A Ce(MoO4)2 nanozyme rich in oxygen vacancy defects, its preparation method and application

Abstract

This invention discloses an oxygen vacancy-deficient Ce(MoO4)2 nanozyme, its preparation method, and its applications. The nanozyme is prepared by completely dissolving molybdenum salt, cerium salt, and urea in an ethanol-water solution, followed by ultrasonication, solvothermal reaction, and calcination to obtain Ce(MoO4)2 nanoparticles. These Ce(MoO4)2 nanoparticles are then etched with hydrogen peroxide. The preparation method of this invention is simple and mild, and the resulting material exhibits excellent mimicking oxidase activity. It can be used for highly selective colorimetric-photothermal dual-mode determination and identification of phenolic antioxidants in traditional Chinese medicine. This invention provides a new approach for constructing highly catalytically active oxygen vacancy-deficient Ce(MoO4)2 bimetallic mimicking oxidases and also provides a simple, self-calibrating, and portable method for the determination and identification of phenolic antioxidants in traditional Chinese medicine.

Description

Technical Field

[0001] This invention belongs to the field of nanozyme technology, specifically relating to an oxygen vacancy-defective Ce(MoO4)2 nanozyme, as well as the preparation method and application of the nanozyme. Background Technology

[0002] Natural enzymes are highly efficient catalysts capable of catalyzing various reactions under mild conditions, exhibiting superior catalytic activity and specificity, and have been widely used in medicine, agriculture, and the environment. However, natural enzymes suffer from drawbacks such as high preparation costs, poor stability, and demanding storage conditions, which significantly limit their widespread application in various fields. In recent years, researchers have discovered some nanomaterials with good enzyme-like catalytic activity, which scientists have termed "nanozymes." Compared to natural enzymes, nanozymes have attracted widespread attention in drug analysis, biocatalysis, and disease diagnosis and treatment due to their high stability, low preparation costs, and tunable catalytic activity. However, it is undeniable that most reported nanozymes not only have lower catalytic activity than natural enzymes but also suffer from limitations such as high preparation costs, limited functionality, and slow catalytic reaction rates. Therefore, there is an urgent need to develop nanozymes with high catalytic activity, environmental friendliness, tunable active sites, and fast reaction rates.

[0003] Currently, research on nanozymes mainly focuses on redox mimics and hydrolysis mimics. In contrast, redox mimics have great application potential in fields such as biosensing, disease diagnosis and treatment, and environmental remediation. These nanozymes mainly include peroxide mimics, oxidases, and laccases. Studies have revealed that oxidases have more advantages than peroxide mimics in the field of biosensing and detection, mainly in the following aspects: (1) When using peroxide mimics as catalysts, a strong oxidant H2O2 is required to achieve the catalytic reaction, while hydrogen peroxide easily oxidizes the analyte, resulting in poor accuracy of the detection results; (2) Most nanozymes have both peroxide mimic and hydrogen peroxide mimic activities, which makes the reaction substrate H2O2 easily decomposed, resulting in large measurement errors and difficulty in reproducibility; (3) Oxidases use dissolved oxygen to catalyze the color development of the substrate, without the need to add the oxidant hydrogen peroxide, making the operation simple and the conditions mild. Since the advent of nanozymes, noble metal nanoparticles, metal oxides, metal sulfides, metal-organic frameworks, and carbon nanomaterials have been successively proven to have enzyme-like catalytic activities. Studies have shown that the catalytic activity of nanozymes can be modulated by controlling the size, morphology, surface modification, lattice defects, surface valence state, and composition of nanomaterials. Although nano-oxidative enzymes have been reported, synthesizing nano-oxidative enzymes with fast reaction rates and high catalytic activity using multivalent transition metals and variable-valence rare earth elements remains extremely challenging.

[0004] Natural polyphenols are widely found in plants. Modern scientific research shows that natural polyphenols not only possess excellent antioxidant and antitumor activities, but their main pharmacological effects include delaying aging, preventing cardiovascular diseases, anti-cancer, anti-radiation, and anti-AIDS. Accurate assessment of phenolic antioxidant levels is crucial for food and pharmaceutical quality control. Current methods for determining phenolic compounds mainly include the potassium permanganate method, spectroscopic analysis, and chromatographic analysis. However, these methods suffer from drawbacks such as long processing times, expensive equipment, and the inability to identify phenolic antioxidants. In particular, the chemical structures of phenolic antioxidants are extremely similar, making accurate identification or differentiation difficult with existing detection techniques. Therefore, there is an urgent need to develop portable analytical platforms with high selectivity, high sensitivity, and fast analysis speed for the simultaneous determination and identification of phenolic antioxidants. This has extremely important practical significance and scientific value for the quality control of phenolic foods and drugs, and for in vivo drug analysis.

[0005] The rise of nanozyme sensing arrays has brought new opportunities for the simultaneous determination and rapid identification of similar compounds. Currently, V-doped Co@carbon nanozymes have been used for colorimetric determination and differentiation of phenolic antioxidants, while MnCo@C nanozymes have been used for the determination of phenolic compounds. In addition, NiMoO6@Co3O4, FeCo nanoparticle-doped porous carbon, and layered hydroxide nanozymes have been successively used to determine or differentiate biothiols. Although nanozymes have been reported in the determination and differentiation of phenolic antioxidants, they suffer from limitations such as low catalytic activity, high preparation cost, limited testing modes, and slow analysis speed. Therefore, there is an urgent need to develop nano-oxidases with high catalytic activity, tunable active sites, and fast reaction rates for the sensitive and rapid determination and / or differentiation of phenolic antioxidants.

[0006] Compared to single-mode sensing technologies, nanozyme-based dual-mode sensing platforms offer a wider linear range and higher detection sensitivity, and are particularly capable of self-calibrating the test signal. Currently, dual-mode detection technologies such as colorimetric-fluorescence, colorimetric-electrochemical, colorimetric-photothermal, and colorimetric-Raman spectroscopy have been applied to the determination of various target compounds. In contrast, the colorimetric-temperature combination mode offers significant advantages in high sensitivity, low cost, and portability. Although there have been reports on the application of colorimetric-temperature dual-mode sensing technology in toxicology analysis, there is still a lack of research reports on its use for the simultaneous determination and differentiation of phenolic antioxidants. Summary of the Invention

[0007] To address the bottleneck issue of the difficulty in identifying phenolic antioxidants, this invention provides a method for preparing oxygen vacancy-defect-rich Ce(MoO4)2 nanozymes. This method uses cerium and molybdenum salts as metal precursors. First, a CeMo bimetallic gel is prepared using a sol-gel assembly technique. Then, Ce(MoO4)2 is obtained through hydrothermal crystallization. Finally, the oxygen vacancy-defect-rich Ce(MoO4)2 nanozyme is obtained by sequentially performing thermal annealing and H2O2 etching surface reshaping techniques.

[0008] To achieve the above objectives, the oxygen-vacancy-defect-rich Ce(MoO4)2 nanozyme of the present invention is prepared by the following method:

[0009] Step 1: Urea, cerium salt, and molybdenum salt are completely dissolved in deionized water and sonicated for 20-40 minutes. Then, a 15%-40% (v / v) ethanol aqueous solution is added, and sonication continues for 60-80 minutes. The resulting mixture is then transferred to a polytetrafluoroethylene reactor and solvothermal reacted at 140-180°C for 5-12 hours. The product after reaction is washed with deionized water and anhydrous ethanol, dried, and finally calcined at 200-350°C in air for 2-3 hours to obtain Ce(MoO4)2 nanoparticles. The molar ratio of Mo(VI) in the molybdenum salt to Ce(III) in the cerium salt and urea is 1:1:5-42.

[0010] Step 2: Mix Ce(MoO4)2 nanoparticles with a hydrogen peroxide solution with a mass concentration of 0.1% to 1% H2O2, sonicate for 60 to 120 minutes, and then transfer to a polytetrafluoroethylene reactor. Perform hydrothermal reaction at 50 to 90 °C for 8 to 12 hours. Wash the obtained product sequentially with deionized water and anhydrous ethanol, and then dry to obtain oxygen vacancy-deficient Ce(MoO4)2 nanozyme.

[0011] In step 1 above, the preferred molar ratio of Mo(VI) in the molybdenum salt to Ce(III) in the cerium salt and urea is 1:1:15 to 25.

[0012] In step 1 above, the molybdenum salt is preferably sodium molybdate dihydrate, and the cerium salt is cerium nitrate hexahydrate.

[0013] In step 1 above, it is even more preferable to transfer the obtained mixture into a hydrothermal reactor and hydrothermally react at 160-170 °C for 10 hours.

[0014] In step 1 above, it is further preferred to calcine at 250 °C for 2 hours in an air atmosphere.

[0015] In step 2 above, the preferred mass concentration of H2O2 in the hydrogen peroxide solution is 0.6% to 1%, and the concentration of Ce(MoO4)2 nanoparticles in the hydrogen peroxide solution is 0.5 to 2 mg / mL.

[0016] In step 2 above, it is further preferred to perform a hydrothermal reaction at 70 °C for 10 hours.

[0017] The oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared by this invention has excellent oxidase-like catalytic activity and can be used for the determination and identification of phenolic antioxidants such as gallic acid, kaempferol, caffeic acid, quercetin and catechin in the traditional Chinese medicine sea buckthorn by colorimetric-temperature dual-mode sensor array.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention uses cerium and molybdenum salts as metal precursors and Mo and Ce as multivalent transition metals. The valence transitions between these multivalent elements exhibit excellent catalytic activity. CeMo gel is prepared using sol-gel assembly technology, followed by a solvothermal reaction to obtain the bimetallic oxide Ce(MoO4)2. Finally, a highly catalytically active oxygen-vacancy-deficient Ce(MoO4)2 nanozyme is prepared through sequential thermal annealing and H2O2 surface etching. Utilizing the excellent oxidative enzyme-like catalytic activity of this nanozyme, this invention constructs a colorimetric-photothermal dual-mode sensor and a gel-based photothermal-smartphone dual-mode sensor. The fabricated sensors have successfully achieved effective differentiation and identification of quercetin, gallic acid, kaempferol, caffeic acid, and catechins in the traditional Chinese medicine Hippophae rhamnoides, demonstrating high sensitivity and good selectivity. In summary, the oxygen-vacancy-deficient Ce(MoO4)2 nanozyme constructed in this invention provides a portable, low-cost, self-calibrating, and versatile sensing array platform for the identification and quality control of genuine and counterfeit traditional Chinese medicine. Attached Figure Description

[0020] Figure 1 These are UV-Vis spectra of the catalytic activity of Ce(MoO4)2 nanoparticles prepared under different urea concentrations.

[0021] Figure 2 These are UV-Vis spectra of the catalytic activity of Ce(MoO4)2 nanoparticles prepared under different calcination temperature conditions.

[0022] Figure 3 The images show the UV-Vis spectra of the catalytic activity of oxygen vacancy-deficient Ce(MoO4)2 nanozymes prepared by hydrogen peroxide treatment with different mass fractions.

[0023] Figure 4 It is the oxygen vacancy defect type Ce(MoO) prepared in Example 1 4-x Scanning electron microscope image of 2 nanozymes.

[0024] Figure 5 This is an X-ray powder diffraction pattern of Ce(MoO4)2 nanoparticles and oxygen vacancy-deficient Ce(MoO4)2 nanozymes prepared in Example 1.

[0025] Figure 6 This is a fitted X-ray photoelectron spectrum of the O element in Ce(MoO4)2 nanoparticles prepared in Example 1.

[0026] Figure 7 This is a fitted X-ray photoelectron spectroscopy (XPS) spectrum of the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1.

[0027] Figure 8 This is a steady-state kinetic curve of TMB for the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1.

[0028] Figure 9 This is the UV-Vis absorption spectrum of tetramethylbenzidine catalyzed by the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1.

[0029] Figure 10 This is a colorimetric curve of the concentration-absorbance of kaempferol prepared in Example 1 using the oxygen vacancy-deficient Ce(MoO4)2 nanozyme.

[0030] Figure 11 This is a linear curve of the colorimetric analysis of kaempferol using the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1.

[0031] Figure 12 This is a linear curve of the concentration-temperature change of kaempferol in the photothermal analysis of the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1.

[0032] Figure 13 This is a colorimetric score diagram of the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1, which distinguishes single phenolic antioxidants.

[0033] Figure 14 This is a score diagram showing the photothermal differentiation of single phenolic antioxidants by the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1.

[0034] Figure 15 This is a score chart showing the colorimetric differentiation of a mixture of triphenolic antioxidants using the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1.

[0035] Figure 16 This is a score diagram showing the photothermal differentiation of a mixture of triphenolic antioxidants by the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1.

[0036] Figure 17 This is a bar chart showing the selectivity of phenolic antioxidants analyzed by colorimetric method using oxygen vacancy-deficient Ce(MoO4)2 nanozymes prepared in Example 1.

[0037] Figure 18 This is a selective score diagram of the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1, which distinguishes phenolic antioxidants from common reducing molecules using a colorimetric method.

[0038] Figure 19 This is a score graph showing the oxygen vacancy-deficient Ce(MoO4)2 nanozyme gel kit prepared in Example 1 used to distinguish phenolic antioxidants in a smartphone / photothermal dual-mode system.

[0039] Figure 20 This is a score chart showing the colorimetric method for distinguishing phenolic antioxidants in the traditional Chinese medicine sea buckthorn using the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1.

[0040] Figure 21 This is a score chart showing the photothermal differentiation of phenolic antioxidants in the traditional Chinese medicine sea buckthorn using the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1. Detailed Implementation

[0041] The present invention will be described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0042] To obtain oxygen vacancy-deficient Ce(MoO4)2 nanozymes with optimal catalytic performance, the optimal urea dosage and calcination temperature were first optimized to screen for Ce(MoO4)2 nanoparticles with the best catalytic activity. Subsequently, the Ce(MoO4)2 nanoparticles were etched with hydrogen peroxide of different mass concentrations to obtain the best-performing oxygen vacancy-deficient Ce(MoO4)2 nanozyme. Specific experiments are as follows:

[0043] 1. Determine the optimal dosage of urea.

[0044] 0.87 g (2 mmol) of cerium nitrate hexahydrate and 0.48 g (2 mmol) of sodium molybdate dihydrate were added sequentially to 100 mL of deionized water, followed by the addition of 0.3 g, 0.6 g, 0.9 g, 1.2 g, 1.8 g, and 2.5 g of urea, respectively. The mixture was sonicated for 30 minutes, and then 50 mL of 25% ethanol aqueous solution was added, followed by sonication for another 70 minutes. The resulting mixture was then transferred to a polytetrafluoroethylene reactor and heated and stirred at 160 °C for 10 hours under sealed conditions. The product was washed sequentially with deionized water and ethanol, dried at 80 °C, and then calcined at 250 °C for 2 hours in air to obtain Ce(MoO4)2 nanoparticles.

[0045] 2. Determine the optimal calcination temperature

[0046] 1.8 g of urea, 0.87 g (2 mmol) of cerium nitrate hexahydrate and 0.48 g (2 mmol) of sodium molybdate dihydrate were added sequentially to 100 mL of deionized water and sonicated for 30 minutes. Then, 50 mL of 25% ethanol aqueous solution was added and sonicated for another 70 minutes. The resulting mixture was then transferred to a polytetrafluoroethylene reactor and heated and stirred at 160 °C for 10 hours under sealed conditions. The product was washed sequentially with deionized water and ethanol, dried at 80 °C, and then calcined at 200 °C, 250 °C, 350 °C, 450 °C and 550 °C for 2 hours in air to obtain Ce(MoO4)2 nanoparticles.

[0047] Depend on Figures 1-2 It can be seen that, under the condition that other synthesis conditions remain unchanged, the Ce(MoO4)2 nanoparticles exhibit the highest oxidase-like catalytic activity when the amount of urea is 1.8 g and the calcination temperature is 250 ℃.

[0048] 3. Determine the optimal concentration of hydrogen peroxide.

[0049] Ce(MoO4)2 nanoparticles were prepared under the optimal conditions described above. 100 mg of Ce(MoO4)2 nanoparticles were mixed with 100 mL of H2O2 aqueous solutions with mass concentrations of 0.1%, 0.3%, 0.6%, and 1%, respectively. After sonication for 90 minutes, the mixture was transferred to a polytetrafluoroethylene (PTFE) reactor and reacted under sealed conditions at 70°C with stirring for 10 hours. After the reaction was complete, the resulting products were washed sequentially with deionized water and ethanol, and dried at 80°C to obtain oxygen vacancy-deficient Ce(MoO4)2 nanozymes.

[0050] Depend on Figure 3 It is evident that treating Ce(MoO4)2 nanoparticles with 0.6% H2O2 by mass concentration yields oxygen vacancy-deficient Ce(MoO4)2 nanozymes with optimal performance. From... Figure 3 It was also found that, under the same experimental conditions, the absorbance of Ce(MoO4)2 nanoparticles after oxidizing tetramethylbenzidine (TMB) was 0.364, while the absorbance of Ce(MoO4)2 nanoparticles (i.e., oxygen vacancy defective Ce(MoO4)2) after oxidizing TMB with 0.6% H2O2 was 1.188. It can be seen that the catalytic activity can be increased by about 3.3 times after H2O2 treatment.

[0051] Example 1

[0052] Step 1: 1.80 g (30 mmol) of urea, 0.87 g (2 mmol) of cerium nitrate hexahydrate and 0.48 g (2 mmol) of sodium molybdate dihydrate were added sequentially to 100 mL of deionized water and sonicated for 30 minutes. Then, 50 mL of 25% ethanol aqueous solution was added and sonicated for another 70 minutes. The resulting mixture was then transferred to a polytetrafluoroethylene reactor and heated and stirred at 160 °C for 10 hours under sealed conditions. The product was washed sequentially with deionized water and ethanol, dried at 80 °C, and then calcined at 250 °C for 2 hours in air to obtain Ce(MoO4)2 nanoparticles.

[0053] Step 2: Mix 100 mg of Ce(MoO4)2 nanoparticles with 100 mL of 0.6% hydrogen peroxide solution, sonicate for 90 minutes, then transfer to a polytetrafluoroethylene reactor. Heat and stir at 70°C for 10 hours under sealed conditions. After the reaction is complete, wash the product sequentially with deionized water and ethanol, and dry at 80°C to obtain oxygen vacancy-deficient Ce(MoO4)2 (denoted as Ce(MoO4)2). 4-x 2) Nanozymes.

[0054] The micro- and meso-morphological characteristics, pore structure, and oxygen vacancy level of the oxygen-vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1 were characterized using field emission scanning electron microscopy, X-ray powder diffraction, an ASAP 2020 physical adsorption analyzer, and X-ray photoelectron spectroscopy. Figure 4 It can be seen that the prepared oxygen vacancy-deficient Ce(MoO4)2 nanozyme exhibits a cauliflower-like structure. (From...) Figure 5 It is known that Ce(MoO4)2 nanoparticles exhibit XRD diffraction peaks of (112), (004), (200), (220), (204), (116), (303), and (316) within the diffraction angle range of 20–80°. These diffraction peaks are typical diffraction peaks of Ce(MoO4)2. However, the XRD diffraction peaks of oxygen vacancy-deficient Ce(MoO4)2 nanozymes include (112), (200), (204), and (303). These diffraction data reveal that H2O2 treatment can cause some of the inherent diffraction peaks of Ce(MoO4)2 nanoparticles to disappear, thereby causing changes in crystal structure and the generation of structural and atomic defects. Figure 6 and Figure 7This indicates that Ce(MoO4)2 nanoparticles exhibit an oxygen vacancy fitting curve near 531.4 eV, while the Ce(MoO4)2 nanoparticles treated with H2O2 (i.e., oxygen vacancy-deficient Ce(MoO4)2 nanozymes) not only show a Ce-O bond fitting peak at 529.7 eV, but also exhibit a strong oxygen vacancy fitting curve near 531.4 eV, indicating that oxygen vacancy-deficient Ce(MoO4)2 nanozymes have been successfully prepared.

[0055] As shown in Table 1, the specific surface area of ​​the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1 is 44.5 m². 2 / g, total pore volume is 0.19 cm³ 3 / g and an average mesopore size of 9.9 nm. Simultaneously, experiments were conducted using the Michaelis equation, and the Michaelis constant (Kmax) and maximum reaction rate constant (Vmax) of the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1 in the TMB and acetate-sodium acetate buffer system were calculated, as shown in Table 1.

[0056] Table 1. Oxygen vacancy defective Ce(MoO) prepared in Example 1 4-x Texture properties and enzyme catalytic kinetic parameters of 2 nanozymes

[0057]

[0058] Note: In the table [a] It is the surface area of ​​BET; [b] It is the total orifice capacity; [c] It is the average mesopore size (BJH method).

[0059] Using tetramethylbenzidine (TMB) as the chromogenic substrate, the same concentration of the oxygen vacancy-deficient Ce(MoO4)2 nanozyme or control prepared in Example 1 was placed with TMB in an acetate buffer system (0.2 M, pH = 4.0) and reacted in a dark room at 40 °C for 15 minutes. The color change was observed, and the 550–750 nm UV-Vis absorption spectrum was scanned. Figure 8 As can be seen, the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1 exhibits a fast reaction rate and good affinity for the substrate TMB as an oxidation mimic enzyme. Figure 9 It can be seen that the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared under optimal conditions has excellent oxidative enzyme catalytic activity. When TMB is mixed with the oxygen vacancy-deficient Ce(MoO4)2 nanozyme at room temperature, it can instantly change from colorless to blue solution, indicating that the nanozyme has the advantages of high catalytic activity and fast reaction rate.

[0060] Example 2

[0061] The oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1 was used for the detection of phenolic antioxidants using a colorimetric-photothermal dual-mode sensing array.

[0062] 1. Colorimetric detection of phenolic antioxidants

[0063] The detection performance of the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1 for phenolic antioxidants was tested using ultraviolet-visible spectrophotometry. This study was based on the phenomenon that the oxygen vacancy-deficient Ce(MoO4)2 nanozyme catalyzes the formation of blue oxidized tetramethylbenzidine from the colorless substrate under H2O2-free conditions. Phenolic antioxidants inhibit the oxidase-like activity of the oxygen vacancy-deficient Ce(MoO4)2 nanozyme, resulting in a decrease in the amount of blue oxidized tetramethylbenzidine produced with increasing phenolic compounds. The blue solution gradually fades, and the corresponding absorbance value gradually decreases, thus enabling the detection of phenolic antioxidants.

[0064] Experimental Method: First, 50 μL of 10.2 mM TMB in N,N-dimethylformamide solution was added to 2.4 mL of an acetate-sodium acetate buffer system (pH 4.0, 0.2 M). Then, 150 μL of an aqueous solution of 5 mg / mL oxygen vacancy-deficient Ce(MoO4)2 nanozyme was added. Subsequently, different concentrations of phenolic antioxidants (kaempferol as an example) in N,N-dimethylformamide were added to the above mixture. The mixture was reacted under ambient temperature and dark conditions for 8 minutes. Finally, the absorbance value at 652 nm was measured. Figures 10-11 It can be seen that as the concentration of the phenolic antioxidant kaempferol increases, the difference in absorbance between the blank group (no phenolic antioxidant added) and the experimental group (different concentrations of phenolic antioxidants) gradually increases, and the linear fit between the concentration of the phenolic antioxidant kaempferol and the change in absorbance is good (R0). 2 =0.9789), and the limit of detection was 0.69 μM.

[0065] 2. Photothermal method for detecting phenolic antioxidants

[0066] Based on the photothermal effect of oxidized tetramethylbenzidine, the detection performance of the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1 for phenolic antioxidants was tested using thermal infrared imaging technology. Detection principle: The oxygen vacancy-deficient Ce(MoO4)2 nanozyme can catalyze TMB to oxidized tetramethylbenzidine in the absence of H2O2, while phenolic antioxidants can inhibit the formation of oxidized tetramethylbenzidine. The less oxidized tetramethylbenzidine is formed, the smaller the temperature rise under constant laser irradiation conditions, and vice versa, thus enabling the detection of phenolic antioxidants.

[0067] Experimental Methods: 125 μL of an aqueous solution of 2.4 mg / mL oxygen-vacancy-deficient Ce(MoO4)2 nanozyme, 100 μL of a 6 mM TMB solution in N,N-dimethylformamide, and 100 μL of a 0.3 mM kaempferol solution in N,N-dimethylformamide were added to a sodium acetate-acetic acid buffer system (200 mM, pH 4.0), for a total volume of 3 mL. The resulting mixture was then irradiated under an 808 nm near-infrared laser (2.42 W) for 8 minutes, and the temperature was recorded in real time using a thermal imager. Figure 12 It can be seen that as the concentration of the phenolic antioxidant kaempferol increases, the temperature change (the temperature difference between the control group and the experimental group) gradually decreases, and there is a good linear relationship between the temperature change and the kaempferol concentration (R0). 2 =0.9953), and the detection limit was 1.34 μM, indicating that the oxygen vacancy-deficient Ce(MoO4)2 nanozyme can sensitively and accurately determine antioxidant phenolic compounds.

[0068] 3. Colorimetric-photothermal dual-mode signal output distinguishes phenolic antioxidants.

[0069] (1) Colorimetric differentiation test

[0070] The effect of the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1 on distinguishing similar phenolic compounds was studied by ultraviolet spectrophotometry. Experimental method: 100 μL of aqueous solution of oxygen vacancy-deficient Ce(MoO4)2 nanozyme (final concentration of system was 80 μg / mL), 50 μL of TMB (final concentration of system was 0.05 mM) and 2.75 mL of sodium acetate-acetic acid buffer system (0.2 M, pH 4.0) were mixed. Then, 100 μL of N,N-dimethylformamide solution of five phenolic antioxidants (quercetin, kaempferol, gallic acid, caffeic acid and catechin) at different concentrations (final concentrations of system was 5 μM, 10 μM and 20 μM) were added to the above mixture. 100 μL of N,N-dimethylformamide was added to the blank group. The total volume of the reaction system was 3 mL. The resulting mixture was then placed in a constant temperature water bath at 25 °C for 8 minutes. The UV-Vis absorption spectrum at 340–800 nm was scanned, and the absorbance values ​​at 370 nm, 450 nm, and 652 nm were recorded.

[0071] Using a data matrix of "5 replicates × 5 phenolic antioxidants × 3 absorbance values", analytical data for different concentrations of the five phenolic antioxidants were fitted. Figure 13It can be seen that when the minimum concentration of different phenolic antioxidants is 5 μM, the fitted data points of the same type of phenolic antioxidants are distributed in the same cluster, and the fitted data points of different types of phenolic antioxidants are significantly distinguished. This indicates that the colorimetric method has a good effect on distinguishing phenolic antioxidants with similar structures, and the minimum distinguishing concentration is 5 μM.

[0072] (2) Photothermal method for differentiation

[0073] To overcome the shortcomings of single-sensor mode, which is susceptible to interference from external factors and prone to false positives due to lack of self-correction, a colorimetric-photothermal dual-mode approach is proposed to differentiate phenolic antioxidants, potentially improving the limitations of single-mode identification. The effectiveness of thermal infrared imaging in differentiating phenolic antioxidants using the oxygen-vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1 was investigated. The experimental method was similar to the colorimetric method, except that a mixture containing different concentrations of different phenolic antioxidants was irradiated with an 808 nm laser (2.42 W) for 8 minutes, and the temperature values ​​at time points 4, 6, and 8 minutes were recorded. Using a matrix of "3 replicates × 5 types of phenolic antioxidants × 3 temperature values," principal component analysis was used to calculate and effectively identify phenolic antioxidants. Figure 14 It can be seen that the photothermal method has a good effect on distinguishing the five phenolic antioxidants, and the lowest distinguishing concentration is 6 μM.

[0074] (3) Differentiation test of phenolic antioxidant mixture

[0075] Considering that actual samples may contain multiple phenolic antioxidants, identification tests were conducted using binary (quercetin, caffeic acid) and ternary (quercetin, caffeic acid, catechin) phenolic antioxidant systems as the research subjects. The experimental method was the same as the colorimetric-photothermal single-mode differentiation method, except that the total concentration of phenolic antioxidants was fixed at 10 μM, and the colorimetric-photothermal differentiation experiment was performed by changing the ratio of phenolic antioxidants in the binary and ternary systems. Figures 15-16 It can be seen that mixtures of binary and trihydric phenolic antioxidants with different concentration ratios can be well distinguished by the colorimetric-photothermal sensing array, indicating that colorimetry and photothermal methods can effectively identify binary and trihydric phenolic antioxidants.

[0076] (4) Anti-interference test

[0077] To investigate the specificity of the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1 for analyzing phenolic antioxidants, common metal ions (Na+, Na ... + Ca 2+ Mn 2+The experiment involved amino acids (glycine, arginine), macromolecules (glucose), and common reducing substances (dopamine, glutathione, ascorbic acid, citric acid). The specific method is as follows: 100 μL of an aqueous solution of oxygen vacancy-deficient Ce(MoO4)2 nanozyme (final concentration of the system is 80 μg / mL), 50 μL of TMB (final concentration of the system is 0.05 mM) and 2.65 mL of sodium acetate-acetic acid buffer system (0.2 M, pH 4.0) were mixed. Then, 100 μL of phenolic antioxidants (except for catechins, the final concentration of the system is 30 μM, and the final concentration of the other phenolic antioxidants is 10 μM) and 100 μL of interfering metal ions (final concentration of the system is 600 μM), amino acids and glucose (final concentration of the system is 300 μM) and general reducing substances (final concentration of the system is 10 μM) were added. After mixing evenly, the resulting mixture (total volume is 3 mL) was placed in a constant temperature water bath at 25 ℃ for 8 minutes. The absorbance values ​​at 370 nm, 450 nm and 652 nm were measured, and the relative activity values ​​of various interfering substances and the blank control group were calculated. Meanwhile, principal component analysis was used to differentiate between interfering substances and phenolic antioxidants.

[0078] Depend on Figure 17 It can be seen that, apart from common reducing agents, other interfering substances have almost no impact on the analysis of phenolic antioxidants. Common reducing agents possess certain antioxidant activity and inhibit the oxidase activity of oxygen vacancy-deficient Ce(MoO4)2 nanozymes, but their antioxidant activity differs from that of phenolic antioxidants, and the degree of inhibition of catalytic activity also varies. Figure 18 Principal component analysis results show that general reducing substances have almost no interference with the identification of phenolic antioxidants and can be well distinguished from each other, indicating that oxygen vacancy-deficient Ce(MoO4)2 nanozymes have good selectivity in the analysis of phenolic antioxidants.

[0079] (5) Construction of gel reagent kit and differentiation of phenolic antioxidants by smartphone / photothermal method

[0080] To broaden the application prospects of oxygen vacancy-deficient Ce(MoO4)2 nanozymes in colorimetric-temperature dual-mode identification of phenolic antioxidants, a kit for gel-encapsulated oxygen vacancy-deficient Ce(MoO4)2 nanozymes was further constructed and a differentiation experiment was conducted.

[0081] Preparation of the gel kit: Dissolve 500 mg of agar powder in 50 mL of deionized water and stir in a 90 °C water bath. Then add 21.5 mg of the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1, stir for 30 minutes and sonicate for 40 minutes. When the temperature of the mixture drops to about 60 °C, pipette 700 μL of the mixture into a 48-well plate and cure at room temperature for 2 hours for later use.

[0082] A smartphone-based photothermal assay for distinguishing phenolic antioxidants using a gel-gel kit: First, 600 μL of acetate-sodium acetate buffer solution (pH 4.0) was added to the prepared gel plate. Then, 100 μL of 60 mM TMB in N,N-dimethylformamide solution and 100 μL of phenolic antioxidant N,N-dimethylformamide solution were added (final concentration of the system was 10 μM). A blank control group was prepared with 100 μL of N,N-dimethylformamide. The gel plate containing the mixture was then irradiated with an 808 nm near-infrared laser (2.42 W) for 8 minutes, and the temperature values ​​at 4, 6, and 8 minutes were recorded. Simultaneously, a smartphone app was used to record color images after 8 minutes of irradiation, and the R, G, and B color parameter values ​​were analyzed. A data matrix of "3 replicates × 5 phenolic antioxidants × 6 analytical values" (3 temperature values ​​+ 3 color values) was constructed using the temperature changes and chromaticity values ​​(RGB) at the corresponding time points, and fitted using principal component analysis. Figure 19 It can be seen that the photothermal-smartphone dual-mode sensing system can distinguish each phenolic antioxidant very well. There is almost no overlap between the distinguishing clusters of each phenolic compound, indicating that the gel kit can successfully achieve the goal of distinguishing phenolic antioxidants using photothermal-smartphone, with a minimum distinguishing concentration of 10 μM.

[0083] 4. Identification test of phenolic antioxidants in the traditional Chinese medicine sea buckthorn

[0084] To explore the application potential of the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in Example 1 in the identification of phenolic antioxidants in traditional Chinese medicine, a method for distinguishing phenolic antioxidants in sea buckthorn was established using colorimetric-temperature dual-mode methods, with sea buckthorn as the research object.

[0085] Experimental method: First, 10 g of sea buckthorn was placed in a round-bottom flask, and 180 mL of n-hexane was added as the extraction solvent. The flask was sealed and ultrasonically treated in a 45 ℃ ultrasonic water bath (120 W) for 1.5 hours to remove fat. Then, it was washed with n-hexane and 75% ethanol in sequence, filtered, and dried to obtain defatted sea buckthorn powder. Next, 6 g of defatted sea buckthorn powder was mixed with 36 mL of dimethylacetamide and ultrasonicated at a constant power of 260 W and a temperature of 50 ℃ for 3 hours. The supernatant was collected and filtered through a 0.45 μM microporous membrane to obtain sea buckthorn extract containing phenolic antioxidants. The extract was diluted 60 times with dimethylacetamide for later use.

[0086] Five phenolic antioxidants of equal amounts were added to diluted sea buckthorn extract using a spiking method to prepare two sample test solutions (0.15 mM and 0.18 mM) (where the diluted sea buckthorn extract was used as one type of phenolic antioxidant). The test was then performed according to the steps in Experiment 3 above, which involved colorimetric-photothermal dual-mode signal output to distinguish the phenolic antioxidants (3). Similarly, using absorbance change and temperature difference as response signals, a data matrix of "3 replicates × 6 types of phenolic antioxidants (including one type of diluted sea buckthorn extract) × 3 absorbance change values ​​or 3 temperature difference values" was constructed. Figures 20-21 It is evident that both colorimetric and photothermal sensor arrays can distinguish different types of phenolic antioxidants. Each phenolic compound clusters well into a single class with almost no overlap, achieving satisfactory discrimination capability. These results fully demonstrate that the oxygen vacancy-deficient Ce(MoO4)2 nanozyme can effectively identify phenolic antioxidants in the traditional Chinese medicine Hippophae rhamnoides using a colorimetric-photothermal dual-mode sensor. This sensor also holds promise for accurately identifying phenolic antioxidants in pharmaceuticals, food, and biofluids.

[0087] The above results demonstrate that the oxygen vacancy-deficient Ce(MoO4)2 nanozyme prepared in this invention has excellent oxidative enzyme catalytic activity and can be used to construct a low-cost, highly sensitive, self-calibrating, multi-mode, and portable sensing array for distinguishing phenolic antioxidants in traditional Chinese medicine.

Claims

1. A method for preparing oxygen vacancy-defect-rich Ce(MoO4)2 nanozymes, characterized in that: Includes the following steps: Step 1: Urea, cerium salt, and molybdenum salt are completely dissolved in deionized water and sonicated for 20-40 minutes. Then, a 15%-40% (v / v) ethanol aqueous solution is added, and sonication continues for 60-80 minutes. The resulting mixture is then transferred to a polytetrafluoroethylene reactor and solvothermal reacted at 140-180°C for 5-12 hours. The product after reaction is washed with deionized water and anhydrous ethanol, dried, and finally calcined at 200-350°C in air for 2-3 hours to obtain Ce(MoO4)2 nanoparticles. The molar ratio of Mo(VI) in the molybdenum salt to Ce(III) in the cerium salt and urea is 1:1:5-42. Step 2: Mix Ce(MoO4)2 nanoparticles with a hydrogen peroxide solution with a mass concentration of 0.6%–1% H2O2, sonicate for 60–120 minutes, and then transfer to a polytetrafluoroethylene reactor. Perform a hydrothermal reaction at 50–90 °C for 8–12 hours. Wash the resulting product sequentially with deionized water and anhydrous ethanol, and then dry to obtain oxygen vacancy-deficient Ce(MoO4)2 nanozyme.

2. The method for preparing oxygen-vacancy-defective Ce(MoO4)2 nanozymes according to claim 1, characterized in that: In step 1, the molar ratio of Mo(VI) in the molybdenum salt to Ce(III) and urea in the cerium salt is 1:1:15 to 25.

3. The method for preparing oxygen-vacancy-defective Ce(MoO4)2 nanozymes according to claim 2, characterized in that: In step 1, the molybdenum salt is sodium molybdate dihydrate, and the cerium salt is cerium nitrate hexahydrate.

4. The method for preparing oxygen-vacancy-defective Ce(MoO4)2 nanozymes according to claim 1, characterized in that: In step 1, the resulting mixture is transferred to a hydrothermal reactor and hydrothermally reacted at 160–170 °C for 10 hours.

5. The method for preparing oxygen-vacancy-defective Ce(MoO4)2 nanozymes according to claim 1, characterized in that: In step 1, the sample is calcined at 250 °C for 2 hours in air.

6. The method for preparing oxygen-vacancy-defective Ce(MoO4)2 nanozymes according to claim 1, characterized in that: In step 2, the concentration of the Ce(MoO4)2 nanoparticles in the hydrogen peroxide solution is 0.5–2 mg / mL.

7. The method for preparing oxygen-vacancy-defective Ce(MoO4)2 nanozymes according to claim 1, characterized in that: In step 2, the hydrothermal reaction is carried out at 70 °C for 10 hours.

Images