A cerium-based nanoscale enzyme, a preparation method and application thereof
By modifying bovine serum albumin on the surface of cerium-based nanozymes, precise regulation of the activity of cerium-based nanozymes was achieved, the catalytic ability was improved, and high sensitivity and simplicity were demonstrated in glutathione detection.
Patent Information
- Application Number
- CN202310727621.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-20
- Publication Date
- 2025-10-14
- Estimated Expiration
- 2043-06-20
AI Technical Summary
The catalytic ability of existing cerium-based nanozymes needs to be further improved, and there is a problem of insufficient sensitivity in glutathione detection.
Bovine serum albumin (BSA) was used to modify the surface of the cerium-based nanozyme. By adjusting the concentration of macromolecules, pH value, temperature and enzyme dosage, the activity of the cerium-based composite nanozyme was precisely regulated to prepare BSA/CeO2 nanozyme.
The catalytic capacity has been increased by 4 times, achieving high-sensitivity detection of glutathione with a detection range of 0-1mmol/L, which has the advantages of being visible to the naked eye and easy to detect.
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Figure CN116899551B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of nanoscale enzyme and its application, and particularly relates to a cerium-based nanoscale enzyme, a preparation method and application thereof. BACKGROUND
[0002] Natural enzymes are efficient catalysts produced by living cells through long-term evolution in nature, and have the advantages of high catalytic efficiency, good selectivity and strong specificity, and are key substances for information transmission and regulation of metabolism of living organisms. Enzymes are also increasingly widely used in food and drugs, and the demand is increasing. However, in the development and utilization of natural enzymes, there are difficulties in preparation and purification, scarcity of raw materials, high cost, and insufficient thermal stability, which make the development and utilization of enzymes difficult.
[0003] With the rapid progress and development of modern nanotechnology and biotechnology, more and more people know about nanomaterials, and researchers have developed a variety of nanomaterials that mimic enzymes, which have both rational properties of nanomaterials and catalytic functions similar to enzymes, and combine the advantages of natural enzymes and artificial enzymes. In the field of nanoscale enzymes, cerium-based nanoscale enzymes have been widely recognized at home and abroad due to their unique physicochemical properties. Among them, CeO2 has mixed valence of Ce 3+ and Ce 4+ and oxygen vacancies, and different valences can be switched to obtain better catalytic activity. It has oxidation and antioxidant properties for different reaction substrates under different reaction conditions, which makes CeO2 have catalytic properties and a variety of artificial enzyme activities.
[0004] However, compared with natural biological enzymes, the catalytic ability of cerium-based nanoscale enzymes needs to be further improved. Therefore, exploring effective and precise regulation strategies for nanoscale enzyme activity has become the mainstream direction and key problem of current research. SUMMARY
[0005] In view of the above problems in the prior art, the present application provides a cerium-based nanoscale enzyme, a preparation method and application thereof, and specifically provides a precise regulation strategy for improving the activity of cerium oxide, in which bovine serum albumin is used as a modification molecule to modify the surface of cerium oxide, and a BSA / CeO2 composite nanoscale enzyme prepared by the method can realize high-sensitivity detection of glutathione.
[0006] In order to achieve the above purpose, the present application provides a preparation method of a cerium-based nanoscale enzyme, comprising:
[0007] Ce(NO3)3·6H2O is dissolved in deionized water to obtain a solution, then a precipitating agent is added dropwise into the solution, and stirring is performed at room temperature to obtain a precipitate;
[0008] The slurry of the above-mentioned precipitate is aged, centrifuged, washed with hot deionized water until the pH of the supernatant solution is 7, vacuum dried, calcined, and CeO2 nanoscale enzyme powder is obtained.
[0009] In some embodiments, the method further comprises: dissolving Ce(NO3)3·6H2O in deionized water to obtain a solution, adding a precipitant to the solution, stirring at room temperature for 60 min, then adding a bovine serum albumin solution, and continuing to stir for 30 min to obtain a precipitate;
[0010] The slurry of the above-mentioned precipitate is aged, centrifuged, washed with hot deionized water until the pH of the supernatant solution is 7, vacuum dried, calcined, and BSA / CeO2 nanoscale enzyme powder is obtained.
[0011] In some embodiments, the precipitant is NH3·H2O with a concentration of 25%-28%, and the amount of the precipitant added is 0.8 mL.
[0012] In some embodiments, the centrifugation is performed at a speed of 3000 rmp for 5 min.
[0013] In some embodiments, the bovine serum albumin solution has a mass concentration of any value in the range of 2-12 mg / mL.
[0014] Another aspect of the present application provides a cerium-based nanoscale enzyme prepared by the method for preparing a cerium-based nanoscale enzyme according to any of the above-mentioned technical solutions.
[0015] The present application also provides an application of the above-mentioned cerium-based nanoscale enzyme in glutathione detection, which comprises: adding a glutathione solution with different concentrations into a buffer, then adding a 3,3',5,5'-tetramethylbenzidine solution and an aqueous dispersion of the cerium-based nanoscale enzyme, performing a catalytic reaction, recording the absorbance at 652 nm of a detection system, and obtaining a concentration-absorbance curve.
[0016] In some embodiments, the concentration of the glutathione solution is 0-10 mM, and the concentration of the 3,3',5,5'-tetramethylbenzidine solution is 0.02 mM.
[0017] In some embodiments, the pH of the catalytic reaction is 1-6, and the temperature of the catalytic reaction is 10-80℃.
[0018] In some embodiments, the pH of the catalytic reaction is 3, and the temperature of the catalytic reaction is 60℃.
[0019] Compared with the prior art, the present application has the following advantages:
[0020] The application provides a preparation method of cerium-based nanoscale enzyme, and a cerium-based nanoscale enzyme modified by bovine serum albumin (BSA) is prepared by one-step synthesis method, the surface of cerium oxide is functionally modified by taking bovine serum albumin as a modification molecule, and the activity regulation of the cerium-based composite nanoscale enzyme is realized by changing environmental factors, specifically, the catalytic capacity of the cerium-based nanoscale enzyme can be optimized and adjusted by adjusting the concentration, pH value, temperature and enzyme dosage of the macromolecular substance bovine serum albumin, and the precise regulation of the activity of cerium oxide is improved; compared with the pure-phase CeO2 nanoscale enzyme, the catalytic capacity of the BSA / CeO2 nanoscale enzyme prepared by the above method is improved by 4 times.
[0021] The application also provides application of the above cerium-based nanoscale enzyme in glutathione detection, and the cerium-based nanoscale enzyme has the advantages of naked-eye visibility and simple detection, has a good linear relationship in the range of 0-1 mmol / L, and can realize high-sensitivity detection of glutathione. BRIEF DESCRIPTION OF DRAWINGS
[0022] In order to more clearly illustrate the technical solutions in the embodiments of the application, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.
[0023] Figure 1 The ultraviolet-visible spectrum changes of BSA / CeO2 nanoscale enzyme catalyzing TMB for different BSA concentrations;
[0024] Figure 2 The ultraviolet-visible spectrum changes of BSA / CeO2 nanoscale enzyme catalyzing TMB in different pH environments;
[0025] Figure 3 The ultraviolet-visible spectrum changes of BSA / CeO2 nanoscale enzyme catalyzing TMB at different temperatures;
[0026] Figure 4 The standard curve of glutathione;
[0027] Figure 5 The stability analysis of glutathione;
[0028] Figure 6 The selectivity analysis of glutathione. DETAILED DESCRIPTION
[0029] The technical solutions of the application will be described in detail below with reference to the drawings and specific embodiments, so as to further understand the purposes, solutions and effects of the application, but not as a limitation on the protection scope of the claims attached to the application.
[0030] Embodiment 1
[0031] (1) Ce(NO3)3·6H2O was dissolved in deionized water, then 0.8 mL of NH3·H2O with a concentration of 25%-28% was added, stirred at room temperature for 90 min, and a precipitate was obtained;
[0032] (2) The slurry of the precipitate was aged and centrifuged at a speed of 3000 rmp for 5 min, and then washed with hot deionized water until the pH of the upper solution was 7;
[0033] (3) Vacuum drying and calcination were performed to obtain CeO2 nanose powder.
[0034] Example 2
[0035] (1) Ce(NO3)3·6H2O was dissolved in deionized water, then 0.8 mL of NH3·H2O with a concentration of 25%-28% was added, stirred at room temperature for 60 min, and then a bovine serum albumin (BSA) solution with a mass concentration of 8 mg / mL was added, and stirring was continued for 30 min to obtain a precipitate;
[0036] (2) The slurry of the precipitate was aged and centrifuged at a speed of 3000 rmp for 5 min, and then washed with hot deionized water until the pH of the upper solution was 7;
[0037] (3) Vacuum drying and calcination were performed to obtain BSA / CeO2 nanose powder.
[0038] Example 3
[0039] The BSA / CeO2 nanose powder prepared in Example 2 was configured into BSA / CeO2 nanose solutions with concentrations of 0 mg / mL, 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, and 12 mg / mL, respectively, and 400 μL of each solution was taken and placed in a centrifuge tube, and 4 mL of buffer solution (pH = 3) was added. 100 μL of TMB-ethanol solution was added to each solution, and the absorbance at 652 nm was measured by ultraviolet spectrophotometry under room temperature conditions. As shown in Figure 1 BSA exists in small amounts (2-6 mg / mL), the inhibition of cerium nanose activity is greater than the promotion; when the concentration of BSA is higher than 6 mg / mL, the promotion dominates, and the activity of 12 mg / mL BSA modified cerium-based composite nanose is 1.7 times higher than that of pure cerium oxide.
[0040] Example 4
[0041] Take 400 μL of pure CeO2 nanoscale enzyme solution prepared in Example 1 and 400 μL of BSA / CeO2 nanoscale enzyme solution prepared in Example 2, respectively, and add 4 mL of NaAc-HAc buffer solution with different pH (pH is 1, 2, 3, 4, 5, and 6, respectively) to each centrifuge tube. Then, 100 μL of TMB-ethanol solution is added to each solution, and the absorbance at 652 nm is measured by ultraviolet spectrophotometer after oscillation for 3 min at room temperature. As shown in FIG. 6, the BSA / CeO2 nanoscale enzyme is a pH environment-controllable nanoscale enzyme, and its activity is better under acidic conditions than under neutral conditions, and the optimal pH value is 3. Figure 2 As shown in FIG. 6, the BSA / CeO2 nanoscale enzyme is a pH environment-controllable nanoscale enzyme, and its activity is better under acidic conditions than under neutral conditions, and the optimal pH value is 3.
[0042] Example 5
[0043] Take 400 μL of pure CeO2 nanoscale enzyme solution prepared in Example 1 and 400 μL of BSA / CeO2 nanoscale enzyme solution prepared in Example 2, respectively, and add 4 mL of NaAc-HAc buffer solution with different pH (pH is 1, 2, 3, 4, 5, and 6, respectively) to each centrifuge tube. Then, 100 μL of TMB-ethanol solution is added to each solution, and the absorbance at 652 nm is measured by ultraviolet spectrophotometer after oscillation for 3 min at room temperature. As shown in FIG. 6, the BSA / CeO2 nanoscale enzyme is a pH environment-controllable nanoscale enzyme, and its activity is better under acidic conditions than under neutral conditions, and the optimal pH value is 3. Figure 3 As shown in FIG. 6, the BSA / CeO2 nanoscale enzyme is a pH environment-controllable nanoscale enzyme, and its activity is better under acidic conditions than under neutral conditions, and the optimal pH value is 3.
[0044] Example 6
[0045] Take 400 μL of pure CeO2 nanoscale enzyme solution prepared in Example 1 and 400 μL of BSA / CeO2 nanoscale enzyme solution prepared in Example 2, respectively, and add 4 mL of NaAc-HAc buffer solution with different pH (pH is 1, 2, 3, 4, 5, and 6, respectively) to each centrifuge tube. Then, 100 μL of TMB-ethanol solution is added to each solution, and the absorbance at 652 nm is measured by ultraviolet spectrophotometer after oscillation for 3 min at room temperature. As shown in FIG. 6, the BSA / CeO2 nanoscale enzyme is a pH environment-controllable nanoscale enzyme, and its activity is better under acidic conditions than under neutral conditions, and the optimal pH value is 3. Figure 4 As shown in FIG. 6, the BSA / CeO2 nanoscale enzyme is a pH environment-controllable nanoscale enzyme, and its activity is better under acidic conditions than under neutral conditions, and the optimal pH value is 3.
[0046] Example 7
[0047] Take 20 μL of 3 mmol / L glutathione standard solution into a centrifuge tube, add 50 μL of BSA / CeO2 nanoscale enzyme solution configured by BSA / CeO2 nanoscale enzyme prepared in Example 2, 4 mL of buffer solution (pH = 3) and 50 μL of TMB-ethanol solution. Under room temperature conditions, the absorbance at 652 nm was measured, and the absorbance was recorded every 40 min for 240 min. As shown in Figure 5 the catalytic activity of BSA / CeO2 nanoscale enzyme still remained more than 70% after 4 h, indicating that the cerium-based composite nanoscale enzyme prepared by the application has good stability.
[0048] Example 8
[0049] Into 20 μL of 3 mmol / L glutathione / iron chloride / copper oxide / zinc oxide / sodium hydroxide / calcium carbonate mixed solution, add 50 μL of BSA / CeO2 nanoscale enzyme solution configured by BSA / CeO2 nanoscale enzyme prepared in Example 2, 4 mL of buffer solution (pH = 3) and 50 μL of TMB-ethanol solution. Under room temperature conditions, the reaction was carried out for 1 h, and the absorbance at 652 nm was measured. As shown in Figure 6 most of the interference substances can be ignored, indicating that BSA / CeO2 nanoscale enzyme has good selectivity for glutathione and good prospects for the detection of glutathione.
[0050] The above is only a preferred embodiment of the application, and is not intended to limit the application in other forms. Any skilled person in the art can modify or change the above disclosed technical content to equivalent embodiments applied to other fields, but any simple modification, equivalent change and modification made on the basis of the technical essence of the application to the above embodiments still belongs to the protection scope of the technical solution of the application.
Claims
1. An application of a cerium-based nanozyme in glutathione detection, characterized in that: include: Glutathione solutions of varying concentrations were added to a buffer solution, followed by addition of a 3,3',5,5'-tetramethylbenzidine solution and an aqueous dispersion of the cerium-based nanozyme to carry out a catalytic reaction. A concentration-absorbance curve was obtained by recording the absorbance of the detection system at 652 nm. The concentration of the glutathione solution was 0-10 mM, and the concentration of the 3,3',5,5'-tetramethylbenzidine solution was 0.02 mM. The pH of the catalytic reaction was 1-6, and the catalytic reaction temperature was 10-80°C. The preparation method of the cerium-based nanozyme includes: Ce(NO3)3•6H2O was dissolved in deionized water to obtain a solution, a precipitant was added dropwise to the solution, and the solution was stirred at room temperature for 60 min, and then bovine serum albumin solution was added and stirred for 30 min to obtain a precipitate; The precipitated slurry was aged and centrifuged, washed with hot deionized water until the pH of the upper layer solution was 7, and then vacuum dried and calcined to obtain BSA / CeO2 nanozyme powder.
2. The use of the cerium-based nanozyme in glutathione detection according to claim 1, characterized in that: The precipitant is NH 3 • H 2 O with a concentration of 25%-28%, and the amount of the precipitant added is 0.8 mL.
3. The use of the cerium-based nanozyme in glutathione detection according to claim 1, characterized in that: The centrifugal speed was 3000 rpm and the centrifugal time was 5 min.
4. The use of the cerium-based nanozyme in glutathione detection according to claim 1, characterized in that The mass concentration of the bovine serum albumin solution is any value between 2 and 12 mg / mL.
5. The use of the cerium-based nanozyme in glutathione detection according to claim 1, characterized in that: The pH of the catalytic reaction is 3, and the catalytic reaction temperature is 60°C.