Preparation method and application of amino-functionalized cerium oxide nano-enzyme

Abstract

The invention belongs to the technical field of preparation and application of nano materials, and relates to a preparation method and application of amino-functionalized cerium oxide nano enzyme, and the preparation method comprises the following steps: adding 3-aminophenylboronic acid into a cerous nitrate hexahydrate aqueous solution to obtain a mixed solution; dropwise adding a sodium hydroxide aqueous solution into the mixed solution, carrying out hydrothermal reaction, cooling after the reaction is finished, centrifuging, washing and drying to obtain milk white powder, namely the amino-functionalized cerium oxide nano-enzyme. The boron element is introduced in the preparation of the cerium oxide nano-enzyme, and the amino-functionalized cerium oxide nano-enzyme is prepared by adopting a simple one-step hydrothermal synthesis method, so that the nano-enzyme has good phosphatase-like activity, has high selectivity and sensitivity to LPS, and is short in detection time, high in sensitivity, low in detection limit, simple in preparation process and high in preparation efficiency; the product is good in quality, high in stability, environment-friendly and extremely wide in market prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of preparation and application of nanomaterials, and relates to a preparation method and application of an amino-functionalized cerium oxide nanenzyme. BACKGROUND

[0002] As a kind of nanomaterial with natural enzyme catalytic characteristics, nanenzyme has been widely used as a substitute for natural enzyme in sensing. However, nanenzyme lacks specific recognition sites and has weak catalytic activity, so it is urgent to improve its selectivity and catalytic activity.

[0003] At present, molecular imprinting is a commonly used technology, which can establish specific recognition sites on the surface of nanenzyme to simulate enzyme-substrate interaction, but the specific recognition sites constructed by this method can easily cover the active sites on the surface of nanenzyme, resulting in great loss of catalytic activity. In order to improve the catalytic activity of nanenzyme, it is found that smaller size nanenzyme has higher catalytic activity due to the exposure of active sites, but small size nanenzyme is easy to aggregate because of larger surface energy, therefore, a complicated supporting platform for fixing nanenzyme to avoid its aggregation is always inevitable, therefore, an urgent need exists for a method capable of improving the catalytic activity and selectivity of nanenzyme.

[0004] The surface functional groups of nanoscale enzymes play an important role in the catalytic process, so it can be expected that the modification of the surface functional groups of nanoscale enzymes is an effective method to improve their catalytic activity and selectivity. The effectiveness of element doping in improving the catalytic activity and selectivity of nanoscale enzymes has been proven. Cerium dioxide (CeO2) nanoparticles have the characteristics of mimicking enzymes due to their catalytic activity and oxygen storage properties. There have been many studies on metal doping of cerium dioxide nanoscale enzymes, for example, Chinese Patent CN202311719976.X discloses a preparation method of strontium-doped cerium dioxide nanoscale enzymes, which is obtained by mixing strontium ion oleylamine complex with cerium dioxide, heating, standing, and purifying. The doping of strontium further improves the antioxidant performance of cerium dioxide nanoparticles and has a synergistic effect. Chinese Patent CN202011354661.6 discloses an anti-tumor single-atom noble metal-loaded cerium oxide nanoscale enzyme, which is a nanoscale enzyme with cerium oxide nanomaterial as a carrier and loaded with single-atom noble metal. The single-atom noble metal-loaded cerium oxide nanoscale enzyme has peroxidase and oxidase activity, which can catalyze the production of reactive oxygen species (ROS). The water content of active oxygen in normal healthy cells is strictly regulated. When the content of active oxygen is excessive, it not only damages biological macromolecules such as proteins, lipids, and nucleic acids, but also regulates related signaling pathways, promoting cell apoptosis. The nanoscale enzyme has great cytotoxicity to tumor cells and little cytotoxicity to normal cells, and can be used for the treatment of liver cancer, lung cancer, and cervical cancer.

[0005] However, there is no related report on boron-doped cerium oxide nanoscale enzymes. SUMMARY

[0006] The present application aims to overcome the above-mentioned defects in the prior art and provides a preparation method of amino-functionalized cerium oxide nanoscale enzymes. The method uses cerium nitrate and 3-aminophenylboronic acid as precursors to construct nanoscale enzymes through a simple one-step hydrothermal method, which can specifically, quickly, and sensitively detect LPS.

[0007] To achieve the above-mentioned purpose, the present application provides a preparation method of amino-functionalized cerium oxide nanoscale enzymes, and the specific process comprises the following steps:

[0008] 3-aminophenylboronic acid is added to a cerium nitrate hexahydrate aqueous solution, and ultrasonic treatment is performed to obtain a mixed solution. Then, an aqueous sodium hydroxide solution is added dropwise to the mixed solution, and a hydrothermal reaction is performed at a temperature of 100-160℃ for 12-36 hours. After the reaction is completed and the solution is cooled, centrifugation, washing, and drying are performed to obtain a milky white powder, which is the amino-functionalized cerium oxide nanoscale enzyme.

[0009] The concentration of cerium nitrate hexahydrate in the cerium nitrate hexahydrate aqueous solution is 0.01-0.1 M.

[0010] The 3-aminobenzenboronic acid is used in a molar ratio of 2:1 with the cerium nitrate hexahydrate.

[0011] The concentration of the aqueous sodium hydroxide solution is in the range of 1-4M.

[0012] The volume ratio of the aqueous sodium hydroxide solution to the mixed solution is 1:2.

[0013] The washing is performed three times with water and ethanol respectively; the drying temperature is 60°C, and the drying time is 10 hours.

[0014] The amino-functionalized cerium oxide nanoszyme has a rod-like morphology, an average diameter of 50-100nm, and good phosphatase-like activity, and can specifically bind to endotoxin and be applied to endotoxin detection.

[0015] The application also provides application of the amino-functionalized cerium oxide nanoszyme in detection of endotoxin, which can specifically detect endotoxin, and the detection limit is 5.17 ng / mL.

[0016] The detection mechanism of the cerium oxide nanoszyme for endotoxin is as follows: first, the KDO (3-deoxy-d-manno-2-octulosonic acid) residue in the endotoxin specifically binds to the phenylboronic acid phenylboronic acid group on the surface of the cerium oxide nanoszyme, significantly reducing the activity of the nanoszyme; second, the electronic defects of boron make the water combined with Ce ions tend to be in a deprotonated state, and the remaining electrons need to be stabilized when the protons are dissociated, thereby reducing the anions dissociated from the water combined with Ce ions, thereby generating more nucleophilic substances to enhance the catalytic dephosphorylation reaction. Based on the dephosphorylase-like activity and the binding characteristics of the material to endotoxin, the LPS detection specificity of the application is enhanced, the detection time is reduced, and the sensitivity is significantly improved. Experimental results show that, in the presence of other saccharides such as glucose, sucrose, pectin, chitosan, Vc, Ve, EDTA, ATP, dopamine, aspartic acid, serine, phenol, benzyl alcohol and other substances, the detection fluorescence intensity of the LPS solution group is significantly higher than that of other solution groups, the specificity is significantly enhanced, and the deficiency of the traditional Limulus reagent (LAL) method for detecting LPS, which is prone to false positive results for dextran and pectin, is made up. And the detection time of the application is only 10 min, compared with 1 h of the traditional LAL method, the detection time of the application is greatly reduced. In addition, under room temperature conditions, when the LPS concentration is in the range of 20-200 ng / mL at pH=9, the fluorescence intensity difference ΔI and the LPS concentration x conform to the linear equation ΔI=y=462.92+90.09x, and the lowest detection limit is 5.17 ng / mL. When the LPS concentration is expanded to 1 μg / mL, ΔI and the LPS concentration x conform to the equation ΔI=y=38461.9-36707*0.998 x(ΔI=I0-I, I0 is the blank group fluorescence intensity, I is the fluorescence intensity of the endotoxin experimental group), which indicates that the amino-functionalized cerium oxide nanoscale enzyme has high sensitivity for LPS detection.

[0017] Compared with the prior art, the present application first introduces boron element in the preparation of cerium oxide nanoscale enzyme, uses cerium nitrate and triaminobenzene boronic acid as precursors, and only needs to use a simple one-step hydrothermal synthesis method to prepare the amino-functionalized cerium oxide nanoscale enzyme, so that the nanoscale enzyme has good phosphatase-like activity, can be specifically combined with endotoxin LPS and be detected, and the catalytic activity of the nanoscale enzyme is reduced. The amino-functionalized cerium oxide nanoscale enzyme has high selectivity and sensitivity for LPS, has short detection time, high sensitivity, and low detection limit, has simple preparation process, high preparation efficiency, good product quality, strong stability, and environment-friendly characteristics, and has extremely broad market prospect. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 TEM and element mapping characterization results of the amino-functionalized cerium oxide nanoscale enzyme prepared in the present application are shown in the following figure, in which A-C are TEM images, and D is an element mapping image.

[0019] Figure 2 The phosphatase activity detection and the experimental results of the influence of different LPS concentrations on the amino-functionalized cerium oxide nanoscale enzyme are shown in the following figure, in which A is the phosphatase activity detection result, B is the change of emission intensity under different LPS concentrations, C and E are the size change diagrams of the nanoscale enzyme before and after endotoxin treatment, and D is the potential change diagram of the nanoscale enzyme before and after endotoxin treatment.

[0020] Figure 3 The sensitivity detection experimental results of the amino-functionalized cerium oxide nanoscale enzyme for LPS are shown in the following figure, in which A is a linear relationship diagram between the fluorescence intensity difference and the LPS concentration (20-200 ng mL -1 ); B is a linear relationship between the fluorescence intensity difference and the LPS concentration (20-1000 ng mL -1 ); C is an enzyme kinetics curve diagram of the nanoscale enzyme; and D is a specificity experimental result diagram of the nanoscale enzyme. DETAILED DESCRIPTION

[0021] The present application will be further described below by means of specific embodiments and in conjunction with the accompanying drawings.

[0022] Embodiment 1

[0023] The present embodiment relates to a preparation method of an amino-functionalized cerium oxide nanoscale enzyme, and the specific steps are as follows:

[0024] S1, 0.489 g of cerium nitrate hexahydrate was dissolved in 20 mL of deionized water and stirred to dissolve;

[0025] S2, 0.31 g of 3-aminophenylboronic acid (APBA) was added to the solution prepared in S1, and mixed uniformly under ultrasonic for 10 minutes to obtain a mixed solution;

[0026] S3, 0.576 g of sodium hydroxide was dissolved in 10 mL of deionized water to obtain a sodium hydroxide aqueous solution; under the stirring of the mixed solution prepared in step S2, the sodium hydroxide aqueous solution was slowly added dropwise into the mixed solution;

[0027] S4, the solution obtained in step S3 was placed in a polytetrafluoroethylene sealed tank and placed in a hydrothermal reaction kettle, and hydrothermal reaction was carried out at a temperature of 120°C for 24h;

[0028] S5, after the solution after reaction was cooled to room temperature, the polytetrafluoroethylene tank was taken out, and the solution was taken out into a centrifugal tube, and centrifuged at a speed of 8000 rpm for 10 minutes, then the precipitate was washed with water and ethanol three times, and then placed in a 60°C drying box for drying for 10 hours, to obtain a milky white powder, which was an amino-functionalized cerium oxide nanoscale enzyme (denoted as APBA-CeO2).

[0029] TEM and element mapping standard of the prepared cerium oxide nanoscale enzyme were carried out, and the results are shown in Figure 1 A-C. As can be seen from Figure 1 A-C, the cerium oxide nanoscale enzyme has a rod-like morphology, with an average diameter of 50-100 nm. As can be seen from Figure 1 D, the product contains oxygen element, cerium element, boron element and nitrogen element.

[0030] Example 2:

[0031] This example relates to a preparation method of an amino-functionalized cerium oxide nanoscale enzyme, and the specific steps are as follows:

[0032] S1, cerium nitrate hexahydrate was dissolved in 20 mL of deionized water under stirring, and the concentration of cerium nitrate hexahydrate was 0.1 M;

[0033] S2, 4 mmol of 3-aminophenylboronic acid was added to the solution prepared in S1, and mixed uniformly under ultrasonic for 10 minutes to obtain a mixed solution;

[0034] S3, 10 mmol of sodium hydroxide was dissolved in 10 mL of deionized water to obtain a sodium hydroxide aqueous solution; under the stirring of the mixed solution prepared in step S2, the sodium hydroxide solution was slowly added dropwise into the mixed solution;

[0035] S4, the solution prepared in step S3 was placed in a polytetrafluoroethylene sealed tank and placed in a hydrothermal reaction kettle, and hydrothermal reaction was carried out at a temperature of 100°C for 36h;

[0036] S5, after the solution after the reaction is reduced to room temperature, the polytetrafluoroethylene tank is taken out, the solution is taken out to the centrifuge tube, centrifuged at 8000 rpm for 10 minutes, the precipitate is washed with water and ethanol three times respectively, and then put into a 60℃ drying oven for drying for 10 hours, the obtained milky white powder is the amino functionalized cerium oxide nanoscale enzyme (marked as APBA-CeO2).

[0037] Example 3:

[0038] This example relates to a preparation method of an amino functionalized cerium oxide nanoscale enzyme, the specific steps are as follows:

[0039] S1, dissolve cerium nitrate hexahydrate in 20 mL of deionized water and stir to dissolve, the concentration of cerium nitrate hexahydrate is 0.01M;

[0040] S2, 0.4 mmol of 3-aminobenzoic acid is added to the solution prepared in S1, ultrasonic mixing for 10 minutes to obtain a mixed solution;

[0041] S3, dissolve 40 mmol of sodium hydroxide in 10 mL of deionized water to obtain a sodium hydroxide aqueous solution; under the stirring condition of the mixed solution prepared in step S2, slowly add the sodium hydroxide solution dropwise into the mixed solution;

[0042] S4, put the solution prepared in S3 into a polytetrafluoroethylene sealed tank and put it into a hydrothermal reaction kettle, hydrothermal reaction at 160℃ for 12h;

[0043] S5, after the solution after the reaction is reduced to room temperature, the polytetrafluoroethylene tank is taken out, the solution is taken out to the centrifuge tube, centrifuged at 8000 rpm for 10 minutes, the precipitate is washed with water and ethanol three times respectively, and then put into a 60℃ drying oven for drying for 10 hours, the obtained milky white powder is the amino functionalized cerium oxide nanoscale enzyme (marked as APBA-CeO2).

[0044] Example 4:

[0045] This example will prepare the dephosphorylation enzyme activity of the amino functionalized cerium oxide nanoscale enzyme prepared in example 1 and apply it to endotoxin (LPS) detection.

[0046] 1, APBA-CeO2 nanoscale enzyme dephosphorylation activity detection: Figure A is the evaluation of APBA-CeO2 nanoscale enzyme dephosphorylation, under 360nm excitation light, 4-MU (4-methylumbelliferone) obtained after hydrolysis of 4-methylumbelliferone phosphate (4-MUP) has a very strong emission characteristic peak. 50uL of APBA-CeO2 aqueous solution (final concentration 50ug mL -1) and 10 μL 4-methylumbelliferone phosphate (4-MUP) (final concentration 200 mM) into 1 mL system and incubated for 10 min, and the emission intensity at 448 nm under 360 nm excitation light was collected by a microplate reader; at the same time, the emission intensity at 448 nm of pure CeO2 nanoparticles was detected by the above method, and APBA-CeO2 nanoszyme alone and 4-MUP alone were used as control groups, and the results are shown in Figure 2 A. It can be seen from Figure 2 A that APBA-CeO2 nanoszyme alone (curve b) and 4-MUP alone (curve a) have no fluorescence emission, and when APBA-CeO2 and 4-MUP coexist (curve d), a clear fluorescence emission peak at 448 nm can be observed; compared with APBA-CeO2 nanoszyme, the fluorescence emission peak of pure CeO2 nanoparticles without doping (curve c) is weaker, indicating that APBA-CeO2 nanoszyme has good dephosphorylase activity, and the dephosphorylase activity of pure CeO2 nanoparticles is weak.

[0047] 2. Detection of LPS by APBA-CeO2 nanoszyme: 50 uL APBA-CeO2 aqueous solution (final concentration 50 μg mL -1 ), 10 uL 4-methylumbelliferone phosphate (4-MUP) (final concentration 200 mM) and 10 uL LPS aqueous solution with different concentrations (final concentration 0 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 300 ng / mL) were added into Tris-Hcl buffer (pH = 9) to make a total volume of 1 mL of detection system, and after incubation at room temperature for 10 min, the emission intensity at 448 nm under 360 nm excitation light was collected by a microplate reader, and the results are shown in Figure 2 B. It can be seen from Figure 2 B that as the concentration of LPS increases, the emission intensity gradually decreases, indicating that the catalytic activity of APBA-CeO2 nanoszyme gradually decreases as the concentration of LPS increases (from 0 to 300 ng / mL).

[0048] APBA-CeO2 nanoszyme and pure CeO2 nanoparticles with a concentration of 50 μg / mL were treated with 50 ng / mL endotoxin, and the characterization of the hydrodynamic particle size of the two nanoszymes before and after treatment is as follows Figure 2(C, E) show that the particle size of APBA-CeO2nanoparticles increases from 10 nm to 5 μm after endotoxin treatment, indicating that the successful combination of endotoxin and APBA-CeO2nanoparticles leads to further aggregation of nanoparticles, masking the active sites of the nanoreactor, thus leading to a decrease in phosphatase activity. The particle size of pure CeO2nanoparticles treated with endotoxin does not change significantly, indicating that endotoxin cannot be combined with pure CeO2nanoparticles, and the surface of pure CeO2does not have functional groups that can be combined with LPS. Endotoxin has no effect on pure CeO2nanoparticles, and thus the use of pure CeO2nanoparticles to detect LPS according to the above method has no effect on the change in the intensity of the emitted light with the change in the concentration of LPS.

[0049] The zeta potentials of APBA-CeO2nanoparticles and pure CeO2nanoparticles before and after endotoxin treatment were characterized. Since endotoxin itself is electronegative, the zeta potential of APBA-CeO2nanoparticles after endotoxin treatment shifts to the negative electrode. Compared with non-functionalized pure CeO2nanoparticles, APBA-CeO2nanoparticles exhibit more negative zeta potential, as shown in Figure 2 (D), which is related to the rich negative charge carried by 3-aminobenzene boronic acid itself, further indicating the successful amino functionalization of CeO2.

[0050] Example 5:

[0051] In this example, the amino-functionalized cerium oxide nanoreactor prepared in Example 1 was applied to the detection of LPS sensitivity. Under the conditions of the LPS detection method of Example 4, the change in fluorescence intensity I at 448 nm of the experimental group with different concentrations of LPS (0.02, 0.05, 0.1, 0.15, 0.2, 0.3, 0.5, 0.8, 1 μg mL -1 ) was determined, and the fluorescence intensity difference ΔI (ΔI = I0-I, I0is the fluorescence intensity of the blank group, and I is the fluorescence intensity of the endotoxin experimental group) and the functional correction curve of the LPS concentration x were plotted, as shown in Figure 3 . Figure 3 From Figure 3 A, it can be seen that when the LPS concentration is in the range of 20-200 ng mL -1 , the relationship between the fluorescence intensity difference ΔI and the LPS concentration x conforms to the linear equation ΔI = y = 462.92 + 90.09x, and the detection limit (LOD = 3σ / S; σ: standard deviation of the blank sample, S: slope of the correction curve) is 5.17 ng / mL (S / N≥3). From Figure 3 B, it can be seen that when the LPS concentration is expanded to 1 μg / mL, ΔI and LPS concentration x conform to the equation ΔI = y = 38461.9-36707*0.998 x; ΔI = I0- I, I0is the blank group fluorescence intensity, I is the fluorescence intensity of endotoxin experimental group).

[0052] The difference in fluorescence intensity used in this embodiment eliminates the reaction of nanozyme with the reaction substrate 4-MUP, and more intuitively reflects the change in fluorescence intensity of nanozyme affected by endotoxin, showing the sensitivity of the nanozyme.

[0053] Example 6

[0054] In this embodiment, the cerium oxide nanozyme prepared in Example 1 is applied to the specific detection of LPS. Under the LPS detection method conditions of Example 2, the fluorescence intensity of 1 μg / mL of LPS is determined, and LPS is replaced with common monosaccharide and polysaccharide substances such as dextran (D-Glucan), glucose, sucrose, pectin, chitosan, Vc, Ve, EDTA, adenine nucleotide triphosphate (ATP), dopamine, aspartic acid (Asp), serine (Ser), phenol, benzyl alcohol, soybean oil, etc., as shown in FIG. 6. Figure 3 As shown in FIG. 6, the difference in fluorescence intensity of the LPS solution is significantly greater than that of the other groups of solutions, indicating that the APBA-CeO2 nanozyme has good selectivity for LPS.

[0055] Example 7 In this embodiment, the APBA-CeO2 nanozyme prepared in Example 1 is subjected to kinetic analysis detection. Under the 4-MUP detection method conditions of Example 2, as the concentration of the reaction substrate 4-methyl umbelliferyl phosphate (4-MUP) increases (0-10 mM), the catalytic rate of the nanozyme gradually increases and finally tends to be constant (Vmax= 0.018 min-1, Km= 0.62 mM), indicating that the enzyme reaction kinetics conforms to the Michaelis-Menten equation model. Figure 3 C).

Claims

1. A method for preparing an amino-functionalized ceria nanoszyme, characterized in that, The specific steps are as follows: 3-aminobenzene boronic acid is added to a cerium nitrate hexahydrate aqueous solution to obtain a mixed solution; a sodium hydroxide aqueous solution is added dropwise to the mixed solution to perform a hydrothermal reaction; after the reaction is completed and cooled, centrifugation, washing, and drying are performed to obtain a milky white powder, which is an amino-functionalized cerium oxide nanoscale enzyme.

2. The process for the preparation of amino-functionalized ceria nanoszymes according to claim 1, characterized in that, The concentration of cerium nitrate hexahydrate in the cerium nitrate hexahydrate aqueous solution is 0.01-0.1M.

3. The method for preparing amino-functionalized ceria nanoszymes according to claim 1, wherein, The molar ratio of the amount of 3-aminobenzene boronic acid to cerium nitrate hexahydrate is 2:

1.

4. The method for preparing amino-functionalized ceria nanoszymes according to claim 1, wherein, The concentration of the sodium hydroxide aqueous solution is 1-4M.

5. The method for preparing amino-functionalized ceria nanoszymes according to claim 1, wherein, The volume ratio of the sodium hydroxide aqueous solution to the mixed solution is 1:

2.

6. The method for preparing amino-functionalized ceria nanoszymes according to claim 1, wherein, The hydrothermal reaction temperature is 100-160℃, and the reaction time is 12-36 hours.

7. The method for preparing amino-functionalized cerium oxide nanozymes according to claim 1, characterized in that, The washing is performed with water and ethanol three times respectively; the drying temperature is 60℃, and the drying time is 10 hours.

8. The aminofunctionalized ceria nanoszyme produced by the method of any one of claims 1-7, characterized in that, The amino-functionalized cerium oxide nanoscale enzyme has a rod-like morphology, and the average diameter is 50-100nm.

9. The amino-functionalized ceria nanoszyme of claim 8, wherein, It has good dephosphoesterase-like activity.

10. Use of the amino-functionalized ceria nanoszyme according to any one of claims 8-9 for the detection of endotoxins, characterized in that, It can specifically detect endotoxins, and the detection limit is 5.17ng / mL.

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