Hollow bimetallic palladium-platinum nanoszyme and preparation method and application thereof

Abstract

This invention relates to the field of food detection technology, disclosing a hollow bimetallic palladium / platinum nanozyme, its preparation method, and its applications. This invention utilizes the excellent surface area, stability, uniformity, and accessibility of polystyrene microspheres, as well as the advantages of the three-dimensional structure of the microspheres providing a larger surface area than traditional two-dimensional carriers, which is beneficial for catalytic reactions. A three-dimensional hollow bimetallic Pd / Pt nanozyme was designed and synthesized. This nanomaterial exhibits significantly enhanced peroxidase-like catalytic activity and can be used for hydrogen peroxide detection. This invention also provides a colorimetric method for the enzyme-like properties of hydrogen peroxide based on hollow palladium / platinum nanozymes. This method is simple to operate, low in cost, and provides visualized and intuitive detection results, showing broad application prospects in the fields of food, environmental monitoring, and bioanalysis.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, and more specifically, to hollow bimetallic palladium-platinum nanozymes, their preparation methods, and applications. Background Technology

[0002] Hydrogen peroxide (H2O2) is an important food processing aid, widely used for food bleaching, preservation, and sterilization. However, improper use can lead to food residues. Ingesting excessive amounts of residual food can trigger oxidative stress in the human body, producing a large number of free radicals, which can then induce cell damage, cancer, and neurological diseases such as Alzheimer's, seriously endangering human health. Therefore, countries around the world have strict regulations on the amount of H2O2 residues in food: the Food and Agriculture Organization of the United Nations stipulates that the residue in milk should not exceed 0.25%; the US FDA requires that the residue in packaged food be less than 0.5 μg / mL; and my country's GB 2760-2014 stipulates that H2O2, as a processing aid, can be used in various food processing processes and must be removed before the finished product; if complete removal is not possible, the residue should be reduced as much as possible. To prevent illegal use, researchers have developed various H2O2 detection methods, including chemical titration analysis, near-infrared analysis, high-performance liquid chromatography, electrochemical and chemiluminescence methods, etc. Traditional chemical and instrumental analysis methods have drawbacks such as large equipment size, long time consumption, and the need for professional operation, making it difficult to achieve on-site real-time detection of food samples. Therefore, it is of great significance to establish a method for rapid on-site quantitative detection of H2O2 content in food.

[0003] In recent years, nanozymes have emerged as a new class of nanomaterials, possessing unique physicochemical properties, diverse enzyme-like catalytic functions, and excellent stability, making them highly attractive alternatives to natural enzymes. Among them, platinum nanomaterials, due to their outstanding catalytic performance, excellent physicochemical stability, and unique optical properties, have attracted considerable attention as peroxidase-like materials in ultrasensitive colorimetric detection and analysis. However, the catalytic activity of single-metal materials is often insufficient, necessitating the development of strategies to enhance their substrate affinity, specificity, and overall catalytic efficiency. Bimetallic doping has become a common method for improving catalytic performance, fully utilizing the synergistic effects between metals. These bimetallic nanozymes can optimize surface electronic structure, increase active site density, and enhance catalytic kinetics, thereby overcoming the performance bottlenecks of single-metal systems. Although alloying can improve intrinsic catalytic activity, simple adjustment of elemental composition often fails to achieve optimal catalytic performance for bimetallic nanozymes.

[0004] Against this backdrop, three-dimensional hollow nanozymes have emerged as an ideal solution balancing structural stability and catalytic activity. Their unique hollow three-dimensional structure offers dual advantages: it inherits the characteristics of three-dimensional frameworks in inhibiting aggregation and enhancing stability, while the hollow cavity enables high exposure of active sites and enhanced mass transfer, overcoming the instability of two-dimensional nanozymes and the kinetic limitations of traditional spherical three-dimensional nanozymes. This structural design lays a crucial foundation for constructing a new generation of nanozyme sensing platforms with high activity, high stability, and high practicality. Currently, research on bimetallic nanocomposites is widely applied in various fields; however, research on hollow palladium-platinum nanozymes as enzyme mimics has not yet been reported. The hollow structure exposes enzyme catalytic sites, exhibiting stronger enzyme mimicry activity than solid media. Summary of the Invention

[0005] The purpose of this invention is to provide a hollow bimetallic palladium-platinum nanozyme, its preparation method, and its application. To achieve the objective of this invention, in a first aspect, this invention provides a method for preparing hollow bimetallic palladium-platinum nanozymes, comprising the following steps: (1) Under alkaline conditions, dopamine is brought into contact with a polystyrene microsphere template (e.g., monodisperse polystyrene microspheres from Tianjin Bestray Co., Ltd. with a particle size of 0.2 μm). A polydopamine layer is formed on the surface of the polystyrene microsphere template by the self-polymerization reaction of dopamine (e.g., the thickness of the polydopamine layer is about 33.78 nm), and polydopamine-coated polystyrene microspheres are obtained, denoted as PS@PDA; (2) Platinum nanoparticles are loaded as seed crystals onto the surface of the polydopamine-coated polystyrene microspheres, denoted as PS@PDA / Pt (for example, the Pt mass percentage content is about 1.57 wt% as determined by EDS). (3) In a reaction system containing palladium precursor and platinum precursor, a palladium-platinum alloy layer (e.g., the thickness of the palladium-platinum alloy layer is about 65 nm) is grown on the surface of the seed crystal by seed-mediated in-situ growth at 65 °C to obtain solid bimetallic palladium-platinum nanomaterials (Pd / Pt-SNPs). (4) The solid bimetallic palladium-platinum nanomaterial is etched with an organic solvent to remove the polystyrene microsphere template and obtain hollow bimetallic palladium-platinum nanozymes (Pd / Pt-HNPs).

[0006] Further, in step (2), the platinum nanoparticles are platinum nanoparticles coated with polyvinylpyrrolidone (PVP@Pt).

[0007] Further, in step (3), the palladium precursor is sodium tetrachloropalladium and / or potassium tetrachloroplatinate, the platinum precursor is chloroplatinic acid, and the reduction reaction uses ascorbic acid as a reducing agent.

[0008] Furthermore, the reaction system in step (3) comprises PS@PDA / Pt, polyvinylpyrrolidone, sodium tetrachloropalladium, chloroplatinic acid, ascorbic acid and water.

[0009] Polyvinylpyrrolidone was added to the reaction system as a structure directing agent to regulate the morphology of the nanomaterials.

[0010] Preferably, the molar ratio of sodium tetrachloropalladium to chloroplatinic acid is 2:8.

[0011] For example, the above reaction system is prepared as follows: Polyvinylpyrrolidone is prepared with ultrapure water to a mass percentage concentration of 20 wt%. Then, 340 μL of polyvinylpyrrolidone solution is mixed with 240 μL of 100 mM sodium tetrachloroplatinate solution and 960 μL of 100 mM chloroplatinic acid solution, and then 66 mg of ascorbic acid is added.

[0012] Furthermore, in step (4), the organic solvent includes, but is not limited to, tetrahydrofuran and / or chloroform.

[0013] In a second aspect, the present invention provides hollow bimetallic palladium-platinum nanozymes (e.g., with a particle size of 310 ± 1.7 nm) prepared according to the method described.

[0014] Thirdly, the present invention provides the application of the hollow bimetallic palladium-platinum nanozyme in the detection of hydrogen peroxide.

[0015] Fourthly, the present invention provides a method for detecting hydrogen peroxide in a sample, comprising the following steps: 1) In a buffer solution, the hollow bimetallic palladium-platinum nanozyme, the chromogenic substrate, and the sample to be tested are mixed to form a reaction system; 2) The reaction system is incubated under preset conditions. If hydrogen peroxide is present in the sample to be tested, the hollow bimetallic palladium-platinum nanozyme catalyzes the chromogenic substrate to undergo a chromogenic reaction. 3) The presence or concentration of hydrogen peroxide in the sample to be tested is determined by detecting the color change of the reaction system or its corresponding absorbance value.

[0016] Furthermore, the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine (TMB).

[0017] Further, the preset conditions in step 2) include: the pH value of the buffer solution is 3.6-5.6 (preferably pH value is 4.0), and the incubation temperature is 25-35℃.

[0018] Further, step 3) includes: measuring the absorbance value of the reaction system at a wavelength of 652 nm, and calculating the concentration of hydrogen peroxide in the sample to be tested based on a pre-established standard curve of hydrogen peroxide concentration-absorbance.

[0019] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (i) This invention provides for the first time a hollow Pd / Pt-HNPs nanosphere based on polydopamine, and utilizes the enzyme-like activity of the hollow Pd / Pt-HNPs nanosphere to detect H2O2.

[0020] (ii) High sensitivity and wide detection linear range. The detection range of H2O2 in the sample is 2.5 μM-12.8 mM, and the detection limit is 1.14 µM.

[0021] (iii) The operation is simple and easy, the cost is low, and the test results are visualized, intuitive and convenient.

[0022] (iv) This sensing platform can be used to detect H2O2 in actual food samples such as milk, dried tofu, and pickled chicken feet. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the principle of the hollow bimetallic palladium-platinum nanozyme, its preparation method, and its application according to the present invention.

[0024] Figure 2 This is a scanning electron microscope image of Pd / Pt-HNPs prepared in a preferred embodiment of the present invention.

[0025] Figure 3 This is a transmission electron microscope image of the PS@PDA prepared in a preferred embodiment of the present invention.

[0026] Figure 4 The FTIR analysis results of PS and PS@PDA prepared in a preferred embodiment of the present invention are shown.

[0027] Figure 5 The XRD analysis results of PS@PDA / Pt and Pd / Pt-SNPs in the preferred embodiment of the present invention are shown.

[0028] Figure 6 XPS characterization images of Pd and Pt in the Pd / Pt-HNPs material prepared in a preferred embodiment of the present invention.

[0029] Figure 7 This is to verify the peroxidase-like activity of Pd / Pt-HNPs in a preferred embodiment of the present invention.

[0030] Figure 8 The figure shows the EPR (electron co-spin vibration) test results during the reaction process of hollow bimetallic palladium-platinum nanozymes Pd / Pt-HNPs in a preferred embodiment of the present invention.

[0031] Figure 9The standard curves for H2O2 detection by hollow bimetallic palladium-platinum nanozymes (Pd / Pt-HNPs) in a preferred embodiment of the present invention are shown. (a) shows the UV-Vis absorption spectra at different H2O2 concentrations (H2O2 concentrations of 0.15, 0.3, 0.6, 1.25, 2.5, 5, 10, 20, 40, 80, 100, 200, 400, 800 μM and 1.6, 3.2, 12.8 mM), and (b) and (c) are the corresponding H2O2 detection standard curves.

[0032] Figure 10 This is an optimization of the pH conditions for hydrogen peroxide detection in a preferred embodiment of the present invention.

[0033] Figure 11 This is a schematic diagram of the coordination interaction between PVP@Pt and PS@PDA in this invention. Detailed Implementation

[0034] This invention provides hollow bimetallic palladium-platinum nanozymes, their preparation methods, and applications. The invention prepares three-dimensional hollow nanospheres (Pd / Pt-HNPs) with highly dispersed Pd / Pt atomic active sites, which exhibit significantly enhanced peroxidase-like catalytic activity. This is attributed to the synergistic catalytic effect of the bimetallic active centers and the hollow three-dimensional structure. This material demonstrates significantly improved peroxidase-like catalytic activity and can be used for the detection of H2O2, further expanding the application range of hollow palladium-platinum nanozymes.

[0035] This invention utilizes the excellent surface area, stability, uniformity, and accessibility of polystyrene microspheres (PS), as well as the advantages of PS's three-dimensional structure, which provides a larger surface area than traditional two-dimensional carriers, thus facilitating catalytic reactions. A three-dimensional hollow bimetallic Pd / Pt nanozyme was rationally designed and synthesized, constructing a colorimetric sensing platform for the ultrasensitive detection of H2O2 in food samples (see schematic diagram). Figure 1 As shown in the figure, a polystyrene (PS) sphere is used as the core, coated with a polydopamine (PDA) interlayer (PS@PDA), and then the good coordination interaction between PVP@Pt and PS@PDA is achieved. Figure 11 Ordered platinum nanoparticles were assembled as seed crystals for in-situ growth (PS@PDA / Pt), providing anchoring sites for subsequent metal deposition. Then, the Pd / Pt precursor was reduced in situ with ascorbic acid (AA) to form solid Pt / Pd nanoparticles (Pd / Pt-SNPs). Finally, the PS template was selectively dissolved and gently removed using tetrahydrofuran (THF) to obtain hollow structures (Pd / Pt-HNPs). This structure exposes a large number of catalytically active sites, thereby enhancing the catalytic performance of the resulting nanomaterials.

[0036] The present invention adopts the following technical solution: This invention provides a hollow bimetallic palladium-platinum nanozyme, its preparation method, and its application, comprising the following steps: (1) Synthesis of polyvinylpyrrolidone (PVP)-coated platinum nanoparticles (PVP@Pt): 8 mL of ethylene glycol solution containing 16 mM chloroplatinic acid (H2PtCl6) and 0.09 g PVP was shaken for 1 minute and then incubated at 110 °C for 3 hours to obtain solution A.

[0037] (2) Dissolve 10 mg of 200 nm diameter PS microspheres in 20 mL of Tris / HCl buffer (pH=8.5, 0.01 M), and mix by shaking and sonication. Then, dissolve 60 mg of dopamine hydrochloride in 0.6 mL of ultrapure water and add it to the mixed solution. Sonicate for 10 minutes and then place the solution on a mixer to react in the dark for 24 h. Centrifuge the obtained solution and discard the supernatant. Wash the precipitate 2-3 times with ultrapure water. Finally, redissolve the precipitate in 4 mL of ultrapure water to obtain solution B.

[0038] (3) Take 0.8 mL of solution B obtained in step (2) and centrifuge at 4℃ (12000 rpm, 10 min). Dissolve the precipitate in 480 μL of ultrapure water with pH=10.0 (adjust pH with 1M NaOH), take 50~200 μL of solution A obtained in step (1) and mix it with the above 480 μL solution, and place it in a rotary mixer to react at room temperature for 12 h.

[0039] (4) After the reaction is complete, centrifuge, remove the supernatant, and leave the precipitate.

[0040] (5) Wash the precipitate obtained in step (4) repeatedly with ultrapure water 2-3 times, and then add a certain volume of ultrapure water (e.g., 0.8 mL of ultrapure water) to obtain solution C.

[0041] (6) First, prepare polyvinylpyrrolidone with ultrapure water to a concentration of 20 wt%. Then, take 340 μL of polyvinylpyrrolidone solution, 100 mM sodium tetrachloropalladium solution, 100 mM chloroplatinic acid solution, and 66 mg of ascorbic acid and add them to solution C. Add ultrapure water to make the total volume of the reaction system 12 mL. The volume ratios of sodium tetrachloropalladium solution and chloroplatinic acid solution are 10:0, 8:2, 4:6, 1:1, 6:4, 2:8, and 0:10, respectively. The total volume of sodium tetrachloropalladium solution and chloroplatinic acid solution in the reaction system is 1200 μL.

[0042] (7) The mixture obtained in step (6) was subjected to hydrothermal reaction at 65 °C for 2.5 h. The resulting solution was centrifuged and the supernatant was discarded. The precipitate was washed with ultrapure water 2-3 times. The precipitate obtained by discarding the supernatant for the last time was the black solid palladium-platinum nanomaterial (Pd / Pt-SNPs).

[0043] (8) Add tetrahydrofuran to the precipitate obtained in step (7) to etch PS microspheres, react overnight, then centrifuge and discard the supernatant. Wash the precipitate with ultrapure water 2-3 times, and finally dissolve it in ultrapure water containing 4wt% PVP. Store it in a refrigerator at 4℃ to obtain a solution of hollow palladium-platinum nanomaterials (i.e., hollow palladium-platinum nanoenzymes, Pd / Pt-HNPs).

[0044] (9) The obtained Pd / Pt-HNPs were prepared into a dispersion with a concentration of 5 μg / mL using ultrapure water. The dispersion was mixed with 0.01 M acetate-sodium acetate buffer at pH 4.0, 0.75 mM TMB and 0.25 μM-12.8 mM H2O2 in a volume ratio of 1:1:1:2. The mixture was incubated for 15 min at a reaction temperature of 35℃. The absorbance of the reaction solution after incubation was measured at 652 nm, and a standard curve of different H2O2 concentrations versus absorbance was plotted.

[0045] (10) Add a chromogenic solution containing 5 μg / mL Pd / Pt-HNPs aqueous solution, 0.01 M acetate-sodium acetate buffer solution at pH 4.0, and 0.75 mM TMB to actual spiked milk samples, pickled chicken feet samples, and dried bean curd samples with H2O2 concentrations of 20-1000 μM. The volume ratio of the actual sample volume to the chromogenic reaction system is 2:3. For example, the actual sample volume is 100 μL, and the chromogenic reaction system volume is 150 μL (in the chromogenic reaction system, 50 μL each of the Pd / Pt-HNPs aqueous solution, 0.01 M acetate-sodium acetate buffer solution at pH 4.0, and 0.75 mM TMB). Incubate the reaction for 15 min, and calculate the concentration of H2O2 in the actual sample based on the standard curve obtained in step (9).

[0046] Furthermore, in step (2), the concentration of solution B is 2.5 mg / mL.

[0047] Further, in step (6), the volume ratio of sodium tetrachloropalladium solution to chloroplatinic acid solution is 2:8.

[0048] Furthermore, in step (8), the concentration of the hollow palladium-platinum nanozyme is 0.25 mg / ml.

[0049] Furthermore, the incubation temperature in step (10) is 25 ℃~35 ℃.

[0050] This invention provides a colorimetric sensing platform for H2O2 based on hollow palladium-platinum nanozymes. It also provides a method for detecting hydrogen peroxide using peroxidase-like properties based on hollow bimetallic Pd / Pt-HNPs nanozymes, applicable to food, environmental, and other monitoring technologies. First, polydopamine-coated polystyrene microspheres (PS@PDA) are synthesized through the oxidative self-polymerization of dopamine on the surface of polystyrene microspheres (PS) under alkaline conditions. Then, uniformly fine platinum nanoparticles (Pt NPs) are adsorbed onto the surface of the polydopamine and noble metal platinum as seeds for in-situ growth (PS@PDA / Pt). Finally, palladium-platinum nanoparticles are further grown on the seed surface using a hydrothermal method to obtain solid bimetallic palladium-platinum nanomaterials (Pd / Pt-SNPs). Finally, the PS spheres are etched with tetrahydrofuran to obtain hollow bimetallic palladium-platinum nanomaterials (Pd / Pt-HNPs). Due to the synergistic catalytic effect between palladium and platinum bimetals and the large specific surface area and easily accessible active sites of its three-dimensional hollow structure, Pd / Pt-HNPs exhibit excellent enzyme-like activity. In the presence of hydrogen peroxide, this material promotes its decomposition to generate hydroxyl radicals (·OH). These ·OH radicals oxidize the chromogenic substrate TMB to oxTMB, causing the reaction system to change from colorless to blue. The colorimetric sensing method of this invention has a linear detection range of 2.5 μM - 12.8 mM for hydrogen peroxide and a detection limit of 1.14 μM. It features high sensitivity, speed, intuitiveness, and low cost, providing new insights for the application of this material in the fields of food, environmental monitoring, and bioanalysis.

[0051] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0052] Terminology of this invention: PS: Monodisperse polystyrene microspheres; PVP: Polyvinylpyrrolidone; PS@PDA: Polydopamine-coated polystyrene microspheres; PVP@Pt: Platinum nanoparticles coated with polyvinylpyrrolidone (PVP); PS@PDA / Pt: Pt seeds grow on the PS@PDA surface; Pd / Pt-SNPs: Solid palladium-platinum bimetallic nanozymes; Pd / Pt-HNPs: Hollow palladium-platinum bimetallic nanozymes.

[0053] The polyvinylpyrrolidone, sodium tetrachloropalladium, and chloroplatinic acid used in the following examples were all purchased from Aladdin Reagent Co., Ltd., with product numbers P434442, S101701, and H164499, respectively.

[0054] Monodisperse polystyrene microspheres were purchased from Tianjin Bestlite Chromatography Technology Development Center, item number: 6-1-0020.

[0055] Magnetic stirrer (Aika), RCT Bask kit; long-shaft rotary mixer (Qun'an), RO-80 model; vacuum drying oven (Linhai Yonghao), DZF-6024 model; plastic vacuum dryer, HP-PC-150 model, purchased from Changde Bickman Biotechnology Co., Ltd.; UV spectrophotometer, Q-6 model, purchased from Shanghai Yuanxi Instrument Co., Ltd.; ultrasonic cleaner, purchased from Kunshan Ultrasonic Instrument Co., Ltd.; electronic balance (Qun'an); high-speed benchtop refrigerated centrifuge, H1750R model, purchased from Hunan Xiangyi Laboratory Instrument Development Co., Ltd.

[0056] Example 1: A hollow bimetallic palladium-platinum nanozyme, its preparation method, and its application. This embodiment provides a hollow bimetallic palladium-platinum nanozyme and its preparation method, including the following steps: Step 1: Synthesis of PVP-coated platinum nanoparticles (PtNPs) Shake 8 mL of ethylene glycol (containing 16 mM H2PtCl6 and 0.09 g PVP) for 1 minute, and react at 110 °C for 3 hours to obtain solution A.

[0057] Step 2: Preprocessing of PS microsphere template 10 mg of 200 nm diameter PS microspheres were dissolved in 20 mL of Tris / HCl buffer (pH=8.5, 0.01 M). After mixing by shaking and sonication, 60 mg of dopamine hydrochloride dissolved in 0.6 mL of ultrapure water was added to the mixture and sonicated for 10 minutes. The mixture was then placed on a mixer and reacted in the dark for 24 h. The resulting solution was centrifuged, the supernatant was discarded, and the precipitate was washed 2-3 times with ultrapure water. Finally, the precipitate was redissolved in 4 mL of ultrapure water to obtain solution B.

[0058] Step 3: Composite of Pt NPs and PS microspheres Take 0.8 mL of solution B obtained in step (2) and centrifuge at 4 °C (12000 rpm, 10 min). Dissolve the precipitate in 480 μL of ultrapure water with pH=10.0 (adjust pH with 1M NaOH). Take 200 μL of solution A and mix it with the above 480 μL solution, and react for 12 hours. Centrifuge and retain the precipitate. Wash and redissolve it in 0.8 mL of ultrapure water to obtain solution C.

[0059] Step 4: Synthesis of bimetallic Pd / Pt nanomaterials First, polyvinylpyrrolidone (PVP) was prepared with ultrapure water to a concentration of 20 wt%. Then, 340 μL of PPVP solution, 1200 μL of a mixed metal precursor (100 mM sodium tetrachloropalladium solution: 100 mM chloroplatinic acid solution = 2:8, v / v), and 66 mg of ascorbic acid were added to solution C, and the volume was adjusted to 12 mL with ultrapure water. The mixture was subjected to hydrothermal reaction at 65 ℃ for 2.5 hours, and the solid Pd / Pt nanomaterials were obtained by centrifugation and washing.

[0060] Step 5: Preparation of hollow Pd / Pt-HNPs The PS template was etched overnight with tetrahydrofuran, centrifuged, washed, and dispersed in ultrapure water containing 4% PVP to obtain 0.25 mg / mL hollow Pd / Pt-HNPs.

[0061] Step 6: Establishment of the H2O2 detection standard curve 5 μg / mL Pd / Pt-HNPs, 0.01M acetate-sodium acetate buffer (pH 4.0), 0.75 mM TMB, and different concentrations of H2O2 (0.25 μM-12.8 mM) were mixed at a volume ratio of 1:1:1:2 to a total volume of 250 μL. The mixture was reacted at 35℃ for 15 minutes, and the absorbance at 652 nm was measured to plot a standard curve.

[0062] Structural and performance testing: The structure and properties of the PS purchased in Example 1, the prepared PS@PDA, PS@PDA / Pt, and the hollow Pd / Pt-HNPs material with excellent peroxidase-like activity were tested. The thickness of the polydopamine layer in PS@PDA is approximately 33.78 nm. Figure 3 The load of Pt in PS@PDA / Pt is shown in Table 1.

[0063] Table 1

[0064] Figure 2 This is a scanning electron microscope image of Pd / Pt-HNPs prepared in a preferred embodiment of the present invention. Figure 3 This is a transmission electron microscope image of the PS@PDA prepared in a preferred embodiment of the present invention. Figure 4 The FTIR analysis results of PS and PS@PDA prepared in a preferred embodiment of the present invention are shown. Figure 5 The XRD analysis results of PS@PDA / Pt and Pd / Pt-SNPs in the preferred embodiment of the present invention are shown. Figure 6The XPS peak profiles of Pt 4f and Pd 3d in the Pd / Pt-HNPs material prepared in the preferred embodiment of the present invention are shown below. Figure 7 This is to verify the peroxidase-like activity of Pd / Pt-HNPs in a preferred embodiment of the present invention. Figure 8 The figure shows the EPR (electron co-spin vibration) test results during the reaction process of hollow bimetallic palladium-platinum nanozymes Pd / Pt-HNPs in a preferred embodiment of the present invention. Figure 9 The standard curves for H2O2 detection using hollow bimetallic palladium-platinum nanozymes (Pd / Pt-HNPs) in a preferred embodiment of the present invention are shown. (a) shows the UV-Vis absorption spectra at different H2O2 concentrations (H2O2 concentrations of 0.15, 0.3, 0.6, 1.25, 2.5, 5, 10, 20, 40, 80, 100, 200, 400, 800 μM and 1.6, 3.2, 12.8 mM), and (b) and (c) show the corresponding H2O2 detection standard curves.

[0065] Test Result Analysis: 1. Schematic diagram of the preparation and detection principle of hollow bimetallic palladium-platinum nanozyme. like Figure 1 As shown, this invention rationally designs and synthesizes a three-dimensional hollow bimetallic Pd / Pt nanozyme, and uses it to construct a colorimetric platform for the ultrasensitive detection of hydrogen peroxide in food samples.

[0066] 2. Scanning electron microscope images of synthesized Pd / Pt-HNPs Figure 2 The successful synthesis of Pd / Pt-HNPs was demonstrated. As shown in the figure, this nanomaterial has a typical hollow nanosphere structure with uniform distribution and a diameter of 300-310 nm.

[0067] 3. Transmission electron microscopy analysis results of PS@PDA Figure 3 The results show that PS@PDA was successfully synthesized, and the thickness of the PDA is approximately 33.78 nm.

[0068] 4. FTIR analysis results of PS and PS@PDA like Figure 4 As shown, its structure was analyzed using Fourier transform infrared (FT-IR) spectroscopy. Compared to pure PS, PS@PDA exhibits higher performance at 3420 cm⁻¹. -1 A relatively broad peak is observed nearby, which is due to the superposition of asymmetric stretching vibrations of phenolic hydroxyl (OH) and amino (NH) groups in PDA, resulting from hydrogen bonding, confirming the successful coating of PDA onto the PS sphere surface. Furthermore, at 1602 cm⁻¹... -1It belongs to the C=C vibration of the benzene ring skeleton, originating from the indole or catechol structure of PDA, and is related to the benzene ring peak of PS (1600 cm⁻¹). -1 The peaks overlap, but the PDA peak is wider or stronger. Looking at the PS@PDA spectrum, the peak at 1289 cm⁻¹... -1 The characteristic peak at 752 cm⁻¹ represents the stretching vibration of the CO single bond in the phenolic hydroxyl group of PDA, providing strong evidence for the formation of PS@PDA. Additionally, the peak at 752 cm⁻¹... -1 and 698 cm -1 The fingerprint peak at the location is a characteristic peak of PS, and the peak intensity decreases significantly after PS@PDA is formed, further confirming the successful coating of PDA on PS spheres.

[0069] 5. XRD analysis results of PS@PDA / Pt and Pd / Pt-SNPs like Figure 5 As shown, X-ray diffraction (XRD) analysis of the crystal structure of the intermediate and final products revealed no obvious diffraction peaks in the PS@PDA / Pt spectrum, indicating that the PDA matrix is ​​amorphous and the Pt nanoparticles are well dispersed, consistent with previously reported systems. In contrast, the XRD pattern of the final product Pd / Pt-SNPs showed four clear diffraction peaks at 39.96°, 46.35°, 67.75°, and 81.70°, corresponding to the (111), (200), (220), and (311) crystal planes of the Pd / Pt-SNPs crystal structure, respectively. Crucially, these diffraction peaks are located between the standard diffraction angles of pure palladium (JCPDS No. 46-1043) and pure platinum (JCPDS No. 04–0802), and no independent diffraction peaks corresponding to a single metallic phase were detected. This result strongly demonstrates that a uniform Pd-Pt alloy structure was formed in the final nanomaterial.

[0070] 6. XPS analysis results of Pd / Pt-HNPs like Figure 6 As shown, XPS analysis confirmed that the material has been successfully loaded with Pd / Pt elements. Detailed analysis of the high-resolution Pt 4f spectrum of Pd / Pt-HNPs revealed four characteristic peaks with different binding energies near 70.80 eV, 74.10 eV, 72.10 eV, and 75.90 eV, which are attributed to metallic Pt (Pt4f). 0 ) and oxidized Pt (Pt 2+The Pd 3d spectrum shows four typical characteristic peaks at 340.10 eV (Pd 3d5 / 2), 334.90 eV (Pd 3d3 / 2), 336.7 eV (Pd 3d5 / 2), and 341.60 eV (Pd3d3 / 2), corresponding to Pd 3d5 / 2 and 341.60 eV, respectively. 0 With Pd 2+ Price state.

[0071] 7. Verification of peroxidase-like activity of hollow Pd / Pt-HNPs like Figure 7 As shown, the catalytic activity of nanozymes is key to verifying their synthesis. The enzyme-like activity of the nanomaterials was investigated by monitoring the catalytic oxidation of the chromogenic substrate TMB. Nanozymes exhibiting peroxide-mimicking enzyme activity can oxidize H2O2 to produce… These active substances can further catalyze the conversion of TMB to oxTMB, thereby initiating a blue reaction and exhibiting a characteristic absorption peak at a wavelength of 652 nm. The type of Pd / Pt-HNPs-like peroxidase (POD) activity was verified through a control experiment: when only 3,3',5,5'-tetramethylbenzidine (TMB) was present in the system, or when TMB coexisted with H2O2 without a catalyst, the solution showed no color change; however, when Pd / Pt-HNPs and TMB were added to the system simultaneously (with H2O2), a significant increase in absorbance was detected. This confirms its applicability as a catalyst for H2O2 detection. The absorbance value of the reaction system increased linearly with increasing Pd / Pt-HNPs concentration, further confirming that its catalytic activity is positively correlated with the nanozyme concentration.

[0072] 8. Catalytic reaction mechanism of hollow Pd / Pt-HNPs The presence of hydroxyl radicals (·OH) in the catalyst was verified by electron paramagnetic resonance (EPR) testing. Figure 8 As shown, a 1:2:2:1 characteristic signal of hydroxyl radical adducts was detected, indicating that in the degradation system, the holes generated in the valence band of Pd / Pt hollow nanospheres (Pd / Pt-HNPs) can be converted into hydroxyl radicals ·OH.

[0073] 9. Standard curve for H2O2 detection using hollow Pd / Pt-HNPs like Figure 9 As shown, under optimal experimental conditions, standard curves were plotted by measuring the relationship between absorbance values ​​and H2O2 concentrations for different concentrations of added hydrogen peroxide.

[0074] The results are shown in the figure: The standard curve fitting regression equation for the detection of hydrogen peroxide by hollow Pd / Pt-HNPs in the linear range of 0.25-100 μM is: y = 0.0396 log (x) + 0.0438, R 2 =0.9882, within the linear range of 100 μM-12.8 mM, y = 0.6337 log(x)-1.134, R 2 =0.9983, with a detection limit of 1.14 μM.

[0075] Example 2: Actual Sample Testing Referring to step 6 in Example 1, spiked food samples (milk, pickled chicken feet, and dried tofu) with H2O2 contents of 20 μM, 100 μM, 500 μM, and 1000 μM, respectively, were mixed with 5 μg / mL Pd / Pt-HNPs, 0.01M acetate-sodium acetate buffer at pH 4.0, and 0.75 mM TMB colorimetric solution at a volume ratio of 2:1:1:1, for a total volume of 250 μL. The mixture was reacted at 35 °C for 15 minutes, and the H2O2 recovery rate was calculated based on the standard curve plotted in Example 1.

[0076] The detection results of H2O2 in actual food samples (milk, pickled chicken feet, dried tofu) are shown in Table 2.

[0077] Table 2

[0078] The experimental results above show that, in the three actual samples (milk, dried tofu, and pickled chicken feet), the spiked recoveries of the target analyte ranged from 91.36% ± 4.13% to 115.71% ± 9.14% within the spiking concentration range of 20.00 μM to 1000.00 μM, with most spiking levels showing recoveries concentrated in the ideal range of 95%–105%. This fully demonstrates that the detection method of this invention maintains extremely high accuracy even when faced with interference from complex food matrices such as proteins and fats, and is not significantly affected by matrix effects. The coefficient of variation (CV) of all actual sample detection results was <15%, and the CV value was less than 10% at most concentration levels, indicating that this method has good repeatability and stability in actual sample detection.

[0079] Example 3: A method for preparing hollow single-metal palladium nanozymes Referring to step 4 in Example 1, 72.08 mg PVP, 1200 μL of mixed metal precursor (100 mM sodium tetrachloropalladium solution: 100 mM chloroplatinic acid solution = 10:0, v / v), and 66 mg ascorbic acid were added to solution C, and ultrapure water was added to 12 mL. The reaction was carried out hydrothermally at 65 °C for 2.5 hours, and the solid Pd / Pt nanomaterials were obtained by centrifugation and washing.

[0080] Example 4: A method for preparing hollow bimetallic palladium-platinum nanozymes Referring to step 4 in Example 1, 72.08 mg PVP, 1200 μL of mixed metal precursor (100 mM sodium tetrachloropalladium solution: 100 mM chloroplatinic acid solution = 8:2, v / v), and 66 mg ascorbic acid were added to solution C, and ultrapure water was added to 12 mL. The mixture was subjected to hydrothermal reaction at 65 °C for 2.5 hours, and the solid Pd / Pt nanomaterials were obtained by centrifugation and washing.

[0081] Example 5: A method for preparing hollow bimetallic palladium-platinum nanozymes Referring to step 4 in Example 1, 72.08 mg PVP, 1200 μL of mixed metal precursor (100 mM sodium tetrachloropalladium solution: 100 mM chloroplatinic acid solution = 6:4, v / v), and 66 mg ascorbic acid were added to solution C, and ultrapure water was added to 12 mL. The reaction was carried out hydrothermally at 65 °C for 2.5 hours, and the solid Pd / Pt nanomaterials were obtained by centrifugation and washing.

[0082] Example 6: A method for preparing hollow bimetallic palladium-platinum nanozymes Referring to step 4 in Example 1, 72.08 mg PVP, 1200 μL of mixed metal precursor (100 mM sodium tetrachloropalladium solution: 100 mM chloroplatinic acid solution = 5:5, v / v), and 66 mg ascorbic acid were added to solution C, and ultrapure water was added to 12 mL. The reaction was carried out hydrothermally at 65 °C for 2.5 hours, and the solid Pd / Pt nanomaterials were obtained by centrifugation and washing.

[0083] Example 7: A method for preparing hollow bimetallic palladium-platinum nanozymes Referring to step 4 in Example 1, 72.08 mg PVP, 1200 μL of a mixed metal precursor (100 mM sodium tetrachloropalladium solution: 100 mM chloroplatinic acid solution = 4:6, v / v), and 66 mg ascorbic acid were added to solution C, and ultrapure water was added to bring the volume to 12 mL. The mixture was subjected to hydrothermal reaction at 65 °C for 2.5 hours, and the solid Pd / Pt nanomaterials were obtained by centrifugation and washing.

[0084] Example 8: A method for preparing hollow monometallic platinum nanozymes Referring to step 4 in Example 1, 72.08 mg PVP, 1200 μL of mixed metal precursor (100 mM sodium tetrachloropalladium solution: 100 mM chloroplatinic acid solution = 0:10, v / v), and 66 mg ascorbic acid were added to solution C, and ultrapure water was added to 12 mL. The reaction was carried out hydrothermally at 65 °C for 2.5 hours, and the solid Pd / Pt nanomaterials were obtained by centrifugation and washing.

[0085] Table 3

[0086] The absorbance of the hollow palladium-platinum nanozymes prepared in Examples 1 and 3-8 was measured, and the results are shown in Table 3. The results indicate that when the volume ratio of sodium tetrachloropalladate solution to chloroplatinic acid solution is 2:8, the absorbance at 652 nm reaches its maximum value (absorbance = 1.934 ± 0.0382), significantly higher than other ratios: 0:10 (absorbance = 1.44525 ± 0.04595), 1:1 (absorbance = 1.01205 ± 0.03055), and 10:0 (absorbance = 0.22585 ± 0.00365). This superior catalytic performance can be attributed to the synergistic effect of the bimetallic structure: Pd... 2+ With Pt 2+ Ions are uniformly deposited on the surface of Pt nanoparticle seeds, forming an alloy structure with enhanced electron transfer kinetics. Furthermore, the elemental doping strategy synergistically accelerates the catalytic process, ultimately resulting in catalytic performance superior to that of single-metal Pt or Pd nanomaterials.

[0087] Example 9: Optimization of pH conditions for hydrogen peroxide detection The pH values ​​of the 0.01M acetate-sodium acetate buffer solution used in step 6 of Example 1 were set to 3.6, 3.8, 4.0, 4.4, 4.8, 5.0, 5.2, and 5.6, respectively. H2O2 was detected according to step 6, and the results are shown in [Figure 1]. Figure 10 The results indicate that Pd / Pt-HNPs exhibit the highest catalytic activity in a buffer solution at pH 4.0, therefore this condition was chosen as the optimal pH for hydrogen peroxide detection.

[0088] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing hollow bimetallic palladium-platinum nanoszymes, characterized in that, Includes the following steps: (1) Under alkaline conditions, dopamine is brought into contact with a polystyrene microsphere template, and a polydopamine layer is formed on the surface of the polystyrene microsphere template by the self-polymerization reaction of dopamine, to obtain polydopamine-coated polystyrene microspheres, denoted as PS@PDA; (2) Platinum nanoparticles are loaded as seed crystals onto the surface of the polydopamine-coated polystyrene microspheres, denoted as PS@PDA / Pt; (3) In a reaction system containing palladium precursor and platinum precursor, a palladium-platinum alloy layer is grown on the surface of the seed crystal by reduction reaction to obtain solid bimetallic palladium-platinum nanomaterials. (4) The solid bimetallic palladium-platinum nanomaterial is etched with an organic solvent to remove the polystyrene microsphere template and obtain a hollow bimetallic palladium-platinum nanozyme.

2. The method of claim 1, wherein, In step (2), the platinum nanoparticles are platinum nanoparticles coated with polyvinylpyrrolidone.

3. The method of claim 1, wherein, In step (3), the palladium precursor is sodium tetrachloropalladium and / or potassium tetrachloroplatinate, the platinum precursor is chloroplatinic acid, and the reduction reaction uses ascorbic acid as a reducing agent.

4. The method of claim 3, wherein, The reaction system in step (3) includes PS@PDA / Pt, polyvinylpyrrolidone, sodium tetrachloropalladium, chloroplatinic acid, ascorbic acid and water.

5. The method of claim 4, wherein, The molar ratio of sodium tetrachloropalladium to chloroplatinic acid is 2:

8.

6. The method according to any one of claims 1-5, characterized in that, In step (4), the organic solvent is tetrahydrofuran and / or chloroform.

7. Hollow bimetallic palladium-platinum nanozyme prepared according to any one of claims 1-6.

8. The application of the hollow bimetallic palladium-platinum nanozyme of claim 7 in the detection of hydrogen peroxide.

9. A method for detecting hydrogen peroxide in a sample, characterized in that, Includes the following steps: 1) In a buffer solution, the hollow bimetallic palladium-platinum nanozyme of claim 7, the chromogenic substrate, and the sample to be tested are mixed to form a reaction system; 2) The reaction system is incubated under preset conditions. If hydrogen peroxide is present in the sample to be tested, the hollow bimetallic palladium-platinum nanozyme catalyzes the chromogenic substrate to undergo a chromogenic reaction. 3) The presence or concentration of hydrogen peroxide in the sample to be tested is determined by detecting the color change of the reaction system or its corresponding absorbance value; Preferably, the chromogenic substrate is TMB.

10. The method according to claim 9, characterized in that, The preset conditions mentioned in step (2) include: the pH value of the buffer solution is 3.6-5.6, and the incubation temperature is 25-35℃; Preferably, the pH value of the buffer solution is 4.0.

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