes of different alloys;

[0027] Figure 6 A comparison graph showing the effect of different sugars on absorbance;

[0028] Figure 7 This is a standard curve for determining glucose. Detailed Implementation

[0029] like Figure 1 As shown, this invention discloses a PtRhRuCu quaternary alloy nanozyme, which is composed of four elements: Pt, Rh, Ru, and Cu. Its nanoparticles have an isolated island-like structure. The atomic percentage composition is Pt: 25%-35%, Rh: 20%-30%, Ru: 1%-6%, Cu: 35%-45%, and the average particle size is 25-30 nm.

[0030] This invention also discloses a method for preparing PtRhRuCu quaternary alloy nanozymes, used to prepare the aforementioned PtRhRuCu quaternary alloy nanozymes. The preparation method includes the following steps:

[0031] S1: Mix K2PtCl4 solution, K3RhCl6 solution, RuCl3 solution and CuCl2 solution to obtain mixed solution I;

[0032] Specifically, equal volumes of K₂PtCl₄ solution, K₃RhCl₆ solution, RuCl₃ solution, and CuCl₂ solution are mixed in deionized water to obtain mixed solution I. The volumes of K₂PtCl₄ solution, K₃RhCl₆ solution, RuCl₃ solution, and CuCl₂ solution are all V₁, and the volume of deionized water is V₂, with V₁:V₂ = 1:5-10. The concentrations of K₂PtCl₄ solution, K₃RhCl₆ solution, RuCl₃ solution, and CuCl₂ solution are all 20-30 mM.

[0033] S2: While stirring, add polyvinylpyrrolidone powder to mixed solution I, heat to 150-220℃, and react at a constant temperature for 6-8 hours to obtain mixed solution II;

[0034] Specifically, polyvinylpyrrolidone powder is added to mixed solution I under vigorous stirring, followed by subsequent heating. In mixed solution I, when the amounts of K2PtCl4 solution, K3RhCl6 solution, RuCl3 solution, and CuCl2 solution added are all 1 ml, the amount of deionized water added is 6-8 ml, and the amount of polyvinylpyrrolidone powder added is 420-460 mg.

[0035] Polyvinylpyrrolidone powder was added to mixed solution I and mixed evenly. The mixture was then transferred to a reaction vessel and placed in an oven to be heated to 150-200℃ and reacted at a constant temperature for 6-8 hours to obtain mixed solution II.

[0036] S3: Cool the mixed solution II to room temperature, centrifuge, wash, separate the solid particles, freeze-dry, and the quaternary alloy nanoenzyme is obtained.

[0037] Specifically, the mixed solution II after the reaction is completed is cooled to room temperature, and then centrifuged and washed with ethanol and deionized water respectively to obtain solid precipitate particles. The centrifugation speed is 13000-15000 rpm / min for 25-30 min. Then, the precipitate is freeze-dried into powder to obtain the PtRhRuCu quaternary alloy nanozyme. The specific process of separating the precipitate by centrifugation and washing with ethanol and deionized water and freeze-drying the precipitate into powder is existing technology and will not be described in detail here.

[0038] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] Example 1

[0040] Preparation of PtRhRuCu quaternary alloy nanoenzymes:

[0041] S1: Mix 1 mL of 20 mM K2PtCl4 solution, 1 mL of 20 mM K3RhCl6 solution, 1 mL of 20 mM RuCl3 solution, and 1 mL of 20 mM CuCl2 solution in 6.6 mL of deionized water.

[0042] S2: Under vigorous stirring, add 440mg of PVP powder (i.e., polyvinylpyrrolidone powder) to the above mixed solution, mix evenly, transfer to a reaction vessel, place in an oven and heat to 200℃ for 6h.

[0043] S3: After the reaction is completed and cooled to room temperature, the precipitates are separated and washed by centrifugation with ethanol and deionized water respectively. The precipitates are then freeze-dried into powder to obtain PtRhRuCu quaternary alloy nanozyme.

[0044] Example 2

[0045] Preparation of PtRhRuCu quaternary alloy nanoenzymes:

[0046] S1: Mix 1 mL of 20 mM K2PtCl4 solution, 1 mL of 20 mM K3RhCl6 solution, 1 mL of 20 mM RuCl3 solution, and 1 mL of 20 mM CuCl2 solution in 6 mL of deionized water.

[0047] S2: Under vigorous stirring, add 420mg of PVP powder (i.e., polyvinylpyrrolidone powder) to the above mixed solution, mix evenly, transfer to a reaction vessel, place in an oven and heat to 150℃ for 8h.

[0048] S3: After the reaction is completed and cooled to room temperature, the precipitates are separated and washed by centrifugation with ethanol and deionized water respectively. The precipitates are then freeze-dried into powder to obtain PtRhRuCu quaternary alloy nanozyme.

[0049] Example 3

[0050] Preparation of PtRhRuCu quaternary alloy nanoenzymes:

[0051] S1: Mix 1 mL of 25 mM K2PtCl4 solution, 1 mL of 25 mM K3RhCl6 solution, 1 mL of 25 mM RuCl3 solution, and 1 mL of 25 mM CuCl2 solution in 7.5 mL of deionized water.

[0052] S2: Under vigorous stirring, add 430mg of PVP powder (i.e., polyvinylpyrrolidone powder) to the above mixed solution, mix evenly, transfer to a reaction vessel, place in an oven and heat to 220℃ for 6.5h.

[0053] S3: After the reaction is completed and cooled to room temperature, the precipitates are separated and washed by centrifugation with ethanol and deionized water respectively. The precipitates are then freeze-dried into powder to obtain PtRhRuCu quaternary alloy nanozyme.

[0054] Example 4

[0055] Preparation of PtRhRuCu quaternary alloy nanoenzymes:

[0056] S1: Mix 1 mL of 30 mM K2PtCl4 solution, 1 mL of 30 mM K3RhCl6 solution, 1 mL of 30 mM RuCl3 solution, and 1 mL of 30 mM CuCl2 solution in 8 mL of deionized water.

[0057] S2: Under vigorous stirring, add 460mg of PVP powder (i.e., polyvinylpyrrolidone powder) to the above mixed solution, mix evenly, transfer to a reaction vessel, place in an oven and heat to 220℃ for 8h.

[0058] S3: After the reaction is completed and cooled to room temperature, the precipitates are separated and washed by centrifugation with ethanol and deionized water respectively. The precipitates are then freeze-dried into powder to obtain PtRhRuCu quaternary alloy nanozyme.

[0059] Example 5

[0060] Preparation of PtRhRuCu quaternary alloy nanoenzymes:

[0061] S1: Mix 1 mL of 20 mM K2PtCl4 solution, 1 mL of 20 mM K3RhCl6 solution, 1 mL of 20 mM RuCl3 solution, and 1 mL of 20 mM CuCl2 solution in 5 mL of deionized water.

[0062] S2: Under vigorous stirring, add 420mg of PVP powder (i.e., polyvinylpyrrolidone powder) to the above mixed solution, mix evenly, transfer to a reaction vessel, place in an oven and heat to 150℃ for 8h.

[0063] S3: After the reaction is completed and cooled to room temperature, the precipitates are separated and washed by centrifugation with ethanol and deionized water respectively. The precipitates are then freeze-dried into powder to obtain PtRhRuCu quaternary alloy nanozyme.

[0064] Comparative Example 1

[0065] Preparation of PtRhRuIr quaternary alloy nanozymes:

[0066] 1 mL of 20 mM K₂PtCl₄ solution, 1 mL of 20 mM K₂PdCl₄ solution, 1 mL of 20 mM RuCl₃ solution, and 1 mL of 20 mM IrCl₃ solution were mixed in 6.6 mL of deionized water. Under vigorous stirring, 440 mg of PVP powder was added to the above solution, mixed thoroughly, and then transferred to a reaction vessel. The mixture was placed in an oven and heated to 200 °C for 6 h. After the reaction was completed and cooled to room temperature, the mixture was centrifuged with ethanol and water respectively, and the precipitate was washed and freeze-dried into powder to obtain the PtRhRuIr quaternary alloy nanozyme.

[0067] Comparative Example 2

[0068] Preparation of PtPdIrCu quaternary alloy nanoenzymes:

[0069] 1 mL of 20 mM K₂PtCl₄ solution, 1 mL of 20 mM K₂PdCl₄ solution, 1 mL of 20 mM IrCl₃ solution, and 1 mL of 20 mM CuCl₂ solution were mixed in 6.6 mL of deionized water. Under vigorous stirring, 440 mg of PVP powder was added to the above solution, mixed thoroughly, and then transferred to a reaction vessel. The mixture was placed in an oven and heated to 200 °C for 6 h. After the reaction was completed and cooled to room temperature, the mixture was centrifuged with ethanol and water, respectively. The precipitates were washed and freeze-dried into powder to obtain the PtPdIrCu quaternary alloy nanozyme.

[0070] Comparative Example 3

[0071] Preparation of PtPdRuCu quaternary alloy nanoenzymes:

[0072] 1 mL of 20 mM K₂PtCl₄ solution, 1 mL of 20 mM K₂PdCl₄ solution, 1 mL of 20 mM RuCl₃ solution, and 1 mL of 20 mM CuCl₂ solution were mixed in 6.6 mL of deionized water. Under vigorous stirring, 440 mg of PVP powder was added to the above solution, mixed thoroughly, and then transferred to a reaction vessel. The mixture was placed in an oven and heated to 200 °C for 6 h. After the reaction was completed and cooled to room temperature, the mixture was centrifuged with ethanol and water, respectively. The precipitates were washed and freeze-dried into powder to obtain the PtPdRuCu quaternary alloy nanozyme.

[0073] Comparative Example 4

[0074] 1.333 mL of a 20 mM K₂PtCl₄ solution, 1.333 mL of a 20 mM K₃RhCl₆ solution, and 1.333 mL of a 20 mM RuCl₃ solution were mixed in 6.6 mL of deionized water. Under vigorous stirring, 440 mg of PVP powder was added to the above solution, mixed thoroughly, and then transferred to a reaction vessel. The mixture was placed in an oven and heated to 200 °C for 6 h. After the reaction was completed and cooled to room temperature, the mixture was centrifuged with ethanol and water, respectively. The precipitates were washed and freeze-dried into powder to obtain the PtPhRu ternary alloy nanozyme.

[0075] The catalytic activities of the PtRhRuCu quaternary alloy nanozymes prepared in the embodiments of the present invention and the PtRhRuIr, PtPdIrCu, PtPdRuCu quaternary alloy nanozymes and PtRhRu ternary alloy nanozymes prepared in the comparative examples are measured below.

[0076] First, prepare solutions of various alloy nanozymes.

[0077] Specifically, the PtRhRuCu quaternary alloy nanozyme prepared in Example 1 was dispersed in water to prepare a PtRhRuCu quaternary alloy nanozyme solution with a concentration of 10 mg / mL; the PtRhRuIr quaternary alloy nanozyme prepared in Comparative Example 1 was dispersed in water to prepare a PtRhRuIr quaternary alloy nanozyme solution with a concentration of 10 mg / mL; the PtPdIrCu quaternary alloy nanozyme prepared in Comparative Example 2 was dispersed in water to prepare a PtPdIrCu quaternary alloy nanozyme solution with a concentration of 10 mg / mL; the PtPdRuCu quaternary alloy nanozyme prepared in Comparative Example 3 was dispersed in water to prepare a PtPdRuCu quaternary alloy nanozyme solution with a concentration of 10 mg / mL for standby. Then, the characteristics of the nanozymes prepared in the examples and comparative examples were detected in turn. It should be noted that: the experimental test methods not detailed below are all unified standards in the field, and the instrument operations involved can be repeated without difference in the industry.

[0078] <OXD Activity Test of PtRhRuCu Quaternary Alloy Nanozyme>

[0079] Take 3 ml of deionized water, and sequentially add 20 μl of 3,3',5,5'-tetramethylbenzidine (TMB) with a concentration of 20 mM and 10 μL of PtRhRuCu quaternary alloy nanozyme solution with a concentration of 10 mg / mL thereto, and then mix the above solutions evenly; let it stand at room temperature (25 °C). Here is a gradual change process, and the color changes from colorless to light blue and then to dark blue. The activity test time is 10 min, and it can be observed that the solution changes from colorless to blue.

[0080] It shows that the quaternary alloy nanoparticle mimetic enzyme of the present invention has characteristics similar to those of peroxidase and can be used as a peroxidase mimetic enzyme.

[0081] <POD Activity Test of PtRhRuCu Quaternary Alloy Nanozyme>

[0082] Take 3 ml of deionized water, and sequentially add 20 μl of 3,3',5,5'-tetramethylbenzidine (TMB) with a concentration of 20 mM, 20 μL of hydrogen peroxide (H2O2) with a concentration of 100 mM, and 10 μl of PtRhRuCu quaternary alloy nanoparticle mimetic enzyme solution with a concentration of 10 mg / mL thereto, and then mix the above solutions evenly; let it stand at room temperature (25 °C). Here is a gradual change process, and the color changes from colorless to light blue and then to dark blue. The activity test time is 10 min, and it can be observed that the solution quickly changes from colorless to blue.

[0083] It shows that the quaternary alloy nanoparticle mimetic enzyme of the present invention has characteristics similar to those of peroxidase and can be used as a peroxidase mimetic enzyme.

[0084] <Activity Comparison between PtRhRu Ternary Alloy Nanozyme and PtRhRuCu Quaternary Alloy Nanozyme>

[0085] Test of peroxidase-mimicking activity: Add TMB to water, mix evenly, and then add the corresponding nanozyme and immediately start testing the absorbance of the solution. Test several different nanozymes under the same conditions. After the test, compare the absorbance values to determine the activity level. The greater the change in absorbance value, the higher the activity.

[0086] Test of catalase-mimicking activity: Add TMB and H2O2 to water, mix evenly, and then add the corresponding nanozyme and immediately start testing the absorbance of the solution. Test several different nanozymes under the same conditions. After the test, compare the absorbance values to determine the activity level. The greater the change in absorbance value, the higher the activity.

[0087] As shown in the Figure 2 attachment, through comparative experiments, it can be seen that: under the condition of no H2O2, with the same reaction time (10 min), the PtRhRuCu quaternary alloy nanozyme has a lower absorbance value of catalytic oxidation of TMB compared to the PtRhRu ternary alloy nanozyme, indicating that the former has lower OXD activity; under the condition of H2O2, with the same reaction time (10 min), the PtRhRuCu quaternary alloy nanozyme has a higher absorbance value of catalytic oxidation of TMB compared to the PtRhRu ternary alloy nanozyme, indicating that the former has higher POD activity. Therefore, the addition of Cu significantly improves the POD activity of the material while inhibiting the OXD activity, indicating that the addition of Cu achieves a directional regulation effect.

[0088] The specific analysis is as follows:

[0089] As shown in the Figure 2 attachment, the abscissa represents the wavelength of light, and the ordinate represents the absorbance value at that wavelength.

[0090] Figure 2The yellow and black curves represent the activity of the OXD-like enzyme, and the red and blue curves represent the activity of the POD-like enzyme. (During the oxidation of TMB to blue, the absorbance value at 650 nm increases). The yellow curve is the result of the PtRhRu ternary alloy nanozyme catalyzing the oxidation of TMB, and the black curve is the result of the PtRhRuCu quaternary alloy nanozyme catalyzing the oxidation of TMB. The absorbance value of the black curve is significantly lower than that of the yellow curve, indicating that the PtRhRuCu quaternary alloy nanozyme has a weaker ability to oxidize TMB under the same conditions. Therefore, the addition of Cu inhibits the OXD-like activity. The red curve is the result of the PtRhRuCu quaternary alloy nanozyme catalyzing the oxidation of TMB under the condition of H2O2, and the blue curve is the result of the PtRhRu ternary alloy nanozyme catalyzing the oxidation of TMB under the condition of H2O2. The absorbance value of the red curve is significantly higher than that of the blue curve, indicating that the POD-like activity of the PtRhRuCu quaternary alloy nanozyme is significantly improved. In summary, the addition of the Cu element realizes the directional regulation of catalytic activity.

[0091] It should be noted that when H2O2 exists alone without adding a nanozyme catalyst, TMB cannot be oxidized to blue.

[0092] <Kinetic curves of the POD-like activity of the PtRhRuCu quaternary alloy nanozyme at different concentrations>

[0093] The kinetic curves were tested using kinetic detection software.

[0094] As Figure 3 shown is the kinetic curve graph of the POD-like activity of the PtRhRuCu quaternary alloy nanozyme at different concentrations. As can be seen from Figure 3 it, the higher the concentration of the PtRhRuCu quaternary alloy nanozyme, the higher the activity.

[0095] <Comparison of the peroxidase-like activity of the PtRhRuCu quaternary alloy nanozyme in different pH environments>

[0096] As Figure 4 shown is the comparison graph of the peroxidase-like activity of the PtRhRuCu quaternary alloy nanozyme in different pH environments. As can be seen from Figure 4 it, in an environment with a pH value of 4, the PtRhRuCu quaternary alloy nanozyme has the best activity.

[0097] <Comparison of the OXD-like and POD-like activities of different multi-component alloy nanozymes>

[0098] As Figure 5The figures show bar charts of the relative OXD and POD activities of four quaternary alloy nanozymes: PtRhRu, PtRhRuIr, PtPdIrCu, and PtPdRuCu. Compared with the PtRhRu ternary alloy nanozyme, the three quaternary alloys in the figures show improved OXD and POD activities, indicating that the quaternary alloy nanozymes prepared in Comparative Examples 1-3 do not possess directional regulatory effects.

[0099] from Figure 5 It can also be seen that the addition of Ir to the PtRhRu ternary alloy increases both OXD and POD activities, indicating that this behavior does not have a directional regulatory effect. Furthermore, the addition of Cu to both PtPdIr and PtPdRu ternary alloys increases both OXD and POD activities, indicating that this behavior also does not have a directional regulatory effect.

[0100] Example 6

[0101] This embodiment discloses the application of PtRhRuCu quaternary alloy nanozyme in glucose concentration detection. The specific application method includes the following steps:

[0102] 1) Plot the working curve: 40 μL of 20 mg·mL⁻¹ solution was used to plot the working curve. -1 Glucose oxidase was mixed with glucose solutions of different concentrations to obtain a series of mixtures with a total constant volume of 3 mL. The concentration range of the glucose solution was 0-0.5 mM. In this example, the selected glucose concentrations were 0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, and 0.50 mM, a total of 11 concentrations, resulting in 11 groups of mixtures with a total volume of 3 mL. The mixtures were incubated at 38 °C for 90 min, and then 20 μL of 20 mM TMB and 10 μL of PtRhRuCu quaternary alloy nanoenzyme solution (1 mg·mL⁻¹) were added to the 11 glucose mixtures, respectively. -1 Finally, after being left at room temperature for 5 minutes, the absorbance at 650 nm was measured. The detected absorbance value at 650 nm was plotted against the glucose concentration to create a working curve.

[0103] 2) Detection of glucose solution concentration: Take deionized water and add 40 μL of 20 mg / mL glucose solution sequentially. -1 Glucose oxidase and 120 μL of the glucose solution to be tested were mixed to obtain a total volume of 3 mL. The mixture was incubated at 38 °C for 90 min, and then 20 μL of 20 Mm TMB and 10 μL of PtRhRuCu quaternary alloy nanozyme solution (1 mg·mL⁻¹) were added. -1Then, mix the above solution evenly, let it stand at room temperature for 5 minutes, and then use a UV-Vis spectrophotometer to detect the absorbance value. Substitute the detected absorbance value at 650nm into the obtained working curve function to calculate the concentration of the glucose solution to be tested.

[0104] It should be noted that when the total volume of the mixture is less than 3 mL, deionized water is used until the volume of the mixture reaches a constant 3 mL. The applicant has previously calculated that the glucose oxidase added in this embodiment can oxidize all the glucose in the glucose solution and produce hydrogen peroxide. The added PtRhRuCu quaternary alloy nanoenzyme solution is sufficient to detect the hydrogen peroxide produced after glucose oxidation, avoiding incomplete glucose oxidation or insufficient PtRhRuCu quaternary alloy nanoenzyme from affecting the experimental results. The calculation method uses existing technology and will not be elaborated further here.

[0105] In the glucose detection specificity experiment, 5mM sucrose, fructose, maltose and lactose were used as glucose substitutes, and the specific detection steps are as described above.

[0106] Appendix Figure 6 The graph shows the effect of different sugars on absorbance, indicating that when using the peroxidase-like properties of PtRhRuCu quaternary alloy nanozymes to detect glucose colorimetrically, the presence of other sugars has little interference with the experimental results and the detection specificity is good.

[0107] Appendix Figure 7 The working curves are plotted based on the detection results of different glucose concentrations. The horizontal and vertical axes represent the glucose concentration and the absorbance of the solution at 650 nm after the reaction, respectively. The working curves show a good linear relationship between the absorbance value at 650 nm and the added glucose concentration within a certain concentration range, indicating reliable detection accuracy.

[0108] As can be seen from the above, the PtRhRuCu quaternary alloy nanozyme prepared by this invention exhibits excellent peroxidase-like activity and can serve as a novel peroxidase mimic. Based on its enzyme-like catalytic characteristics, this invention also provides a colorimetric method for the determination of glucose concentration. The PtRhRuCu quaternary alloy nanozyme of this invention has broad application prospects in immunoassay, biodetection, and clinical diagnostics.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a PtRhRuCu quaternary alloy nanozyme in the detection of glucose concentration, characterized in that, The peroxidase-like activity of PtRhRuCu quaternary alloy nanozymes was utilized to detect hydrogen peroxide produced after glucose oxidase oxidizes glucose, thereby enabling the detection of glucose concentration. The quaternary alloy nanozymes are nanoparticles composed of four elements: Pt, Rh, Ru, and Cu, with an isolated island-like structure.

2. The application of the PtRhRuCu quaternary alloy nanozyme according to claim 1 in the detection of glucose concentration, characterized in that, The specific testing method includes the following steps. 1) Plotting the working curve: Mix glucose oxidase with glucose solutions of different concentrations to obtain multiple mixture solutions with the same total volume; incubate the mixture solutions in a water bath at 37-40℃ for 80-100 min, then add 3,3',5,5'-tetramethylbenzidine and PtRhRuCu quaternary alloy nanozyme solution respectively, mix thoroughly, and let stand at room temperature for 3-6 min; use a UV-Vis spectrophotometer to detect the absorbance value, and plot the detected absorbance value at 650 nm against the glucose concentration to create a working curve; 3) Glucose solution concentration detection: Mix glucose oxidase with the glucose solution to be tested to obtain a mixture solution with the same volume as in step 1). Incubate in a water bath at 37-40℃ for 80-100 min. Then add 3,3',5,5'-tetramethylbenzidine and PtRhRuCu quaternary alloy nanozyme solution, mix well, and let stand at room temperature for 3-6 min. Use a UV-Vis spectrophotometer to detect the absorbance value. Substitute the detected absorbance value at 650 nm into the working curve obtained in step 1) to obtain the concentration of the glucose solution to be tested.

3. The application of the PtRhRuCu quaternary alloy nanozyme according to claim 1 in the detection of glucose concentration, characterized in that, The preparation method of this quaternary alloy nanozyme includes the following steps: S1: Mix K2PtCl4 solution, K3RhCl6 solution, RuCl3 solution and CuCl2 solution to obtain mixed solution I; S2: While stirring, add polyvinylpyrrolidone powder to mixed solution I, heat to 140-240℃, and react at a constant temperature for 5-9 h to obtain mixed solution II; S3: Cool the mixed solution II to room temperature, centrifuge, wash, separate the solid precipitate, freeze dry, and the PtRhRuCu quaternary alloy nanozyme is obtained.

4. The application of the PtRhRuCu quaternary alloy nanozyme according to claim 3 in the detection of glucose concentration, characterized in that, In step S1, equal volumes of K2PtCl4 solution, K3RhCl6 solution, RuCl3 solution and CuCl2 solution are mixed in deionized water to obtain mixed solution I. The volumes of K2PtCl4 solution, K3RhCl6 solution, RuCl3 solution and CuCl2 solution are all V1, and the volume of deionized water is V2, where V1:V2=1:5-10.

5. The application of the PtRhRuCu quaternary alloy nanozyme according to claim 4 in the detection of glucose concentration, characterized in that, The concentrations of K2PtCl4 solution, K3RhCl6 solution, RuCl3 solution, and CuCl2 solution are all 15-30 mM.

6. The application of the PtRhRuCu quaternary alloy nanozyme according to claim 4 in the detection of glucose concentration, characterized in that, In the mixed solution I, V1 is 1 ml, V2 is 6-8 ml, and the amount of polyvinylpyrrolidone powder added is 420-460 mg.

7. The application of the PtRhRuCu quaternary alloy nanozyme according to claim 3 in the detection of glucose concentration, characterized in that, In step S3, the centrifugation speed is 12000-15000 rpm / min, and the duration is 25-30 min.

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