Platinum-loaded cerium-based nanoscale enzyme, and preparation method and application thereof

Abstract

The application discloses a kind of platinum supported cerium-based nanoscale enzyme and its preparation and application, the nanoscale enzyme includes cerium oxide nanorod carrier and the Pt active component loaded on its surface, the Pt active component is dispersed in the form of single atom or nanocluster on the surface of cerium oxide nanorod, the surface of cerium oxide nanorod has oxygen defect, by first loading platinum monolayer on the surface of cerium dioxide nanorod, then by reduction treatment, platinum atom forms platinum nanocluster on the surface of cerium dioxide and obtains.The nanoscale enzyme of the application effectively adjusts the oxygen vacancy and Pt site on the surface of cerium dioxide, has good peroxidase-like characteristics, can be used to construct TMB colorimetric system for colorimetric analysis of thiol reducing substances, and further used for disease screening, significantly improves the distinguishing effect.

Description

Technical Field

[0001] This invention relates to a nanoenzyme material, its preparation method and application, and particularly to a platinum-supported cerium-based nanoenzyme, its preparation method and application. Background Technology

[0002] Thiols in the body, such as glutathione (GSH), homocysteine ​​(Hcy), and cysteine ​​(Cys), are small-molecule reducing substances containing sulfhydryl groups (–SH) produced during metabolism. They play an important role in maintaining redox balance and participating in cellular metabolism, and abnormal levels are often closely related to the occurrence and development of various diseases. For example, abnormal GSH concentrations are closely associated with neurodegenerative diseases, tumors, and metabolic diseases such as diabetes, while elevated Hcy levels are often associated with cardiovascular diseases such as atherosclerosis and hypertension. Therefore, developing efficient, sensitive methods that can differentiate between multiple endogenous thiols is of great significance for disease diagnosis and bioanalysis.

[0003] Currently, the main methods for detecting endogenous thiols include electrochemical analysis, high-performance liquid chromatography, capillary electrophoresis, chemical probes, and ultraviolet spectrophotometry. Most of these methods are based on the reactive properties of thiol groups; however, due to the high structural similarity among different thiol molecules, their selective recognition capabilities are limited, making it difficult to accurately distinguish between multiple thiols.

[0004] In recent years, colorimetric array detection strategies based on multidimensional response signals have provided a novel approach for distinguishing structurally similar thiol molecules. By constructing multi-response systems and extracting differentiated signal features, specific identification of different thiols can be achieved. However, constructing stable, efficient signal units with tunable response characteristics remains a key challenge for this method.

[0005] Nanozymes, as a class of nanomaterials with catalytic activities similar to natural enzymes, have shown broad application prospects in the field of bioassay due to their good stability, tunable surface structure, and excellent catalytic performance. In particular, nanozymes with peroxidase-like activity can catalyze the generation of reactive oxygen species (ROS) from hydrogen peroxide, which further oxidize chromogenic substrates to produce detectable colorimetric signals. Different thiol molecules, due to differences in reducing power, reaction kinetics, and interactions with the nanozyme surface, can exert varying degrees of regulatory influence on the nanozyme catalytic process, thereby forming distinguishable response signals.

[0006] Among numerous nanozyme systems, cerium-based nanomaterials stand out due to their unique Ce... 3+ / Ce 4+Reversible redox pairs and abundant surface oxygen vacancies exhibit excellent catalytic performance in the generation of reactive oxygen species (ROS), and are widely used in peroxidase-like catalytic reactions. However, single cerium-based materials still have certain limitations in electron transport efficiency and hydrogen peroxide activation ability, restricting their application in highly sensitive detection systems. To further improve catalytic performance, introducing noble metal components to construct composite nanozymes has become an effective strategy. Among them, platinum (Pt) has significant advantages in promoting electron transfer and accelerating H2O2 decomposition due to its excellent electronic structure and catalytic activity. In existing technologies, introducing a platinum monolayer on the surface of cerium dioxide and introducing Pt into the CeO2 system can enhance the ROS generation ability by regulating the surface electronic structure through metal-support interactions. Although the introduction of a platinum monolayer has improved the peroxidase activity of cerium dioxide to some extent, its peroxidase activity still needs to be improved. Summary of the Invention

[0007] Objectives of the Invention: The first objective of this invention is to provide a platinum-loaded cerium-based nanozyme that enhances the activity of cerium oxide peroxidases; the second objective of this invention is to provide a method for preparing the platinum-loaded cerium-based nanozyme; and the third objective of this invention is to provide applications of the platinum-loaded cerium-based nanozyme.

[0008] Technical solution: The platinum-supported cerium-based nanozyme of the present invention is characterized in that the nanozyme comprises a cerium oxide nanorod carrier and a Pt active component loaded on its surface. The Pt active component is dispersed on the surface of the cerium oxide nanorod in the form of single atoms or nanoclusters. The surface of the cerium oxide nanorod has oxygen vacancies. It is obtained by first loading a platinum monoatom layer on the surface of the cerium dioxide nanorod, and then reducing it to form platinum nanoclusters on the surface of the cerium dioxide.

[0009] During the reduction process, platinum atoms float to the surface of the crystal lattice, forming clusters and creating oxygen defects.

[0010] Preferably, the loading of the Pt active component is 0.5~1.5 wt% (calculated based on the amount of platinum precursor added). The lower the platinum loading, the easier it is to form a platinum monolayer, and the less likely it is to form clusters during subsequent reduction; the higher the platinum loading, the easier it is to generate large-sized nanoparticles.

[0011] Preferably, the size of the nanoclusters is 1.5~3 nm. If the nanoclusters are too small, they lack stability and are prone to agglomeration to form large particles. If the nanoclusters are too large, fewer platinum sites are exposed, resulting in poor activity.

[0012] Preferably, the cerium oxide nanorods have a one-dimensional rod-like structure with a length of 100-400 nm and a diameter of 10-40 nm.

[0013] The method for preparing platinum-supported cerium-based nanozymes according to the present invention includes the following steps:

[0014] (1) Loading a platinum monolayer onto the surface of cerium oxide nanorods;

[0015] (2) After reduction treatment, platinum atoms float out on the crystal lattice surface to form nanoclusters, thus obtaining the nanozyme.

[0016] Preferably, the reduction treatment is a heat treatment reduction in a mixture of hydrogen and an inert gas. More preferably, the heat treatment reduction temperature is 300~400℃; and the heat treatment reduction time is 0.5~2 hours.

[0017] Preferably, the preparation method of the cerium oxide nanorods is as follows: cerium precursor is dissolved in deionized water, NaOH solution is added to adjust the pH, and the mixture is continuously stirred at room temperature to obtain a homogeneous mixed solution. The mixed solution is then transferred to a hydrothermal reactor for hydrothermal reaction. After the reaction is complete, the mixture is cooled, the product is separated, washed, dried, and finally calcined to obtain cerium oxide nanorods (CeNR).

[0018] Preferably, the hydrothermal reaction temperature is 120~180℃ and the reaction time is 12~24 h.

[0019] Preferably, the NaOH solution concentration is 200~300 mM, and the pH is adjusted to 13~14.

[0020] Preferably, the calcination temperature is 450~650℃ and the time is 2~4 h.

[0021] Preferably, the method for loading a platinum monolayer on the surface of cerium oxide nanorods is as follows: using platinum nitrate solution as a precursor solution, the Pt precursor solution is added dropwise to the cerium oxide nanorod support, thoroughly impregnating and mixing it evenly, drying the impregnated sample, and finally calcining to obtain the Pt / CeNR-IWI material.

[0022] Preferably, the calcination temperature is 500~600℃, and the heating rate is 2~10℃·min. -1 The time is 1 to 5 hours.

[0023] The application of the platinum-supported cerium-based nanozyme described in this invention in peroxidase-like reactions.

[0024] Preferably, the peroxidase-like reaction is for detecting thiols.

[0025] Mechanism of invention: This invention prepares cerium-based nanozymes with controlled platinum sites through three methods: modifying the surface of cerium dioxide with a platinum monolayer, loading platinum nanoclusters on the surface of cerium dioxide, and reducing monoatoms on the surface of cerium dioxide to form platinum nanoclusters. It is found that after platinum atoms are incorporated into the cerium dioxide lattice, under H2 reduction, the platinum atoms float to the surface of the lattice, generating more oxygen vacancies, which improves the electron transfer ability and peroxidase-like activity of the nanozyme. This is suitable for constructing a TMB colorimetric system for colorimetric differentiation of thiols.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The present invention introduces a platinum monoatom layer on the surface of cerium dioxide nanorods, and then uses reduction treatment to make platinum atoms float on the lattice surface to form clusters, thereby generating more oxygen vacancies, promoting electron transfer, and improving the peroxidase-like activity of cerium dioxide; (2) The nanoenzyme of the present invention, when the platinum content is 1wt%, has a Michaelis constant (K) of peroxidase. m ) reached 9.377×10 -8 mM, maximum reaction rate (V max (3) The preparation method is simple and easy to industrialize; (4) The colorimetric system constructed by the nanozyme of the present invention can accurately distinguish endogenous thiols and patients with related diseases. Attached Figure Description

[0027] Figure 1 This is a flowchart illustrating the preparation process of the platinum-supported cerium-based nanozyme of the present invention.

[0028] Figure 2 Transmission electron microscopy (TEM) and high-angle annular dark-field scanning TEM images of the samples prepared in Comparative Examples 1-3 and Example 1 are shown below: (a) TEM image of CeNR; (b) high-resolution TEM image of CeNR; (c) TEM image of Pt / CeNR-IWI; (d) TEM image of Pt / CeNR-IWI in high-resolution mode; (e) TEM image of Pt / CeNR-EG; (f) TEM image of Pt / CeNR-EG in high-resolution mode; (g) TEM image of Pt / CeNR-IWI-350a; (h) TEM image of Pt / CeNR-IWI-350a in high-resolution mode.

[0029] Figure 3 Transmission electron microscopy elemental analysis diagram of Pt / CeNR-IWI-350a prepared in Example 1;

[0030] Figure 4 XRD diffraction patterns of the samples prepared in Example 1 and Comparative Examples 1-3;

[0031] Figure 5X-ray photoelectron energy maps of the samples prepared in Example 1 and Comparative Examples 1-3;

[0032] Figure 6 The UV spectra of the peroxidase-like properties of the samples prepared in the examples and comparative examples 1-3 are shown.

[0033] Figure 7 The graph shows the effect of reaction time and concentration on the performance of Pt / CeNR-IWI-350a type peroxidase.

[0034] Figure 8 Figure 1 shows the effect of pH on the activity of Pt / CeNR-IWI-350a type peroxidase.

[0035] Figure 9 Figure 1 shows the effect of temperature on the activity of Pt / CeNR-IWI-350a type peroxidase.

[0036] Figure 10 Lineweaver-Burk equation curves fitted to the reaction substrate H2O2 for the study of Pt / CeNR-IWI-350a enzyme activity;

[0037] Figure 11 The graph shows the quantitative analysis standard curve of GSH by the colorimetric sensing liquid described in Example 5; a is the UV-Vis spectral response curve of the colorimetric sensing liquid to GSH, and b is the quantitative analysis standard curve of GSH.

[0038] Figure 12 The LDA diagrams of the colorimetric array described in Example 6 for different concentrations of GSH, Hcy, and Cys are shown below; a is the differentiation diagram for GSH, b is the differentiation diagram for Hcy, and c is the differentiation diagram for Cys.

[0039] Figure 13 The following are LDA diagrams of different thiols at the same concentration using the colorimetric array described in Example 6: a is the 1 μM resolution diagram, b is the 2 μM resolution diagram, c is the 5 μM resolution diagram, d is the 10 μM resolution diagram, e is the 20 μM resolution diagram, f is the 50 μM resolution diagram, and g is the 100 μM resolution diagram.

[0040] Figure 14 The LDA plots of the colorimetric array described in Example 6 for pneumonia patients (PP), lung cancer patients (LC), and healthy individuals (HP). Detailed Implementation

[0041] The technical solution of the present invention will be further described below with reference to the embodiments.

[0042] Example 1

[0043] like Figure 1As shown, the platinum-supported cerium-based nanozyme of the present invention is prepared by the following steps:

[0044] (1) Dissolve 2 mM CeCl3·7H2O precursor salt in 40 mL of deionized water, then add 2 mL of NaOH solution (240 mM), adjust the pH of the system to 12, and stir continuously for 30 min;

[0045] (2) The mixed solution was transferred to a hydrothermal reactor and reacted at 140°C for 16 h. After the hydrothermal reactor cooled to room temperature, the product was collected by centrifugation and washed three times with deionized water and ethanol respectively to obtain a gel.

[0046] (3) After drying the obtained gel at 100°C overnight, it was transferred to a muffle furnace and calcined at 550°C for 4 hours at a heating rate of 10°C·min. -1 Cerium dioxide nanorods CeNR were obtained;

[0047] (4) Measure 0.097 mL of platinum nitrate solution (with a Pt content of 18.02% and a density of 1.714 g / cm³). -3 Dilute to prepare a Pt precursor salt solution with a total volume of 0.450 mL; weigh 3 g of cerium dioxide nanorods prepared in step (3), add platinum nitrate solution dropwise onto the cerium dioxide nanorod support, impregnate evenly, put the powder into an oven to dry at 120°C for 20 min, and then put it into a muffle furnace to calcine at 550°C for 2 h, with a heating rate of 5°C·min. -1 Platinum single-atom-supported cerium dioxide nanorods Pt / CeNR-IWI were obtained;

[0048] (5) The platinum single-atom-loaded cerium dioxide nanorods Pt / CeNR-IWI prepared in step (4) were transferred into a tube furnace and reduced for 1 h under a 10% H2 / Ar atmosphere and a heating temperature of 350℃. Platinum atoms formed platinum nanoclusters on the surface of cerium dioxide, and the Pt active component was dispersed on the surface of cerium oxide nanorods as single atoms or nanoclusters to obtain nanoenzymes Pt / CeNR-IWI-350a (Pt loading was 1wt%).

[0049] Example 2

[0050] Based on Example 1, in step (4), the amount of platinum nitrate solution was changed to 0.048 mL, while the other conditions remained unchanged, to obtain a nanozyme with a Pt loading of 0.5 wt%.

[0051] Example 3

[0052] Based on Example 1, in step (4), the amount of platinum nitrate solution was changed to 0.146 mL, while the other conditions remained unchanged, to obtain a nanozyme with a Pt loading of 1.5 wt%.

[0053] Comparative Example 1

[0054] Based on Example 1, only steps (1) to (3) are performed to obtain cerium dioxide nanorods CeNR.

[0055] Comparative Example 2

[0056] Based on Example 1, only steps (1) to (4) were performed to obtain platinum single-atom-supported cerium dioxide nanorods Pt / CeNR-IWI.

[0057] Comparative Example 3

[0058] In this comparative example, platinum nanoparticles were directly loaded onto the surface of cerium dioxide nanorods. The preparation method included the following steps:

[0059] (1) Weigh 100 mg of PVP into a three-necked flask, add 15 mL of ethylene glycol solution, transfer the three-necked flask to a microwave reactor, preheat for 15 min, and when the reaction temperature stabilizes at 150°C, mix 1.28 mL of ethylene glycol solution containing (0.04 M) chloroplatinic acid and 1.03 mL of ethylene glycol solution containing (0.25 M) NaOH into the three-necked flask, and continue heating for 30 min. After the reaction is complete, cool in an ice-water bath for 30 min, wash with acetone and centrifuge, add ethanol for ultrasonic dispersion, add n-hexane for washing and centrifugation twice to obtain Pt nanoparticles;

[0060] (2) 0.03 g of Pt nanoparticles were dispersed in 10 ml of anhydrous ethanol and loaded onto 3 g of cerium dioxide nanorods prepared in Comparative Example 1. The impregnation was uniform. The powder was then dried in an oven at 120°C for 20 min and then calcined in a muffle furnace at 350°C for 2 h with a heating rate of 5°C·min. -1 Pt / CeNR-EG was prepared.

[0061] Example 5

[0062] Based on Example 1, a TMB colorimetric system was constructed using Pt / CeNR-IWI-350a for the quantitative detection of thiols.

[0063] Prepare a 100 μg / mL nanozyme solution of Pt / CeNR-IWI-350a and mix well by sonication. Take a 1.5 mL centrifuge tube and add 40 μL of Pt / CeNR-IWI-350a nanozyme solution, 140 μL of 0.2 M NaAc-HAc buffer, 10 μL of 10.0 mM TMB solution, 10 μL of 100 mM H2O2 solution to construct the colorimetric sensing solution, and 10 μL of the test solution containing thiols. After reacting for 25 min, measure the absorbance and compare with the standard curve.

[0064] Example 6

[0065] Based on Example 5, a colorimetric array was constructed on a 96-well plate using the colorimetric system for the differentiation of thiols and diseases.

[0066] Take a 96-well plate and add 40 μL of a colorimetric sensing solution consisting of 100 μg / mL Pt / CeNR-IWI-350a nanozyme solution, 140 μL of 0.2 M NaAc-HAc buffer, 10 μL of 10.0 mM TMB solution, and 10 μL of 100 mM H2O2 solution, along with 10 μL of the test solution, to each well to construct a colorimetric array. Record absorbance data every 5 minutes using a microplate reader, and perform 5 measurements to construct a 6×5 dataset. Analyze the dataset using the LDA algorithm and finally plot a cluster distribution map for differentiation.

[0067] Structural characterization

[0068] The structures of CeNR, Pt / CeNR-IWI, Pt / CeNR-EG, and Pt / CeNR-IWI-350a prepared in Comparative Examples 1-3 and Example 1 were characterized, and the results are as follows: Figures 2-5 .

[0069] Depend on Figure 2 The TEM images (a, b) show that the cerium dioxide nanorods are 200 nm long and 20 nm in diameter.

[0070] Depend on Figure 2As shown in the HADDF-STEM images (c~h), the Pt / CeNR-IWI (c, d) surface exhibits obvious platinum single atoms, successfully loading a platinum single-atom layer onto cerium dioxide nanorods; the Pt / CeNR-EG (e, f) surface is loaded with a large number of platinum nanoparticles with a particle size of 2~3 nm; the Pt / CeNR-IWI-350a (g, h) surface has clusters of 2~3 nm and 1.5~2 nm, and also contains some platinum single atoms. This is because platinum clusters grow on the cerium dioxide lattice surface based on platinum single atoms, resulting in nanoclusters of different sizes, as well as some platinum single atoms that have not floated out of the lattice. In summary, both Example 1 and Comparative Example 3 successfully loaded platinum nanoclusters onto cerium dioxide nanorods.

[0071] Depend on Figure 3 It can be seen that the Pt / CeNR-IWI-350a prepared in Example 1 is composed of a uniform mixture of Ce and O elements, and contains a small amount of Pt element loading, which is consistent with the above electron microscopy results.

[0072] Depend on Figure 4 The X-ray diffraction patterns show that Pt / CeNR-IWI-350a, Pt / CeNR-IWI, and Pt / CeNR-EG prepared by Example 1, Comparative Examples 2 and 3 exhibit diffraction peaks at 28.6°, 33.4°, 47.6°, and 56.5°, corresponding to their four crystal planes (111), (200), (220), and (311), respectively, which are the same as CeO2. This indicates that the synthesized nanozyme system retains the cubic fluorite structure of CeO2.

[0073] Depend on Figure 5 The X-ray photoelectron spectra show that the samples Pt / CeNR-IWI-350a, Pt / CeNR-IWI, and Pt / CeNR-EG prepared by Example 1, Comparative Examples 2 and 3 exhibit significant peaks of Pt 4f, Ce 3d, and O 1s, confirming that Pt was successfully deposited into CeO2. Figure 5 In (a), the XPS spectra of Pt 4f in Pt / CeNR-EG and Pt / CeNR-IWI-350a show obvious peaks at 70.9, 72.2, and 74.24 eV, respectively, corresponding to Pt 4f. 4+ Pt 2+ and Pt 0 Pt / CeNR-IWI shows extremely low peak intensity for Pt. 4+ Characteristic peaks, not showing Pt 0 Typical characteristic peaks. Figure 5 (b) The high-resolution XPS spectrum of Ce 3d shows that Ce has +4 and +3 valence states, which is important for Ce. 3+ and Ce 4+Calculation of the fitted peak area of ​​Ce on the surface of CeO2 nanostructure 3+ and Ce 4+ The content of Ce in CeNR, Pt / CeNR-IWI, Pt / CeNR-EG and Pt / CeNR-IWI-350a 3+ The percentages were 23.8%, 24.1%, 24.7%, and 28.0%, respectively. This gradual increase indicates that Pt doping can accelerate the deposition of Ce on the CeO2 surface. 4+ To Ce 3+ The transformation, and Ce 4+ Converted to Ce 3+ The ion conversion rate increases sequentially, resulting in more oxygen vacancies. Figure 5 (c) The high-resolution XPS spectrum of O 1s shows significant peaks at 528.6 and 530.7 eV, corresponding to lattice oxygen O, respectively. α and oxygen deficiency O β O in CeNR, Pt / CeNR-IWI, Pt / CeNR-EG and Pt / CeNR-IWI-350a β and O γ The total percentages were 28.6%, 29.7%, 30.5%, and 31.9%, respectively, indicating that the Pt / CeNR-IWI-350a material has the most oxygen vacancies, which is consistent with the aforementioned Ce... 4+ Converted to Ce 3+ The highest ion conversion rate is consistent.

[0074] Performance testing

[0075] 1. Test on the oxidation performance of TMB catalyzed by peroxidase-like enzymes

[0076] The Pt / CeNR-IWI-350a, CeNR, Pt / CeNR-IWI, and Pt / CeNR-EG prepared in Example 1 and Comparative Examples 1-3 were prepared into nanozyme solutions of 100 μg / mL and mixed by sonication. In a 1.5 mL centrifuge tube, 40 μl of nanozyme solution, 150 μL of 0.2 M NaAc-HAc buffer, 10 μL of 100 mM H2O2 solution, and 10 μl of 10.0 mM TMB solution were added, respectively. After reacting for 25 min, the absorbance of the solution was measured.

[0077] Depend on Figure 6It can be seen that the peroxidase-like activity of Pt / CeNR-IWI prepared by loading platinum single atoms onto CeNR was improved, and the peroxidase-like activity of the corresponding nanozyme was further improved by converting platinum single atoms into platinum nanoclusters. In particular, Pt / CeNR-IWI-350a, which generates more oxygen vacancies by reducing surface Pt single atoms, has the best peroxidase-like activity. Therefore, Pt / CeNR-IWI-350a was used to construct the colorimetric analysis system in subsequent experiments.

[0078] 2. Performance testing of nanozymes resembling peroxidases

[0079] The peroxidase-like activity of Pt / CeNR-IWI-350a prepared in Example 1 was tested.

[0080] (1) Effect of reaction time on reaction kinetics

[0081] Test method: Pt / CeNR-IWI-350a nanozyme solutions were prepared at concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, and mixed by sonication. In a 1.5 mL centrifuge tube, 40 μL of Pt / CeNR-IWI-350a nanozyme solution, 150 μL of 0.2 M NaAc-HAc buffer, 10 μL of 10.0 mM TMB solution, and 10 μL of 100 mM H2O2 solution were added respectively to obtain reaction solutions (corresponding to nanozyme concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, and 40 μg / mL). Absorbance was recorded every 5 min of reaction, and six times after 30 min of reaction. Test results are as follows: Figure 7 As shown.

[0082] Depend on Figure 7 It can be seen that by changing the concentration of nanozyme, the reaction rate is significantly increased. At 20 μg / ml, the reaction rate of nanozyme is moderate and reaches the plateau phase of the reaction at 25 min, and the reaction rate is significantly slowed down.

[0083] (2) Effect of pH on the activity of Pt / CeNR-IWI-350a type peroxidase

[0084] Test method: Pt / CeNR-IWI-350a was prepared into a nanozyme solution of 100 μg / mL. 40 μL of Pt / CeNR-IWI-350a nanozyme solution, 150 μL of 0.2 M NaAc-HAc buffer, 10 μL of 10.0 mM TMB solution, and 10 μL of 100 mM H2O2 solution were added to a 1.5 mL centrifuge tube to obtain the reaction solution (corresponding to a nanozyme concentration of 20 μg / mL). The pH was adjusted to 1, 2, 3, 4, 5, 6, 7, and 8, respectively. The absorbance of the solution was measured after reacting at room temperature for 25 min.

[0085] Depend on Figure 8 It can be seen that when the pH value increases from 1 to 3, the peroxidase-like activity of nanozymes is significantly increased, and when the pH value decreases from 4 to 8, the peroxidase-like activity of nanozymes is significantly decreased. This indicates that the peroxidase-like activity of nanozymes is most effective in weakly acidic conditions.

[0086] (3) Effect of temperature on the activity of Pt / CeNR-IWI-350a type peroxidase

[0087] Test method: Pt / CeNR-IWI-350a was prepared into a nanozyme solution of 100 μg / mL and mixed by sonication. 40 μL of Pt / CeNR-IWI-350a nanozyme solution, 150 μL of 0.2 M NaAc-HAc buffer, 10 μL of 10.0 mM TMB solution, and 10 μL of 100 mM H2O2 solution were added to a 1.5 mL centrifuge tube to obtain the reaction solution (corresponding to a nanozyme concentration of 20 μg / mL). The reaction temperatures were controlled at 0℃, 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, and 70℃, and the absorbance of the solution was measured after 25 min of reaction.

[0088] Depend on Figure 9 It can be seen that the reaction rate increases when the temperature is increased from 0℃ to 30℃, and the peroxidase-like activity of the nanozyme decreases when the temperature is increased from 40℃ to 70℃. This is because the reaction rate increases with increasing temperature, but when the temperature is too high, hydrogen peroxide decomposes itself, which hinders the oxidation process of TMB.

[0089] (4) Study on the effect of substrate concentration on the enzymatic reaction kinetics of Pt / CeNR-IWI-350a

[0090] Test method: Pt / CeNR-IWI-350a was prepared into a 100 μg / mL nanozyme solution and sonicated to mix well. In a 1.5 mL centrifuge tube, 40 μl of Pt / CeNR-IWI-350a nanozyme solution, 150 μL of 0.2 M NaAc-HAc buffer, 10 μL of 100 mM H2O2 solution, and 10 μL of TMB solution at different concentrations were added to achieve TMB substrate concentrations of 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.8 mM, and 1.0 mM, respectively, to obtain reaction solutions (corresponding to a nanozyme concentration of 20 μg / mL). After reacting for 5 min, the absorbance of the solution was measured, and the reaction rate was calculated. A Lineweaver-Burk equation curve was plotted, fitting the reciprocal of the substrate concentration to the reciprocal of the reaction rate.

[0091] Depend on Figure 10 The maximum reaction rate (Vt) of Pt / CeNR-IWI-350a can be obtained. max ) reached 1.09×10 -7 M / s, Michael constant (K) m The concentration was 5.83 mM, which is close to the Michaelis constant (K0) of the natural enzyme horseradish peroxidase for H2O2. m 4.3 mM, maximum reaction rate (V max 1.10×10 -5 mM / s.

[0092] (5) Application of Pt / CeNR-IWI-350a colorimetric sensing liquid in quantitative analysis of thiols

[0093] Using the colorimetric sensing solution prepared in Example 5, 10 μL of GSH solution with concentrations of 0 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, and 100 μM was added, and the absorbance after 25 min of reaction was measured to plot the colorimetric standard line. Figure 11 As shown.

[0094] Depend on Figure 11 It can be seen that the change in GSH concentration and absorbance shows a good linear relationship in the range of 5 mM to 35 mM (R0). 2 The detection limit (LOD) is >0.998, with a detection limit of 0.137 μM, indicating a low detection limit that can detect trace amounts of GSH.

[0095] (6) Application of the colorimetric array constructed by Pt / CeNR-IWI-350a in the differentiation of thiol substances and diseases

[0096] Using the colorimetric array prepared in Example 6, 10 μL of GSH, Hcy, and Cys at concentrations of 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM were added for LDA analysis.

[0097] Depend on Figure 12 It can be seen that the colorimetric array constructed by Pt / CeNR-IWI-350a divides the GSH concentrations of 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM and 100 μM shown in Figure (a) into 7 independent regions. Each region contains 6 parallel experimental results. The GSH samples of different concentrations are independently distributed, and finally the identification of different concentrations of GSH is realized. Similarly, Figures (b and c) show that the colorimetric array constructed by Pt / CeNR-IWI-350a can identify Hcy and Cys at different concentrations.

[0098] Depend on Figure 13 It can be seen that the colorimetric array constructed by Pt / CeNR-IWI-350a distinguished and identified GSH, Hcy, and Cys at a concentration of 1 μM as shown in Figure (a). The various thiols at 1 μM were divided into 3 independent regions, each containing 6 parallel experimental results. The samples of different types of thiols were independently distributed and did not interfere with each other, thus achieving the identification of different thiols at a concentration of 1 μM. Similarly, Figure (bg) shows that at 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM, the colorimetric array constructed by Pt / CeNR-IWI-350a achieved the distinction and identification of different thiols at the same concentration.

[0099] Significant differences were found in the content and proportion of thiols in the serum of healthy individuals (HP), pneumonia patients (PP), and lung cancer patients (LC). Using the colorimetric array prepared in Example 6, 10 μL of serum from each of the three groups was added for LDA analysis.

[0100] Depend on Figure 14 It can be seen that the LDA analysis map achieved by the colorimetric array constructed by Pt / CeNR-IWI-350a divides healthy people, pneumonia patients, and lung cancer patients into three separate regions. The results of serum samples of the same type are slightly different, but there are significant differences between them and those of different types of patients. The colorimetric array constructed by Pt / CeNR-IWI-350a has achieved significant differentiation and identification of serum from patients with different types of diseases.

Claims

1. A platinum-supported cerium-based nanozyme, characterized in that, The nanozyme comprises a cerium oxide nanorod carrier and a Pt active component loaded on its surface. The Pt active component is dispersed on the surface of the cerium oxide nanorod in the form of single atoms or nanoclusters. The surface of the cerium oxide nanorod has oxygen vacancies. It is obtained by first loading a platinum single-atom layer on the surface of the cerium dioxide nanorod, and then reducing it to form platinum nanoclusters on the surface of the cerium dioxide.

2. The platinum-supported cerium-based nanozyme according to claim 1, characterized in that, The loading of the Pt active component is 0.5~1.5 wt%.

3. The platinum-supported cerium-based nanozyme according to claim 1, characterized in that, The size of the nanoclusters is 1.5~3 nm.

4. The platinum-supported cerium-based nanozyme according to claim 1, characterized in that, The cerium oxide nanorods have a one-dimensional rod-like structure with a length of 100-400 nm and a diameter of 10-40 nm.

5. A method for preparing platinum-supported cerium-based nanozymes according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Loading a platinum monolayer onto the surface of cerium oxide nanorods; (2) After reduction treatment, platinum atoms float out on the crystal lattice surface to form nanoclusters, thus obtaining the nanozyme.

6. The method for preparing platinum-supported cerium-based nanozymes according to claim 5, characterized in that, The reduction process is a thermal reduction in a mixture of hydrogen and inert gas.

7. The method for preparing platinum-supported cerium-based nanozymes according to claim 6, characterized in that, The heat treatment reduction temperature is 300~400℃.

8. The method for preparing platinum-supported cerium-based nanozymes according to claim 6, characterized in that, The heat treatment reduction time is 0.5 to 2 hours.

9. The application of the platinum-supported cerium-based nanozyme according to any one of claims 1 to 4 in a peroxidase-like reaction.

10. The application according to claim 9, wherein the peroxidase-like reaction is for the detection of thiols.

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