A nanocomposite material with lignin peroxidase-like activity, its preparation method, and its application in resveratrol detection

Abstract

The present invention discloses a nanocomposite material having lignin peroxidase-mimicking activity, a preparation method thereof, and its application in the detection of veratrol. The nanocomposite material comprises a carbon support and composite microspheres supported on the support. The carbon support is a sheet-like structure formed by stacking and intersecting carbon nanowires, and the composite microspheres comprise CoO and V2O3. The present invention utilizes the enzyme-mimicking activity of the nanocomposite material to catalyze hydrogen peroxide to rapidly oxidize veratrol to produce veratraldehyde at room temperature. The ultraviolet-visible spectrum shows an increase in the absorption peak at a wavelength of 310 nm, enabling calculation and evaluation of the resveratrol content with low interference and high sensitivity. Compared to traditional methods for detecting resveratrol using large instruments such as liquid chromatographs, the monitoring method of the present invention is characterized by convenient detection, low cost, good selectivity for resveratrol, and high sensitivity.

Description

Technical Field

[0001] The present invention relates to the fields of nanomaterial preparation and enzymology, and in particular to a nanocomposite material with lignin peroxidase-like activity, a preparation method thereof, and application of the nanocomposite material in resveratrol detection. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Lignin is the most abundant organic renewable resource on Earth after cellulose. It is a complex, stable aromatic polymer compound. my country has abundant straw-like materials, of which lignin accounts for approximately 20%. However, due to the lack of enzymes in animals to degrade lignin, its feed value is low. Cellulose is also used for dry textiles and pulp and paper. Cellulose and cellulose must be released from the confines of lignin, which is then discharged as waste. Therefore, the identification of an enzyme capable of degrading lignin is crucial. Currently, a lignin-degrading enzyme, lignin peroxidase, can be extracted from white-rot fungi, but extraction is complex and the enzyme is unstable. Resveratrol (VA), a small molecule, is also an important raw material for organic synthesis and a pharmaceutical intermediate. Its detection is typically performed using large instruments such as liquid chromatography, which is complex and costly. Summary of the Invention

[0004] To address the above-mentioned issues, the present invention proposes a nanozyme with lignin peroxidase-mimicking activity, a method for its preparation, and its application in resveratrol detection. The nanocomposite material (CoO-V2O3 / C) with lignin peroxidase-mimicking activity prepared by the present invention facilitates the quantitative detection of resveratrol with high sensitivity. To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0005] In a first aspect, the present invention discloses a nanocomposite material having lignin peroxidase-like activity, comprising a carbon support and composite microspheres supported on the support. The carbon support comprises a sheet-like structure formed by stacked and intersecting carbon nanowires, and the composite microspheres comprise CoO and V2O3.

[0006] Furthermore, the particle size of the composite microspheres is about 50 to 300 nm. The composite material has lignin peroxidase-like activity, which can conveniently and quickly detect resveratrol.

[0007] In the second aspect of the present invention, a method for preparing the nanocomposite material having lignin peroxidase-like activity is disclosed: a cobalt source, vanadium acetylacetonate (VO(acac)2) and a carbon source are dissolved in a solvent, and then a solvothermal reaction is carried out. After completion, a solid product is separated to obtain the nanocomposite material.

[0008] Furthermore, the molar ratio of the cobalt source, vanadyl acetylacetonate, and carbon source is in the range of 1 to 3:1:2.

[0009] Furthermore, the cobalt source includes at least one of cobalt nitrate, cobalt sulfate, cobalt chloride, cobalt acetate, etc.

[0010] Furthermore, the carbon source includes 2-methylimidazole, among others. Co atoms coordinate with the nitrogen atoms of the imidazole ring to form an octahedral spatial configuration. The complex molecules form a three-dimensional supramolecular structure through atypical hydrogen bonding and π-π stacking, imparting optical properties and thermal stability to the material. Experimental findings indicate that the addition of a vanadium source in the present invention eliminates the polyhedral structure, while the resulting composite material retains its excellent stability.

[0011] Furthermore, the solvent includes at least one of methanol, deionized water, etc.

[0012] Furthermore, the temperature range of the solvent thermal reaction is 190-210° C., and the time range is 10-12 hours.

[0013] In a third aspect of the present invention, the use of the nanocomposite material having lignin peroxidase-mimicking activity in detecting resveratrol is disclosed.

[0014] Furthermore, the application method includes the following steps: mixing the nanocomposite material with a sample containing resveratrol to form a mixed solution, then adding hydrogen peroxide for reaction, detecting the absorbance of the reaction solution after completion, and achieving quantitative detection of resveratrol through the quantitative relationship between the difference in absorbance values ​​at a wavelength of 310 nm before and after the reaction of the reaction system and resveratrol.

[0015] Furthermore, the concentration ratio of the composite material, resveratrol, and hydrogen peroxide is 6-24 μg / mL: 4 mM: 1 mM.

[0016] Furthermore, the composite material is added to a Tris-HCl buffer solution of resveratrol to form a mixed solution, and then hydrogen peroxide is added dropwise and reacted at 15-30° C. for 0.5-2 hours.

[0017] Compared to existing technologies, the present invention has the following advantages: A composite material with lignin peroxidase-like activity is prepared. Its enzymatic activity can rapidly catalyze hydrogen peroxide to oxidize resveratrol to produce veratraldehyde at room temperature. Its UV-visible spectrum shows an increased absorption peak at a wavelength of 310 nm, enabling calculation and assessment of resveratrol content with minimal interference and high sensitivity. Compared to traditional methods for resveratrol analysis using large instruments such as liquid chromatography, the present invention's monitoring method offers convenient detection, low cost, and excellent selectivity and sensitivity for resveratrol. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute undue limitations thereon. The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0019] Figure 1 This is the XPS test graph of the CoO-V2O3 / C nanocomposite material in the first embodiment of the present invention.

[0020] Figure 2 This is a microscopic morphology test diagram of the CoO-V2O3 / C nanocomposite material in the first embodiment of the present invention.

[0021] Figure 3 This is a diagram showing the effect of lignin peroxidase activity of the CoO-V2O3 / C nanocomposite material in the seventh embodiment of the present invention.

[0022] Figure 4 This is a diagram showing the optimization results of resveratrol detection conditions in the eighth embodiment of the present invention.

[0023] Figure 5 This is a standard working curve diagram obtained by quantitative detection of resveratrol in the ninth embodiment of the present invention.

[0024] Figure 6 This is the specific result of the CoO-V2O3 / C nanocomposite material in detecting resveratrol in the tenth embodiment of the present invention.

[0025] Figure 7 These are the repeatability and stability test results of the CoO-V2O3 / C nanocomposite material in the eleventh embodiment of the present invention. DETAILED DESCRIPTION

[0026] The following description further elaborates on the specific details of the present invention for a full understanding of the present invention. The terms used in the description of the present invention are only used to illustrate the advantages and features of the present invention and are not intended to limit the present invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those understood by those skilled in the art within the technical field of the present invention. Unless otherwise specified, the drugs and reagents used in the following examples of the present invention were used according to the product instructions or in accordance with conventional methods in the art. The present invention will now be further described with reference to the accompanying drawings and specific embodiments.

[0028] First embodiment

[0029] A method for preparing a nanocomposite material having lignin peroxidase-like activity comprises the following steps:

[0030] (1) Cobalt nitrate hexahydrate (Co(NO3)2·6H2O) was dissolved in methanol to obtain solution A.

[0031] (2) Vanadyl acetylacetonate (VO(acac)2) and polyvinyl pyrrolidone were dissolved in methanol to obtain solution B.

[0032] (3) Dissolve 2-methylimidazole in methanol to obtain solution C.

[0033] (4) Solution A and solution B were first mixed in a molar ratio of cobalt nitrate hexahydrate: vanadyl acetylacetonate: carbon source = 3:1:2, and then solution C was added to the mixture of solutions A and B. The mixture was stirred at 25°C for 2 hours. The mixed solution was transferred to a 50 mL polytetrafluoroethylene-lined autoclave and heated at 200°C for 12 hours. After completion, the reaction solution was centrifuged, and the obtained solid product was repeatedly washed with deionized water and ethanol, and then dried in an oven at 70°C for 12 hours to obtain a nanocomposite material.

[0034] The XPS test chart of the nanocomposite material synthesized in this embodiment is as follows: Figure 1 As shown in Figure 2, it can be seen that the main components of the nanocomposite material are CoO, V2O3 and carbon (C). The microscopic morphology of the nanozyme synthesized in this embodiment is shown in Figure 2. Figure 2 As shown, wherein the A and B figures are SEM figures, the C and D figures are TEM figures, the E figure is a HRTEM figure; and the F figure is an EDS mapping image corresponding to the C, O, V, and Co elements. Figure 2 As can be seen in the figure, microspheres composed of a composite of CoO and V2O3 are supported on a sheet-like carbon support formed by stacked and intersecting carbon nanowires. This nanocomposite material (CoO-V2O3 / C) with a unique composition and morphology exhibits lignin peroxidase-like activity, specifically catalyzing the rapid oxidation of resveratrol (VA) to veratraldehyde with hydrogen peroxide at room temperature. The reaction mechanism is shown below.

[0035]

[0036] Second embodiment

[0037] A method for preparing a nanocomposite material having lignin peroxidase-like activity comprises the following steps:

[0038] (1) Cobalt nitrate hexahydrate (Co(NO3)2·6H2O) was dissolved in methanol to obtain solution A.

[0039] (2) Vanadyl acetylacetonate (VO(acac)2) and polyvinyl pyrrolidone were dissolved in methanol to obtain solution B.

[0040] (3) Dissolve 2-methylimidazole in methanol to obtain solution C.

[0041] (4) Solution A and solution B were first mixed in a molar ratio of cobalt nitrate hexahydrate: vanadyl acetylacetonate: carbon source = 2:1:2, and then solution C was added to the mixture of solutions A and B. The mixture was stirred at 25°C for 2 hours. The mixed solution was transferred to a 50 mL polytetrafluoroethylene-lined autoclave and heated at 200°C for 12 hours. After completion, the reaction solution was centrifuged, and the obtained solid product was repeatedly washed with deionized water and ethanol, and then dried in an oven at 70°C for 12 hours to obtain a nanocomposite material.

[0042] Third embodiment

[0043] A method for preparing a nanocomposite material having lignin peroxidase-like activity comprises the following steps:

[0044] (1) Cobalt nitrate hexahydrate (Co(NO3)2·6H2O) was dissolved in methanol to obtain solution A.

[0045] (2) Vanadyl acetylacetonate (VO(acac)2) and polyvinyl pyrrolidone were dissolved in methanol to obtain solution B.

[0046] (3) Dissolve 2-methylimidazole in methanol to obtain solution C.

[0047] (4) Solution A and solution B were first mixed in a molar ratio of cobalt nitrate hexahydrate: vanadyl acetylacetonate: carbon source = 1:1:2, and then solution C was added to the mixture of solutions A and B. The mixture was stirred at 25°C for 2 hours. The mixed solution was transferred to a 50 mL polytetrafluoroethylene-lined autoclave and heated at 200°C for 12 hours. After completion, the reaction solution was centrifuged, and the obtained solid product was repeatedly washed with deionized water and ethanol, and then dried in an oven at 70°C for 12 hours to obtain a nanocomposite material.

[0048] Fourth embodiment

[0049] A method for preparing a nanocomposite material having lignin peroxidase-like activity comprises the following steps:

[0050] (1) Cobalt nitrate hexahydrate (Co(NO3)2·6H2O) was dissolved in a solvent of methanol:water = 1:1 to obtain solution A.

[0051] (2) Vanadyl acetylacetonate (VO(acac)2) and polyvinyl pyrrolidone were dissolved in a solvent of methanol:water = 1:1 to obtain solution B.

[0052] (3) 2-Methylimidazole was dissolved in a solvent of methanol:water = 1:1 to obtain solution C.

[0053] (4) Solution A and solution B were first mixed in a molar ratio of cobalt nitrate hexahydrate: vanadyl acetylacetonate: carbon source = 3:1:2, and then solution C was added to the mixture of solutions A and B. The mixture was stirred at 25°C for 2 hours. The mixed solution was transferred to a 50 mL polytetrafluoroethylene-lined autoclave and heated at 200°C for 12 hours. After completion, the reaction solution was centrifuged, and the obtained solid product was repeatedly washed with deionized water and ethanol, and then dried in an oven at 70°C for 12 hours to obtain a nanocomposite material.

[0054] Fifth embodiment

[0055] A method for preparing a nanocomposite material having lignin peroxidase-like activity comprises the following steps:

[0056] (1) Cobalt nitrate hexahydrate (Co(NO3)2·6H2O) was dissolved in methanol to obtain solution A.

[0057] (2) Vanadyl acetylacetonate (VO(acac)2) and polyvinyl pyrrolidone were dissolved in methanol to obtain solution B.

[0058] (3) Dissolve 2-methylimidazole in methanol to obtain solution C.

[0059] (4) Solution A and solution B were first mixed in a molar ratio of cobalt nitrate hexahydrate: vanadyl acetylacetonate: carbon source = 3:1:2, and then solution C was added to the mixture of solutions A and B. The mixture was stirred at 25°C for 2 hours. The mixed solution was transferred to a 50 mL polytetrafluoroethylene-lined autoclave and heated at 190°C for 12 hours. After completion, the reaction solution was centrifuged, and the obtained solid product was repeatedly washed with deionized water and ethanol, and then dried in an oven at 70°C for 12 hours to obtain a nanocomposite material.

[0060] Sixth embodiment

[0061] A method for preparing a nanocomposite material having lignin peroxidase-like activity comprises the following steps:

[0062] (1) Cobalt nitrate hexahydrate (Co(NO3)2·6H2O) was dissolved in methanol to obtain solution A.

[0063] (2) Vanadyl acetylacetonate (VO(acac)2) and polyvinyl pyrrolidone were dissolved in methanol to obtain solution B.

[0064] (3) Dissolve 2-methylimidazole in methanol to obtain solution C.

[0065] (4) Solution A and solution B were first mixed in a molar ratio of cobalt nitrate hexahydrate: vanadyl acetylacetonate: carbon source = 3:1:2, and then solution C was added to the mixture of solutions A and B. The mixture was stirred at 25°C for 2 hours. The mixed solution was transferred to a 50 mL polytetrafluoroethylene-lined autoclave and heated at 210°C for 10 hours. After completion, the reaction solution was centrifuged, and the obtained solid product was repeatedly washed with deionized water and ethanol, and then dried in an oven at 70°C for 12 hours to obtain a nanocomposite material.

[0066] Seventh embodiment

[0067] The verification of the lignin-mimicking peroxidase activity of the CoO-V2O3 / C nanocomposite material includes the following steps:

[0068] Experimental System A: A mixture of Tris-HCl buffer (pH 8), the CoO-V2O3 / C nanocomposite prepared in Example 1, VA, and H2O2 was prepared. The concentration of the Tris-HCl buffer was 100 mM, the concentration of the CoO-V2O3 / C nanocomposite solution was 50 μg / mL, the concentration of VA was 20 mM, and the concentration of H2O2 was 5 mM. The initial absorbance at room temperature was measured, and the absorption spectrum within the range of 300-700 nm was recorded using a UV-visible spectrophotometer.

[0069] Control Experiment b: A mixture of Tris-HCl buffer (pH = 8), the CoO-V2O3 / C nanocomposite prepared in Example 1, and VA was prepared. The concentration of the Tris-HCl buffer was 100 mM, the concentration of the CoO-V2O3 / C nanocomposite was 50 μg / mL, and the concentration of VA was 20 mM. After reacting at room temperature for 30 minutes, the absorbance was measured and the absorption spectrum was recorded in the 300-700 nm range using a UV-visible spectrophotometer.

[0070] Control Experiment C: A mixture of Tris-HCl buffer (pH = 8), the CoO-V2O3 / C nanocomposite prepared in Example 1, VA, and H2O2 was prepared. The concentration of the Tris-HCl buffer was 100 mM, the concentration of the CoO-V2O3 / C nanocomposite was 50 μg / mL, the concentration of VA was 20 mM, and the concentration of H2O2 was 5 mM. After reacting at room temperature for 30 minutes, the absorbance was measured, and the absorption spectrum was recorded in the 300-700 nm range using a UV-visible spectrophotometer.

[0071] like Figure 3 As shown in Figure A, the absorption peak of experimental system a at 310 nm tends to be flat, the control experiment b does not show an increase in the absorption peak at 310 nm, and the control experiment c shows an obvious increase in the absorption peak at 310 nm. This is because CoO-V2O3 / C has the catalytic activity of lignin peroxidase and catalyzes hydrogen peroxide to oxidize VA to generate veratraldehyde. Therefore, by observing the changes in the characteristic absorption peak at 310 nm, it is convenient to judge the lignin peroxidase activity of CoO-V2O3 / C and determine whether VA is present in the sample.

[0072] like Figure 3 As shown in Figure B, the absorption peak of experimental system a at 310 nm shows an obvious upward trend over time, verifying the change in the absorption spectrum of experimental system a after 30 minutes of reaction.

[0073] Eighth embodiment

[0074] Optimization effect diagram of VA detection conditions for CoO-V2O3 / C nanocomposites:

[0075] Experimental system: CoO-V2O3 / C (50 μg / mL) and VA (20 mM) were added to a Tris-HCl buffer solution (pH 8, 100 mM). H2O2 (5 mM) was added to the mixed solution, and the mixture was incubated at room temperature for 30 minutes. The absorbance at 310 nm was measured, and the difference between the absorbance at the beginning of the reaction and the absorbance after 30 minutes of reaction was obtained.

[0076] In the above experimental system, the response time, pH of the buffer solution, concentration of CoO-V2O3 / C nanozyme, and incubation temperature were changed respectively, and the difference in absorbance at 310 nm was measured.

[0077] like Figure 4 As shown, when the response time is 30 min, the pH of the buffer is 8, the concentration of the CoO-V2O3 / C nanocomposite material is 50 μg / mL, and the incubation temperature is 30°C, the absorbance difference of the reaction system meets the detection requirements.

[0078] Ninth embodiment

[0079] Standard working curve for quantitative detection of VA of CoO-V2O3 / C nanocomposite materials: The CoO-V2O3 / C nanocomposite materials obtained in Example 1 were added to Tris-HCl buffer (pH 8, 100mM) containing different concentrations of VA (0.1-20mM). After reacting at room temperature for 30 minutes, the absorbance at 310nm was recorded using a UV-visible spectrophotometer as a function of VA concentration. The standard working curve for VA was then drawn using the difference in absorbance at 310nm, as shown in FIG. Figure 5 As shown, the linear range is 0.1-20 mM, y = 0.02012x + 0.2652 (R 2 =0.996).

[0080] Tenth embodiment

[0081] Specificity of CoO-V2O3 / C nanocomposite for detecting VA: The catalytic reaction system contained CoO-V2O3 / C (50 μg / mL) obtained in Example 1, various substrates (20 mM), H2O2 (5 mM) and Tris-HCl buffer (pH 8, 100 mM). After reacting at room temperature for 30 min, the absorbance at 310 nm was recorded by UV. Figure 6 As shown, it shows that CoO-V2O3 / C nanocomposites have good selectivity for VA.

[0082] Eleventh embodiment

[0083] Stability study of CoO-V2O3 / C nanocomposites: CoO-V2O3 / C nanocomposites (50 μg / mL) stored for different times were added to Tris-HCl buffer solution (pH 8, 100 mM) for reaction, and the absorbance difference of the reaction system at the beginning of the reaction and after 30 min of reaction was measured; CoO-V2O3 / C nanocomposites (50 μg / mL) prepared from different batches were added to Tris-HCl buffer solution (pH 8, 100 mM) for reaction, and the absorbance difference of the reaction system at the beginning of the reaction and after 30 min of reaction was measured;

[0084] like Figure 7 As shown, when the storage time is 30 days, there is no obvious effect on the absorbance value, and the repeatability is high, indicating that the CoO-V2O3 / C nanocomposite material has high stability.

[0085] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

[0086] It should be noted that the above examples are only intended to illustrate the technical solutions of the present invention and are not intended to limit the same. Although the present invention has been described in detail with reference to the examples given, those skilled in the art may modify or replace the technical solutions of the present invention as needed without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of a nanocomposite material having lignin peroxidase-like activity in detecting resveratrol, characterized in that: The nanocomposite material includes a carbon support and composite microspheres supported on the carbon support, wherein: the carbon support is a sheet structure formed by stacking and crossing carbon nanowires, the components of the composite microspheres include CoO and V2O3, and the particle size of the composite microspheres is 50-300 nm; The preparation method of the nanocomposite material is as follows: dissolving a cobalt source, vanadyl acetylacetonate and a carbon source in a solvent, then performing a solvothermal reaction, and separating a solid product after completion to obtain the nanocomposite material; The application method comprises the following steps: mixing the nanocomposite material with a sample containing resveratrol to form a mixed solution, then adding hydrogen peroxide for reaction, detecting the absorbance of the resulting reaction solution after completion, and achieving quantitative detection of resveratrol through the quantitative relationship between the difference in absorbance values ​​at a wavelength of 310 nm before and after the reaction of the reaction system and resveratrol.

2. Use of the nanocomposite material having lignin peroxidase-like activity as claimed in claim 1 for detecting resveratrol, characterized in that: The molar ratio of the cobalt source, vanadyl acetylacetonate and carbon source is in the range of 1 to 3:1:

2.

3. Use of the nanocomposite material having lignin peroxidase-like activity as claimed in claim 1 for detecting resveratrol, characterized in that: The cobalt source is at least one of cobalt nitrate, cobalt sulfate, cobalt chloride and cobalt acetate.

4. Use of the nanocomposite material having lignin peroxidase-like activity as claimed in claim 1 for detecting resveratrol, characterized in that: The carbon source is 2-methylimidazole.

5. Use of the nanocomposite material having lignin peroxidase-like activity as claimed in claim 1 for detecting resveratrol, characterized in that: The temperature range of the solvent thermal reaction is 180-200° C., and the time range is 10-12 hours.

6. Use of the nanocomposite material having lignin peroxidase-like activity as claimed in claim 1 for detecting resveratrol, characterized in that: The solvent is methanol.

7. Use of the nanocomposite material having lignin peroxidase-like activity as claimed in claim 1 for detecting resveratrol, characterized in that: The nanocomposite material is added to a Tris-HCl buffer solution of resveratrol to form a mixed solution, and then hydrogen peroxide is added dropwise and reacted at 15-30° C. for 0.5-2 h. The concentration ratio of the nanocomposite material, resveratrol, and hydrogen peroxide is 6-24 μg / mL: 4 mM: 1 mM.

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