A ceria nanoscale enzyme with asymmetric oxygen vacancies, a preparation method thereof and application thereof in creatinine detection

Abstract

The application provides a ceria nanoscale enzyme with asymmetric oxygen vacancies and a preparation method and application thereof in creatinine detection, and belongs to the technical field of nanoscale enzymes. The preparation method comprises the following steps: dissolving cerium nitrate hexahydrate and nickel nitrate hexahydrate in deionized water, and stirring to obtain a mixed metal salt solution; dissolving L-threonine and dicyandiamide in deionized water to obtain an amino acid aqueous solution, and adding the amino acid aqueous solution into the mixed metal salt solution to obtain a precursor solution; freeze-drying the precursor solution to obtain a solid precursor, then grinding the solid precursor into a powder to obtain solid matter A; calcining the solid matter A, and obtaining nanoscale enzyme particles after natural cooling, namely the ceria nanoscale enzyme with asymmetric oxygen vacancies. The prepared nanoscale enzyme has an ultrahigh specific surface area and rich asymmetric oxygen vacancies, has excellent POD catalytic activity, and has an extremely low detection limit in creatinine colorimetric sensing detection.

Description

Technical Field

[0001] This invention relates to the field of nanozyme technology, specifically to a cerium dioxide nanozyme with asymmetric oxygen vacancies, its preparation method, and its application in creatinine detection. Background Technology

[0002] Creatinine is an important small-molecule metabolite produced by the metabolism of creatine in the body. Its levels in biological samples such as blood and urine are closely related to renal function. Therefore, creatinine detection is of great significance in renal function assessment, chronic kidney disease monitoring, and auxiliary diagnosis of related diseases. Existing methods for creatinine detection mainly include the Jaffe method, enzymatic methods, chromatographic methods, and electrochemical methods. Among these, the Jaffe method is susceptible to interference from coexisting substances; enzymatic methods typically rely on natural enzyme systems and suffer from high cost, limited stability, and stringent storage requirements; while chromatographic methods offer high sensitivity, the instruments are expensive and the operation is complex, hindering rapid and portable detection.

[0003] Nanozymes have gained widespread attention in the field of colorimetric detection in recent years due to their catalytic activity similar to natural enzymes, combined with advantages such as high stability, low cost, and ease of preparation. Among them, nanozymes with peroxidase-like activity can catalyze the oxidation of chromogenic substrates such as TMB, making them suitable for constructing rapid and intuitive visual detection systems. Cerium (Ce)-based oxides, due to their reversible Ce... 3+ / Ce 4+ Valence state transition and strong oxygen vacancy regulation capabilities are important material systems for constructing POD (peroxidase-like) nanozymes.

[0004] Existing Ce-based oxide nanozymes generally suffer from problems such as relatively limited active site types, insufficient regulation of oxygen vacancy local structures, and limited electronic structure optimization. Chinese patent document CN116889887A discloses a method for preparing cerium dioxide composite nanozymes doped with different transition metal ions. This method involves mixing cerium nitrate solution with various metal salt solutions to obtain cerium dioxide composite nanozymes doped with different transition metal ions, including Ni doping. However, this invention mainly focuses on the influence of different transition metal ion doping types and detection conditions on the activity of cerium dioxide nanozymes, without specifically designing for the formation and retention of oxygen vacancies under specific metal (such as Ni) doping conditions, nor providing solutions for regulating precursor dispersion, particle growth, and oxygen vacancy structure. For cerium dioxide nanozymes, oxygen vacancies are one of the important factors affecting their catalytic performance; if the metal components are not uniformly dispersed during preparation, or if significant particle agglomeration and sintering occur during subsequent heat treatment, it may affect the formation of oxygen vacancies and the exposure of active sites.

[0005] In summary, the construction of asymmetric oxygen vacancies in cerium dioxide nanozymes remains challenging. The uniform introduction and stable coordination of dissimilar metals in Ce-based oxide systems are difficult to control, easily affecting the formation of heterostructures in adjacent sites. Furthermore, oxygen vacancies are sensitive to the preparation process, making it difficult to simultaneously control their formation and retention. Therefore, achieving synergistic regulation of dissimilar metal doping and asymmetric oxygen vacancy construction remains a key challenge in current technologies. Summary of the Invention

[0006] The purpose of this invention is to provide a cerium dioxide nanozyme with asymmetric oxygen vacancies, its preparation method, and its application in creatinine detection. The prepared nanozyme has an extremely high specific surface area (99.3 m²). 2 This nanozyme possesses abundant asymmetric oxygen vacancies and exhibits excellent POD catalytic activity. In creatinine colorimetric detection, this nanozyme demonstrates an extremely low detection limit.

[0007] The first aspect of this invention provides a method for preparing cerium dioxide nanozymes with asymmetric oxygen vacancies, comprising the following steps: S.1 Cerium nitrate hexahydrate (Ce(NO3)3·6H2O) and nickel nitrate hexahydrate (Ni(NO3)2·6H2O) were dissolved in deionized water and stirred to obtain a mixed metal salt solution; L-threonine and dicyandiamide were dissolved in deionized water and stirred to obtain an amino acid aqueous solution, which was then slowly added to the mixed metal salt solution and stirred continuously to obtain a precursor solution; S.2 The precursor solution was freeze-dried to obtain a solid precursor, which was then ground into powder to obtain solid A. S.3 Solid A was placed in a tube furnace and calcined under a nitrogen (N2) atmosphere. After natural cooling, Ni-Ov-Ce nanozyme particles were obtained, which are cerium dioxide nanozymes with asymmetric oxygen vacancies.

[0008] Furthermore, the ratio of cerium nitrate hexahydrate, nickel nitrate hexahydrate, and deionized water in step S.1 is 5 mmol: 0.2-0.3 mmol: 35-50 mL.

[0009] Furthermore, the ratio of L-threonine, dicyandiamide, and deionized water in step S.2 is 10 mmol: 20-40 mmol: 15-30 mL.

[0010] Further, the first stirring conditions described in step S.1 are: temperature: room temperature; time: 15-25 min; the second stirring conditions described in step S.1 are: temperature: room temperature; time: 8-13 min; further, the third stirring conditions described in step S.1 are: temperature: 50-65℃; time: 1.5-3 h.

[0011] Furthermore, the freeze-drying conditions described in step S.2 are: temperature: -80℃; time: 24h.

[0012] Furthermore, the calcination conditions described in step S.3 are as follows: heating rate: 2-3℃ / min; calcination temperature: 500-600℃; holding time: 2-5h.

[0013] A second aspect of the present invention provides a cerium dioxide nanozyme with asymmetric oxygen vacancies prepared according to the above-described preparation method.

[0014] The third aspect of the present invention provides the application of cerium dioxide nanozymes with asymmetric oxygen vacancies as described in the second aspect in a biosensor for creatinine detection.

[0015] Based on the above technical solution, the present invention has the following beneficial effects: (1) This invention proposes a method that uses Ni as a heterometallic control unit, combined with the coordination and dispersion effect of L-threonine and the confinement / reducing atmosphere control effect of dicyandiamide, to achieve synergistic regulation of uniform Ni introduction, grain size control, and asymmetric oxygen vacancy construction. Specifically, L-threonine can promote the uniform dispersion of Ce and Ni metal ions in the precursor, improving the effective introduction of Ni into the local structure of cerium dioxide; the introduction of Ni can change the original Ce–O–Ce symmetric coordination environment, inducing the formation of Ni–O. v –Ce-type asymmetric oxygen vacancies; dicyandiamide forms a confined and locally reducing environment during calcination in an inert atmosphere, inhibiting particle sintering and promoting the generation and stable retention of oxygen vacancies. The above synergistic effect results in nanozymes with small grain size, high specific surface area, and abundant asymmetric oxygen vacancies.

[0016] (2) This invention employs a synergistic approach of "manufacturing + stabilizing" oxygen vacancies, resulting in cerium dioxide nanozymes with a high concentration of oxygen vacancies. The introduction of Ni promotes the formation of oxygen vacancies; dicyandiamide can create a relatively reducing local environment during nitrogen calcination, promoting the release of lattice oxygen and further increasing the number of oxygen vacancies; simultaneously, the dispersing effect of L-threonine on metal ions and the confinement effect generated by the thermal decomposition of dicyandiamide can inhibit the agglomeration and sintering of particles during high-temperature calcination, reducing the loss of already formed oxygen vacancies. The above-mentioned "manufacturing" and "stabilizing" effects work together to facilitate the generation and retention of high-concentration oxygen vacancies.

[0017] (3) Preparation of Ni-O by the present invention V -Ce nanozymes possess an extremely high specific surface area (99.3 m²). 2With abundant asymmetric oxygen vacancies and a g / g content, this nanozyme exhibits excellent POD catalytic activity. In creatinine colorimetric detection, it achieved an extremely low detection limit (LOD 0.506 μM), demonstrating not only its excellent intrinsic catalytic activity but also its high sensitivity and broad application prospects in the field of biosensing. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The image shows the X-ray diffraction (XRD) test pattern of the sample from Example 1. Figure 2 The image shows the scanning electron microscope and elemental mapping (SEM EDS-mapping) images of the sample from Example 1. Figure 3 High-resolution transmission electron microscope (TEM) image of the sample from Example 1; Figure 4 Electron paramagnetic resonance (EPR) test images of the samples from Example 1 and Comparative Examples 1-3; Figure 5 Electron spin resonance (ESR) test images of samples from Example 1 and Comparative Examples 1-3 ( Figure 5 In this context, 'a' represents the detection of ·OH. Figure 5 b in O2 - (Detection) Figure 6 The UV-Vis absorption spectra of peroxidase-like (POD) activity of samples from Examples 1-3 and Comparative Examples 1-3 are shown. Figure 7 This is a calibration line graph for detecting different concentrations of creatinine in the sample of Example 1. Detailed Implementation

[0020] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. However, the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention. The equipment, instruments, and reagents used in the embodiments of the present invention can all be obtained through commercial channels or by using existing solutions in the prior art.

[0021] Example 1: (1) 5.0 mmol Ce(NO3)3·6H2O and 0.25 mmol Ni(NO3)2·6H2O were added to 40 mL of deionized water and stirred at room temperature for 20 min until completely dissolved (first stirring) to obtain a mixed metal salt solution. 10.0 mmol L-threonine and 30.0 mmol dicyandiamide were added to 20 mL of deionized water and stirred at room temperature for 10 min until completely dissolved (second stirring). The mixture was then slowly added to the above mixed metal salt solution and stirred at 60°C for 2 h (third stirring) to obtain a homogeneous precursor solution.

[0022] (2) The above precursor solution was transferred to a freeze-drying container and freeze-dried at -80°C for 24 h to obtain a solid precursor. The obtained solid precursor was then ground into powder to obtain solid A.

[0023] (3) Solid A was placed in a quartz boat in a tubular furnace and heated to 550°C at 3°C / min under a N2 atmosphere. The temperature was held for 3 hours and then naturally cooled to obtain Ni-O. V -Ce nanoparticles, referred to as the sample in Example 1.

[0024] The XRD tests were performed on the Sample 1 obtained in step (3) (see test results). Figure 1 SEM EDS-mapping test (test results can be found in...) Figure 3 TEM test (test results are available in [link]). Figure 3 EPR test (test results are available in [link]). Figure 4 ) and BET test (the test results are shown in Table 1).

[0025] Depend on Figure 1 The XRD test results of the sample in Example 1 show that the characteristic diffraction peaks in the figure are consistent with those of the standard card PDF#97-005-9961, indicating that the main crystalline phase of the obtained sample is cerium dioxide. No obvious independent characteristic diffraction peaks of Ni, NiO or other nickel-based crystalline phases were observed in the spectrum, indicating that Ni species may exist in a highly dispersed state or be incorporated into the local structure of cerium dioxide. Figure 2 The SEM EDS-mapping image of the sample from Example 1 is shown. The sample from Example 1 exhibits a nanoparticle structure. Figure 2 a), and O ( Figure 2 b) Ce ( Figure 2 c) Ni ( Figure 2 d) Element is uniformly distributed. (From...) Figure 3 The TEM image of the sample in Example 1 shows that the grain size of the sample is about 50~90nm. The smaller grain size helps to improve the dispersibility of the material. Figure 4The EPR test results show a strong EPR signal peak at g=2.003, indicating the presence of abundant oxygen vacancies in the sample. Table 1 shows the BET test results of the sample from Example 1, with a specific surface area of ​​99.3 m². 2 / g. In summary, the structural features of the sample in Example 1 are attributed to the following factors: firstly, L-threonine can react with Ni 3+ Ce 3+ Coordination ensures uniform dispersion of the two metal ions in the precursor, facilitating Ni's entry into the cerium dioxide framework during subsequent calcination, rather than forming an independent nickel phase. Secondly, Ni doping transforms the environment of some lattice oxygen from a relatively simple Ce–O–Ce configuration into a heterogeneous coordination environment influenced by both Ni and Ce. Due to the differences in valence state and bonding characteristics between Ni and Ce, the electron distribution and coordination state around oxygen vacancies are no longer symmetrical after formation, making it easier to form asymmetric oxygen vacancies. Finally, the pyrolysis of dicyandiamide under an inert atmosphere forms a framework and provides a localized environment conducive to defect formation and retention. Combined with freeze-drying to maintain the dispersion state of the precursor, oxygen vacancies tend to form and remain stable in the Ni–Ce ortho-position region. Ultimately, the combined effects of these factors favor the construction of asymmetric oxygen vacancies.

[0026] The ESR test was performed on the sample of Example 1 obtained in step (3), and the test results are shown in the figure below. Figure 5 .

[0027] Depend on Figure 5 It can be seen that the hydroxyl radicals (·OH) and superoxide anions (·O2) in the sample of Example 1 are... - The strongest ESR signal indicates its superior ability to generate reactive oxygen species (ROS). This is because, compared to symmetric oxygen vacancies, asymmetric oxygen vacancies establish stronger local charge polarization, which is conducive to electron transfer and the adsorption and activation of molecules such as O2 and H2O2, ultimately promoting the formation of ·OH and ·O2. - The generation of .

[0028] The nanozyme activity of the sample obtained in step (3) of Example 1 was evaluated (test results are shown in [link]). Figure 6 The testing method is as follows.

[0029] Test method: Sample suspension (2 mg / mL), TMB (1 mM), and H2O2 (10 mM) solutions from Example 1 were prepared using deionized water. Then, 10 μL of sample suspension from Example 1, 10 μL of H2O2, and 10 μL of TMB were mixed in 2970 μL of sodium acetate-acetic acid (NaAc-HAc, pH=4.5, 0.2 M) buffer solution and incubated at room temperature for 20 min. The absorbance of the reaction solution was measured at 652 nm using a UV-Vis spectrophotometer.

[0030] Figure 6 This demonstrates the effects of nanozymes and the superoxide radicals (·O2) they catalyze in the presence of H2O2 and the nanozyme sample from Example 1. - The enzyme synergistically catalyzes the decomposition of H₂O₂, generating hydroxyl radicals (·OH), which in turn oxidize TMB to ox-TMB, causing the solution color to change from colorless to blue, with an absorption peak appearing at 652 nm. The color intensity is positively correlated with enzyme activity, with the sample in Example 1 exhibiting the strongest catalytic activity. This is attributed to the strongest ROS generation capacity of the sample in Example 1.

[0031] The creatinine quantitative detection and analysis were performed on the sample of Example 1 obtained in step (3). The detection method is as follows, and the test results are shown in the figure. Figure 7 .

[0032] Test method: 80 μL of PBS (0.015 M, pH=7.5) was mixed with 10 μL of creatine oxidase (0.1 mg / mL), 10 μL of creatinine oxidase (0.2 mg / mL), 10 μL of sarcosine oxidase (0.2 mg / mL), and 20 μL of creatinine (0~550 μM), and incubated at 37 °C for 30 min. Then, 2850 μL of NaAc-HAc buffer (0.2 M, pH=4.5), 10 μL of the sample suspension from Example 1 (2 mg / mL), and 10 μL of TMB (1 mM) were added to the above solution, and incubated at room temperature for 20 min. Finally, the absorbance of the reaction solution was measured at 652 nm using a UV-Vis spectrophotometer.

[0033] Figure 7 The UV absorption spectra of the CR / TMB / H2O2 / Example 1 sample system at different creatinine (CR) concentrations are presented. The results show that the absorbance of the TMB solution gradually increases with increasing creatinine concentration from 0.1 μM to 500 μM. Within the range of 0.1–100 μM, the UV absorbance exhibits a good linear relationship with creatinine concentration, with the linear equation y = 0.1317x + 0.2544, and the limit of detection (LOD) is as low as 0.506 μM (LOD = 3σ / k, where σ is the standard deviation of the blank response and k is the slope of the linear equation), demonstrating that this material has excellent sensitivity for creatinine detection based on colorimetric sensing.

[0034] Example 2: (1) 5.0 mmol Ce(NO3)3·6H2O and 0.2 mmol Ni(NO3)2·6H2O were added to 35 mL of deionized water and stirred at room temperature for 15 min until completely dissolved to obtain a mixed metal salt solution. Separately, 10.0 mmol L-threonine and 20.0 mmol dicyandiamide were added to 15 mL of deionized water and stirred at room temperature for 8 min until completely dissolved. Then, they were slowly added to the above mixed metal salt solution and stirred at 50°C for 3 h to obtain a homogeneous precursor solution.

[0035] (2) The above precursor solution was transferred to a freeze-drying container and freeze-dried at -80°C for 24 h to obtain a solid precursor. The obtained solid precursor was then ground into powder to obtain solid A.

[0036] (3) Solid A was placed in a quartz boat in a tubular furnace and heated to 500°C at 3°C / min under a N2 atmosphere. The temperature was held for 5 hours and then naturally cooled to obtain Ni-O. V -Ce nanoparticles, referred to as Sample 2 of Example.

[0037] The XRD test (results are shown in Table 1) and BET test (results are shown in Table 2) were performed on the sample of Example 2 obtained in step (3). Figure 3 ) and grain size statistical analysis (test results are shown in Table 1).

[0038] As shown in Table 1, the sample of Example 2 is mainly composed of cerium dioxide, consistent with the sample of Example 1. Furthermore, Table 1 shows that the specific surface area of ​​the sample of Example 2 reaches 97.5 m². 2 / g, with a grain size of approximately 50~90nm.

[0039] The nanozyme activity of the sample obtained in step (3) of Example 2 was evaluated (test results are shown in [link]). Figure 6 The testing method is the same as in Example 1.

[0040] Figure 6 The peroxidase-like activity of the sample from Example 2 was demonstrated. The results indicate that the sample from Example 2 also exhibited strong nanozyme activity.

[0041] Example 3: (1) 5.0 mmol Ce(NO3)3·6H2O and 0.3 mmol Ni(NO3)2·6H2O were added to 50 mL of deionized water and stirred at room temperature for 25 min until completely dissolved to obtain a mixed metal salt solution. Separately, 10.0 mmol L-threonine and 40.0 mmol dicyandiamide were added to 30 mL of deionized water and stirred at room temperature for 13 min until completely dissolved. Then, the solution was slowly added to the above mixed metal salt solution and stirred at 65°C for 1.5 h to obtain a homogeneous precursor solution.

[0042] (2) The above precursor solution was transferred to a freeze-drying container and freeze-dried at -80°C for 24 h to obtain a solid precursor. The obtained solid precursor was then ground into powder to obtain solid A.

[0043] (3) Solid A was placed in a quartz boat in a tubular furnace and heated to 600°C at a rate of 2°C / min under a N2 atmosphere. The temperature was held for 2 hours and then allowed to cool naturally to obtain Ni-O. V-Ce nanoparticles, referred to as Sample 3 of Example.

[0044] The XRD test (results are shown in Table 1) and BET test (results are shown in Table 2) were performed on the sample of Example 3 obtained in step (3). Figure 3 ) and grain size statistical analysis (test results are shown in Table 1).

[0045] As shown in Table 1, the sample of Example 3 is mainly composed of cerium dioxide, consistent with the sample of Example 1. Furthermore, Table 1 shows that the specific surface area of ​​the sample of Example 3 reaches 98.0 m². 2 / g, with a grain size of approximately 50~90nm.

[0046] The nanozyme activity of the sample obtained in step (3) of Example 3 was evaluated (test results are shown in [link]). Figure 6 The testing method is the same as in Example 1.

[0047] Figure 6 The peroxidase-like activity of the sample from Example 3 was demonstrated. The results indicate that the sample from Example 3 also exhibited strong nanozyme activity.

[0048] Comparative Example 1: The only difference from Example 1 is that Ni(NO3)2·6H2O is not added in step (1). The rest of the method is exactly the same as in Example 1.

[0049] The comparative example 1 samples obtained in step (3) were subjected to XRD tests (test results are shown in Table 1) and EPR tests (test results are shown in Table 2). Figure 4 BET test (test results are shown in Table 1) and grain size statistical analysis (statistical results are shown in Table 1).

[0050] As shown in Table 1, the sample of Comparative Example 1 is composed of cerium dioxide. Figure 4 The EPR test results of Comparative Example 1 are shown, revealing an EPR signal peak at g=2.003, but its signal intensity is weaker than that of Example 1 sample. This indicates that the oxygen vacancy concentration in the sample is lower than that in Example 1 sample. Table 1 shows that the BET test results of Comparative Example 1 sample show a specific surface area of ​​64.7 m². 2 / g, which is significantly lower than that of the sample in Example 1. Furthermore, Table 1 also shows the statistical results of the grain size of the sample in Comparative Example 1, which shows a grain size of approximately 110-150 nm, significantly larger than that of the sample in Example 1. This is because the low-valence Ni (Ni 2+When cerium dioxide is incorporated, Ni atoms are dispersed near the crystal lattice or grain boundaries, forming localized lattice distortion. This localized distortion increases the grain boundary energy barrier, hindering the migration and aggregation of cerium dioxide nanocrystals during calcination, thus reducing grain size. Furthermore, the proximity of low-valence Ni to Ce alters the local electronic structure and lowers the formation energy of oxygen vacancies, thereby promoting their generation.

[0051] The ESR test was performed on the Comparative Example 1 sample obtained in step (3) (the test results are shown in the figure). Figure 5 ).

[0052] Depend on Figure 5 It can be seen that the ESR signal of hydroxyl radicals (·OH) in the sample of Comparative Example 1 is stronger. Figure 5 (a) in the text, while superoxide anion (·O2) - The ESR signal is very weak. Figure 5 b) indicates that it can only generate ·OH and not ·O2. - This is because, without the introduction of Ni, oxygen vacancies in the system mainly form in a relatively homogeneous Ce-O-Ce local environment, tending to be dominated by ordinary oxygen vacancies. Cerium dioxide itself still possesses a certain amount of Ce... 3+ / Ce 4+ The presence of circulating and ordinary oxygen vacancies allows for a certain degree of adsorption and activation of H₂O₂, thus exhibiting a certain ability to generate ·OH. In contrast, ·O 2- The generation of O2 depends more on the electronic activation process of O2 at defect sites. Since the oxygen vacancies in Comparative Example 1 are mainly relatively uniform Ce–Ov–Ce ordinary oxygen vacancies, the local electronic structure and charge polarization effect are weak, resulting in limited electron transfer efficiency, which is unfavorable for the reduction of O2 to form ·O2. - .

[0053] The nanozyme activity of the Comparative Example 1 sample obtained in step (3) was evaluated (test results are shown in [link to results]). Figure 6 The testing method is the same as in Example 1.

[0054] Figure 6 The peroxidase-like activity of Comparative Example 1 was shown. The results indicate that the peroxidase-like activity of Comparative Example 1 was significantly decreased. This is mainly attributed to a significant decrease in its ROS generation capacity.

[0055] Comparative Example 2: The only difference from Example 1 is that L-threonine is not added in step (1). The rest of the method is exactly the same as in Example 1.

[0056] The comparative example 2 samples obtained in step (3) were subjected to XRD tests (test results are shown in Table 1) and EPR tests (test results are shown in Table 2). Figure 4BET test (test results are shown in Table 1) and grain size statistical analysis (statistical results are shown in Table 1).

[0057] As shown in Table 1, the sample of Comparative Example 2 is composed of cerium dioxide. Figure 4 The EPR test results for Comparative Example 2 show an EPR signal peak at g=2.003, but its signal intensity is weaker than that of Sample 1. This indicates that the oxygen vacancy concentration in the sample is lower than that in Sample 1. Table 1 shows that the specific surface area of ​​Comparative Example 2 sample reaches 50.2 m². 2 / g, significantly lower than that of the sample in Example 1. Furthermore, Table 1 shows the statistical results of the grain size of the sample in Comparative Example 2, which is approximately 130-170 nm, significantly larger than that of the sample in Example 1. This is because the lack of L-threonine reduces the dispersibility of Ni and Ce, weakens the doping effect of Ni, and decreases its inhibitory effect on grain growth. The particles are more prone to agglomeration and sintering during calcination, resulting in increased grain size and decreased specific surface area. Simultaneously, effective Ni doping is more conducive to the formation of Ni-ortho-related asymmetric oxygen vacancies and enhances the system's capacity to accommodate oxygen vacancies. Therefore, the oxygen vacancy concentration in Comparative Example 2 is significantly reduced, making it difficult to form defect-rich structures.

[0058] The comparative sample 2 obtained in step (3) was subjected to ESR testing (the test results are shown in the figure). Figure 5 ).

[0059] Depend on Figure 5 It can be seen that the ·OH and ·O2 of the sample in Comparative Example 2 - The ESR signal is generally positive, indicating that it can generate ·OH and ·O. 2- However, the generation capacity was insufficient. This is because the lack of L-threonine for coordinating and dispersing Ni and Ce reduced the effective doping and uniform dispersion of Ni, resulting in a decrease in the number of Ni-Ce ortho-correlated defect sites in the sample. Therefore, its overall reactive oxygen species generation capacity was weaker than that of Example 1. On the other hand, compared to Comparative Example 1, which did not introduce Ni, Comparative Example 2 still retained some of the Ni-induced electronic structure regulation and defect activation effects, so its superoxide anion generation capacity was stronger than that of Comparative Example 1.

[0060] The nanozyme activity of the comparative example 2 sample obtained in step (3) was evaluated (test results are shown in [link]). Figure 6 The testing method is the same as in Example 1.

[0061] Figure 6 The peroxidase-like activity of Comparative Example 2 was shown. The results indicate that the peroxidase-like activity of Comparative Example 2 was significantly reduced, which is mainly attributed to the weakened ROS generation capacity of Comparative Example 2.

[0062] Comparative Example 3: The only difference from Example 1 is that dicyandiamide is not added in step (1). The rest of the method is exactly the same as in Example 1.

[0063] XRD tests were performed on the comparative sample 3 obtained in step (3) (the test results are shown in the figure). Figure 1 EPR test (test results are available in [link]). Figure 4 BET test (test results are shown in Table 1) and grain size statistical analysis (statistical results are shown in Table 1).

[0064] Table 1 shows that Comparative Example 3 sample is composed of cerium dioxide. Figure 4 The EPR test results of Comparative Example 3 showed an EPR signal peak at g=2.003, but its signal intensity was the weakest. This indicates that the oxygen vacancy concentration in the sample was the lowest. Table 1 shows that the BET test results of Comparative Example 3 indicated a specific surface area of ​​39.60 m². 2 / g, significantly lower than that of the sample in Example 1. Furthermore, Table 1 shows the statistical results of the grain size of the sample in Comparative Example 2, which is approximately 150-200 nm, significantly larger than that of the sample in Example 1. This is because dicyandiamide can form a confined framework structure through thermal decomposition during calcination, which helps to inhibit particle migration and agglomeration, thereby limiting grain growth. Simultaneously, the reducing gas produced by its decomposition can locally form a reducing atmosphere, which is conducive to the formation of oxygen vacancies. Without dicyandiamide, the material is more prone to sintering and densification during high-temperature calcination, causing continuous grain growth and ultimately resulting in a decrease in specific surface area. Moreover, the processes of lattice oxygen migration and defect formation are suppressed, leading to a significant decrease in oxygen vacancy concentration.

[0065] The ESR tests were performed on the comparative sample 3 obtained in step (3), and the test results are shown in the figure below. Figure 5 .

[0066] Depend on Figure 5 It can be seen that the ·OH and ·O of the comparative example 3 sample 2- The moderate ESR signal indicates that it can generate ·OH and ·O. 2- However, the generation capacity is insufficient. This is because the decrease in oxygen vacancy concentration and the reduction in exposed active sites on the surface weaken the adsorption and activation capacity of H2O2 and O2, and limit the efficiency of surface electron transfer to the adsorbed substrate. Therefore, its ·OH and ·O 2- The generation capacity is significantly reduced.

[0067] The nanozyme activity of the comparative sample 3 obtained in step (3) was evaluated (the test results are shown in the figure). Figure 6 The testing method is the same as in Example 1.

[0068] Figure 6The peroxidase-like activity of Comparative Example 3 was shown. The results indicate that the peroxidase-like activity of Comparative Example 3 was significantly decreased, which is also attributed to the reduced ROS generation capacity.

[0069] Table 1 shows the XRD and BET test results and grain size statistical analysis of Examples 1-3 and Comparative Examples 1-3. The test results are expressed as phase composition, specific surface area (m²) and other parameters. 2 The formation of g) and grain size (nm) is evident.

[0070] Table 1 Example 1 Cerium dioxide 99.3 50~90 Example 2 Cerium dioxide 97.5 50~90 Example 3 Cerium dioxide 98.0 50~90 Comparative Example 1 Cerium dioxide 64.7 110~150 Comparative Example 2 Cerium dioxide 50.2 130~170 Comparative Example 3 Cerium dioxide 39.6 150~200 A comparison of Comparative Example 1 and Examples 1-3 shows that the introduction of low-valence Ni can modulate the local electronic structure and reduce the formation energy of oxygen vacancies, thus promoting oxygen vacancy generation. Simultaneously, the proximity of Ni and Ce can form a heterogeneous metal-oxygen coordination environment, constructing an asymmetric oxygen vacancy structure. This asymmetric oxygen vacancy enhances surface electron transfer capabilities and improves ROS generation. A comparison of Comparative Example 2 and Examples 1-3 shows that L-threonine can coordinate with metal ions, inhibiting the local enrichment of metal ions in solution. During calcination, a uniformly distributed metal precursor is more conducive to the effective entry of Ni into the cerium dioxide framework, thereby improving the effective doping of Ni and forming a Ni-Ce adjacent structure, providing a structural basis for constructing asymmetric oxygen vacancies. A comparison of Comparative Example 3 with Examples 1-3 reveals that dicyandiamide possesses dual functions of structure regulation and atmosphere regulation. Firstly, the thermal decomposition of dicyandiamide forms a confined framework structure, effectively inhibiting the migration and sintering of nanoparticles at high temperatures. Secondly, the released reducing gas creates a relatively reducing atmosphere, promoting the migration of lattice oxygen and accelerating the extraction of oxygen atoms from the lattice, thereby promoting oxygen vacancy formation. Ni doping, L-threonine, and dicyandiamide work synergistically in the material preparation process from three levels: electronic structure regulation, metal dispersion control, and defect formation environment adjustment, respectively, jointly promoting the construction of asymmetric oxygen vacancy nanostructures.

Claims

1. A method for preparing cerium dioxide nanozymes with asymmetric oxygen vacancies, characterized in that, Includes the following steps: S.1 Cerium nitrate hexahydrate and nickel nitrate hexahydrate were dissolved in deionized water and stirred to obtain a mixed metal salt solution; L-threonine and dicyandiamide were dissolved in deionized water and stirred to obtain an amino acid aqueous solution, which was then slowly added to the mixed metal salt solution and stirred continuously to obtain a precursor solution. S.2 The precursor solution was freeze-dried to obtain a solid precursor, which was then ground into powder to obtain solid A. S.3 Solid A was placed in a tube furnace and calcined under a nitrogen atmosphere. After natural cooling, Ni-Ov-Ce nanozyme particles were obtained, which are cerium dioxide nanozymes with asymmetric oxygen vacancies.

2. The preparation method according to claim 1, characterized in that, In step S.1, the ratio of cerium nitrate hexahydrate, nickel nitrate hexahydrate, and deionized water is 5 mmol: 0.2-0.3 mmol: 35-50 mL.

3. The preparation method according to claim 1, characterized in that, In step S.2, the ratio of L-threonine, dicyandiamide, and deionized water is 10 mmol: 20-40 mmol: 15-30 mL.

4. The preparation method according to claim 1, characterized in that, In step S.1, the first stirring conditions are: room temperature and 15-25 min; the second stirring conditions are: room temperature and 8-13 min; and the third stirring conditions are: 50-65℃ and 1.5-3 h.

5. The preparation method according to claim 1, characterized in that, In step S.2, the freeze-drying conditions are: temperature -80℃; time 24h.

6. The preparation method according to claim 1, characterized in that, In step S.3, the calcination conditions are as follows: heating rate is 2-3℃ / min; calcination temperature is 500-600℃; and holding time is 2-5h.

7. A cerium dioxide nanozyme with asymmetric oxygen vacancies, characterized in that, The cerium dioxide nanozyme with asymmetric oxygen vacancies is prepared using any one of the preparation methods described in claims 1-6.

8. The application of a cerium dioxide nanozyme with asymmetric oxygen vacancies as described in claim 7, characterized in that, Cerium dioxide nanozymes with asymmetric oxygen vacancies are used in a biosensor for creatinine detection.

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