Nanometer material with specific oxidase activity and preparation method and application thereofTechnical Field
The invention relates to the technical field of enzyme-like active materials, in particular to a nano material with specific oxidase activity, a preparation method and application thereof.
Background
Nanoenzymes are nanomaterials that mimic the activity of natural enzymes. Since the first report in 2007 that Fe3O4 Nanoparticles (NPs) have Peroxidase (POD) like activity, a large number of nanomaterials have been prepared as nanoenzymes, which are derived from noble metals, transition metal oxides, metal-organic framework structures or carbon materials. The nano enzyme has low preparation cost, various structures and high catalytic activity and stability, and is widely applied to the fields of biosensing, disease treatment and environmental protection. However, nanoezymes still suffer from several disadvantages compared to natural enzymes, including low activity due to low density of active sites, low specificity due to inherently heterogeneous atomic composition and complex structure.
Disclosure of Invention
The invention aims to overcome one or more defects in the prior art and provides a novel method for preparing a nano material with specific oxidase activity.
The invention also provides the nano material with the specific oxidase activity prepared by the method.
The invention also provides application of the nano material with the specific oxidase activity prepared by the method in pesticide residue detection, wherein the pesticide residue detection comprises detection of pyrithione (also called chlorpyrifos, chemical name is O, O-diethyl-O- (3, 5, 6-trichloro-2-pyridyl) thiophosphoric acid, molecular formula is C9H11Cl3NO3 PS, white crystallization is realized, the nano material has slight thiol smell, and the nano material is a non-systemic broad-spectrum insecticidal and acaricidal agent and has higher volatility in the soil).
In order to achieve the above purpose, the invention adopts a technical scheme that: a method for preparing a nanomaterial having specific oxidase activity, the method comprising:
(1) Uniformly mixing graphite-like carbon nitride (also called g-C3N4), chitosan, an iron source and water, removing water from the uniformly mixed system, and drying to obtain a precursor;
(2) Heating the precursor in inert atmosphere until the temperature is 750-850 ℃, and sintering at the maintained temperature to obtain an intermediate;
(3) Etching the intermediate by adopting an acidic solution to separate the nano material with specific oxidase activity.
According to some preferred aspects of the present invention, in step (1), the graphite-like phase carbon nitride, the chitosan and the iron source are fed in a mass ratio of 1:0.05-0.80:0.005-0.06.
Further, in the step (1), the feeding mass ratio of the graphite-like phase carbon nitride to the chitosan to the iron source is 1:0.08-0.75:0.01-0.035.
In some specific aspects of the invention, in the step (1), the feeding mass ratio of the graphite-like phase carbon nitride to the chitosan to the iron source is 1:0.06-0.13:0.012-0.020.
According to some preferred and specific aspects of the invention, the iron source is ferric nitrate or ferric nitrate nonahydrate in hydrated form.
In the present invention, according to a specific aspect of the present invention, the precursor is in a powder form.
According to some preferred aspects of the invention, in step (1), the mixing is carried out at 20-30 ℃.
According to a specific aspect of the present invention, in step (1), the mixing process is performed at room temperature.
According to some preferred aspects of the invention, in step (2), the sintering temperature is 780-820 ℃. According to a specific aspect of the invention, in step (2), the sintering temperature is 800 ℃.
According to some preferred aspects of the invention, in step (2), the rate of temperature increase is from 1 to 10 ℃/min. Further, in the step (2), the heating rate is 4-6 ℃/min.
According to some preferred aspects of the invention, in step (2), the sintering time is 0.2 to 3 hours. Further, in the step (2), the sintering time is 0.5-2h.
According to some preferred and specific aspects of the invention, in step (2), the inert atmosphere is formed by passing nitrogen.
In the present invention, according to a specific aspect of the present invention, the intermediate is in the form of black powder.
According to some preferred aspects of the invention, in step (3), the acidic solution is hydrochloric acid.
In some embodiments of the invention, in step (3), the acidic solution is hydrochloric acid, and the concentration of the hydrochloric acid is 0.5-5mol/L. Further, in the step (3), the acid solution is hydrochloric acid, and the concentration of the hydrochloric acid is 1-3mol/L.
According to some preferred aspects of the invention, in step (3), the etching treatment with the acid solution is carried out for a period of 8-20 hours.
In some embodiments of the present invention, in step (3), the etching treatment with the acid solution is performed for 8 to 16 hours.
According to some preferred and specific aspects of the invention, embodiments of preparing the nanomaterial with specific oxidase activity comprise:
(a) Mixing and stirring graphite-like phase carbon nitride, chitosan and water to obtain a uniform solution;
(b) Dissolving an iron source in water, and then adding the water into the solution in the step (a) to obtain a mixed solution;
(c) Stirring and mixing the mixed solution in the step (b) at room temperature;
(d) Removing water from the uniformly mixed system in the step (c), and drying the obtained solid to obtain a precursor;
(e) Raising the temperature of the precursor in the step (d) from room temperature to 750-850 ℃ in inert gas at a constant temperature raising rate, and maintaining the temperature at the temperature for sintering to obtain an intermediate;
(f) Etching the intermediate in the step (e) in an acid solution, centrifuging to obtain a precipitate, cleaning the precipitate, and drying the cleaned precipitate to obtain the nano material with specific oxidase activity.
The invention provides another technical scheme that: the nanometer material with specific oxidase activity prepared by the preparation method.
According to some preferred aspects of the present invention, in the nanomaterial with specific oxidase activity, more than 50% of iron is uniformly distributed on nitrogen-doped carbon in the form of single atoms, and the active site is-N-Fe-N- (also referred to as FeN2 site).
The invention provides another technical scheme that: the application of the nano material with the specific oxidase activity in pesticide residue detection comprises the detection of the clopyralid.
In the invention, the method for simulating the oxidase activity of the nano material with the specific oxidase activity prepared by the preparation method comprises the following steps: oxidase activity was evaluated based on the chromogenic reaction in which TMB (3, 3', 5' -tetramethylbenzidine) was oxidized to blue oxTMB (Chinese name: oxidized 3,3', 5' -tetramethylbenzidine). The prepared nanomaterial and TMB were added to the NaAc-HAc buffer and the mixture was reacted. After completion of the reaction, a supernatant of the reaction mixture was obtained by centrifugation, and its absorbance value was measured at 652 nm.
In the invention, the method for simulating the peroxidase activity of the nano material with the specific oxidase activity prepared by the preparation method comprises the following steps: peroxidase activity was evaluated based on the chromogenic reaction of ABTS (2, 2' -biazo-bis-3-ethylbenzothiazoline-6-sulfonic acid) oxidized to ABTS in the oxidized state by peroxidase in the presence of hydrogen peroxide. The prepared nanomaterial, ABTS, and H2O2 (hydrogen peroxide ) were added to the NaAc-HAc buffer and the mixture was reacted. After completion of the reaction, a supernatant of the reaction mixture was obtained by centrifugation, and its absorbance value was measured at 420 nm.
In the invention, the nano material with specific oxidase activity prepared by the preparation method is applied to chlorpyrifos detection. The method for applying the nanomaterial with specific oxidase activity to chlorpyrifos detection, which is prepared by the preparation method, comprises the following steps: acetylcholinesterase was mixed with chlorpyrifos at various concentrations (1.5 ng/mL,10ng/mL,20ng/mL,30ng/mL,40ng/mL,50 ng/mL), then acetylcholine was added and incubated, then the prepared nanomaterial was added and incubated. Finally, TMB and NaAc-HAc buffer solution are added for reaction. The supernatant of the reaction mixture was obtained by centrifugation and the absorbance was measured at 652 nm.
The simulation test results show that: in the oxidation reaction of catalytic TMB, the product oxTMB has a higher absorption peak at 652nm, which indicates that the nano material prepared by the invention has excellent oxidase activity. The nano material prepared by the invention basically has no peroxidase activity in the peroxidase activity test, which indicates that the nano material prepared by the invention has specific oxidase activity. The method is applied to the detection of chlorpyrifos, the detection range is 1-50ng/mL, the detection limit is 0.25ng/mL, the detection limit is lower than most of nano-enzymes, and the nano-materials prepared by the method have application prospects in the field of pesticide residue detection.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:
(1) The invention prepares a nano material with specific oxidase activity, and the FeN2 structure of the material is similar to the active center of natural enzyme in the aspects of electronics, geometry and chemical structure. Compared with the reported oxidase nano-enzyme, the nano-enzyme has excellent Km (Mi constant) and Vmax (maximum rate), which shows that the nano-material prepared by the invention has better oxidase-like activity;
(2) The specificity of the nano material prepared by the invention is superior to that of nano enzymes derived from other single-atom iron-based materials, and the nano material only shows oxidase activity and the specificity of natural enzymes;
(3) The nano material prepared by the invention is also applied to the detection of chlorpyrifos, has higher detection sensitivity than most nano enzymes, and has wide application prospect.
Drawings
FIG. 1 is a preparation flow chart of Fe@CN-800-A and Fe@CN-700;
FIG. 2 is a graph of synchrotron radiation test spectra of example 1 for preparing nanomaterials with specific oxidase activity;
FIG. 3 is a graph showing the evaluation of oxidase activity, peroxidase activity and catalase activity of Fe@CN-800-A and Fe@CN-700;
FIG. 4 is a graph showing the result of detecting the concentration of chlorpyrifos in the pesticide by Fe@CN-800-A.
Detailed Description
The above-described aspects are further described below in conjunction with specific embodiments; it should be understood that these embodiments are provided to illustrate the basic principles, main features and advantages of the present invention, and that the present invention is not limited by the scope of the following embodiments; the implementation conditions employed in the examples may be further adjusted according to specific requirements, and the implementation conditions not specified are generally those in routine experiments.
All starting materials are commercially available or prepared by methods conventional in the art, not specifically described in the examples below.
G-C3N4 is prepared by continuously calcining melamine under nitrogen at a rate of 5 ℃/min to 550 ℃ for 2 hours; chitosan was purchased from Alatine under the trade designation C105802-100g.
Example 1
The preparation process of the nanomaterial (Fe@CN-800-A) having specific oxidase activity is briefly described as follows. Firstly, the Fe@CN-800-A is mainly prepared by adopting an impregnation method and a high-temperature carbonization method. The combination of the two methods has the advantages of simple preparation process, short synthesis period, easy operation of preparation and the like. In the reaction process, g-C3N4 and chitosan can serve as a carbon source and a nitrogen source at the same time, and the rich nitrogen source can improve the dispersibility of iron atoms so as to obtain a more uniform system.
The nano material with specific oxidase activity (Fe@CN-800-A for short) prepared by the method is prepared by the following steps (the schematic process is shown in figure 1):
(1) 1g of g-C3N4, 0.12g of chitosan and 40mL of water were mixed and vigorously stirred to obtain a homogeneous solution;
(2) 0.03g of ferric nitrate nonahydrate is dissolved in 2mL of water and added to the solution in the step (1);
(3) Stirring the mixed solution in the step (2) for 24 hours at room temperature;
(4) Spin-evaporating the system obtained in the step (3) to remove water, and drying the obtained solid at constant temperature (the drying temperature is 60 ℃) to obtain precursor powder;
(5) Heating the precursor powder dried in the step (4) from room temperature to 800 ℃ at a heating rate of 5 ℃/min in a nitrogen atmosphere, and keeping the temperature for 1h to obtain black powder;
(6) Stirring the black powder in the step (5) in 2mol/L HCl solution for 12h, centrifuging to obtain precipitate, cleaning the precipitate, and drying the cleaned precipitate at constant temperature (the drying temperature is 60 ℃), thereby finally obtaining the nano material with specific oxidase activity.
The synchronous radiation test spectrogram of the nano material with specific oxidase activity (Fe@CN-800-A for short) is shown in a figure 2, wherein a line 1 represents the synchronous radiation test spectrogram of the Fe@CN-800-A; simulating a synchronous radiation test signal peak of Fe@CN-800-A, and exploring a main existence form of iron;
The lines of the line 2, the line 3 and the line 4 respectively represent signal peaks of Fe-Cl bond, fe-Fe bond and Fe-N bond, which are results obtained by simulating synchronous radiation test signal peaks of Fe@CN-800-A, and the circle (fit) is the confluence of the signal peaks of the line 2, the line 3 and the line 4, and can be well overlapped with the signal peak of the Fe@CN-800-A of the line 1, which proves that the simulation is effective;
Simulation results show that iron exists in the form of Fe-Cl bond, fe-Fe bond and Fe-N bond in Fe@CN-800-A, wherein the Fe-N bond exists in the form of FeN2.
Example 2
The nano material with specific oxidase activity (Fe@CN-800-A for short) prepared by the method is prepared by the following steps (the schematic process is shown in figure 1):
(1) 1g of g-C3N4, 0.10g of chitosan and 40mL of water were mixed and vigorously stirred to obtain a homogeneous solution;
(2) 0.04g of ferric nitrate nonahydrate is dissolved in 2mL of water and added to the solution in the step (1);
(3) Stirring the mixed solution in the step (2) at room temperature for reaction for 24 hours;
(4) Spin-evaporating the reaction system in the step (3) to remove water, and drying the obtained solid at constant temperature (the drying temperature is 40 ℃) to obtain precursor powder;
(5) Heating the precursor powder dried in the step (4) from room temperature to 780 ℃ at a heating rate of 5 ℃/min in a nitrogen atmosphere, and keeping the temperature for 1h to obtain black powder;
(6) Stirring the black powder in the step (5) in 2mol/L HCl solution for 12h, centrifuging to obtain precipitate, cleaning the precipitate, and drying the cleaned precipitate at constant temperature (the drying temperature is 40 ℃), thereby finally obtaining the nano material with specific oxidase activity.
Example 3
The nano material with specific oxidase activity (Fe@CN-800-A for short) prepared by the method is prepared by the following steps (the schematic process is shown in figure 1):
(1) 1g of g-C3N4, 0.75g of chitosan and 40mL of water were mixed and vigorously stirred to obtain a homogeneous solution;
(2) 0.02g of ferric nitrate nonahydrate is dissolved in 2mL of water and added to the solution in the step (1);
(3) Stirring the mixed solution in the step (2) at room temperature for reaction for 24 hours;
(4) Spin-evaporating the reaction system in the step (3) to remove water, and drying the obtained solid at constant temperature (the drying temperature is 50 ℃) to obtain precursor powder;
(5) Heating the precursor powder dried in the step (4) from room temperature to 790 ℃ at a heating rate of 5 ℃/min in a nitrogen atmosphere, and keeping the temperature for 1h to obtain black powder;
(6) Stirring the black powder in the step (5) in 2mol/L HCl solution for 12h, centrifuging to obtain precipitate, cleaning the precipitate, and drying the cleaned precipitate at constant temperature (the drying temperature is 50 ℃), thereby finally obtaining the nano material with specific oxidase activity.
Comparative example 1: preparation of Fe@CN-700
(1) 1G of g-C3N4, 0.12g of chitosan and 40mL of water were mixed and vigorously stirred to obtain a uniform solution;
(2) 0.03g of ferric nitrate nonahydrate is dissolved in 2mL of water and added to the solution in the step (1);
(3) Stirring the mixed solution in the step (2) at room temperature for reaction for 24 hours;
(4) Spin-evaporating the reaction system in the step (3) to remove water, and drying the obtained solid at constant temperature (the drying temperature is 60 ℃) to obtain precursor powder;
(5) Heating the precursor powder dried in the step (4) from room temperature to 700 ℃ at a heating rate of 5 ℃/min in a nitrogen atmosphere, and keeping the temperature for 1h to obtain black powder;
(6) Stirring the black powder in the step (5) in 2mol/L HCl solution for 12h, centrifuging to obtain precipitate, cleaning the precipitate, and drying the cleaned precipitate at constant temperature (the drying temperature is 60 ℃), thereby finally obtaining the nano material (Fe@CN-700).
Example 4
In the following, the oxidase activity of the material is tested without adding hydrogen peroxide; hydrogen peroxide is added, and the peroxidase activity of the material is tested; if hydrogen peroxide is not added, the absorbance is provided, or the color of the substrate is changed, which indicates that the material has oxidase activity; if the absorbance is not available when the hydrogen peroxide is not added, the material has peroxidase activity when the absorbance is added with the hydrogen peroxide.
Evaluation of the mimic oxidase Activity
In order to examine the activity of the nanomaterial with specific oxidase activity (Fe@CN-800-A) provided by the invention, the inventors made further experimental verification based on Fe@CN-800-A prepared in example 1, and the related experimental procedures are briefly described below.
Oxidase activity was evaluated based on the chromogenic reaction in which TMB can be oxidized to blue oxTMB by oxidase. 30. Mu.LFe@CN-800-A in water (1 mg/mL) and 10. Mu.L TMB (10 mM) were added to 0.96mLNaAc-HAc buffer (0.1M, pH 4.0) and the mixture was allowed to react at 37℃for 5min. After completion of the reaction, the supernatant of the reaction mixture was obtained by centrifugation (8000 rpm,2 min), and the absorbance value thereof was detected at 652nm (FIG. 3 (a)).
Oxidase activity was evaluated based on the chromogenic reaction in which TMB can be oxidized to blue oxTMB by oxidase. mu.L of the Fe@CN-700 aqueous solution prepared in comparative example 1 (concentration: 1 mg/mL) and 10. Mu.L of TMB (10 mM) were added to 0.96mLNaAc-HAc buffer (0.1M, pH 4.0), and the mixture was reacted at 37℃for 5 minutes. After completion of the reaction, the supernatant of the reaction mixture was obtained by centrifugation (8000 rpm,2 min), and the absorbance value thereof was detected at 652nm (FIG. 3 (a)).
Assessment of mimic peroxidase Activity
Peroxidase activity was evaluated based on the chromogenic reaction of ABTS oxidized to oxidized ABTS by peroxidase in the presence of hydrogen peroxide. mu.L of Fe@CN-800-A in water (1 mg/mL), 50. Mu.L of ABTS (10 mM) and 100. Mu. L H2O2 (10 mM) were added to 0.82mLNaAc-HAc buffer (0.1M, pH 4.0) and the mixture was allowed to react at 37℃for 5min. After completion of the reaction, the supernatant of the reaction mixture was obtained by centrifugation (8000 rpm,2 min), and the absorbance value thereof was detected at 420nm (FIG. 3 (c)).
Mu.L of the Fe@CN-700 aqueous solution prepared in comparative example 1 (concentration: 1 mg/mL), 50. Mu.L of ABTS (10 mM) and 100. Mu. L H2O2 (10 mM) were added to 0.82mLNaAc-HAc buffer (0.1M, pH 4.0), and the mixture was allowed to react at 37℃for 5 minutes. After completion of the reaction, the supernatant of the reaction mixture was obtained by centrifugation (8000 rpm,2 min), and the absorbance value thereof was detected at 420nm (FIG. 3 (b)).
Further, FIG. 3 (d) shows a comparison of the capacities of Fe@CN-700 and Fe@CN-800-A for decomposing hydrogen peroxide to generate oxygen, and the ordinate shows the amount of oxygen generated, and the larger the ordinate, the more hydrogen peroxide is decomposed to generate oxygen.
The result shows that the FeN2 site in the Fe@CN-800-A shows higher oxidase activity and specificity than the FeN4 site in the Fe@CN-700, the energy barrier of the FeN2 active site for catalyzing oxygen adsorption and hydrogen peroxide decomposition is lower than that of the FeN4 site, and the Fe@CN-800-A shows single oxidase activity.
Application example 1 detection of Chlorpyrifos concentration
Mu.L of acetylcholinesterase (40 mU/mL) was mixed with 10. Mu.L of chlorpyrifos at various concentrations (1.5 ng/mL,10ng/mL,20ng/mL,30ng/mL,40ng/mL,50 ng/mL), followed by addition of 50. Mu.L of acetylcholine (10 mM) and incubation for 40min, followed by addition of 20. Mu.L of Fe@CN-800-A aqueous solution (1 mg/mL concentration) and further incubation for 10min. Finally, 30. Mu.L of TMB and 0.84mLNaAc-HAc buffer (0.1M, pH 4.0) were added and reacted for 3min. The supernatant of the reaction mixture was obtained by centrifugation, and the absorbance value was measured at 652nm (a graph showing the result of chlorpyrifos concentration of the pesticide shown in FIG. 4 was obtained).
The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.