Iron-based nano-enzyme based on polydopamine as well as preparation method and application of iron-based nano-enzyme

Abstract

The invention belongs to the field of composite nano materials, and particularly discloses an iron-based nano enzyme based on polydopamine as well as a preparation method and application of the iron-based nano enzyme based on polydopamine, and PDA-coated Fe nano enzyme with high catalytic activity and stability is prepared by chelating polydopamine microspheres with ferrous ions. The nano-enzyme can catalyze the chromogenic reaction of TMB and HO, and is used for ratio colorimetric method detection of nitrite. The PDA-coated Fe microspheres with uniform particle size and good dispersity are obtained by optimizing the preparation process, the utilization rate of iron atoms is remarkably increased, and oxidative inactivation is avoided. The detection method is high in sensitivity and strong in anti-interference capability, the detection limit is as low as 1.0 [mu] M, and the linear range is 5-100 [mu] M. The nano enzyme is low in preparation cost, simple and convenient to operate and high in environmental adaptability, can be applied to the fields of environmental monitoring, food safety, biomedical detection and the like, and provides an efficient and reliable solution for on-site rapid detection.

Description

Technical Field

[0001] The present invention belongs to the field of composite nanomaterials, and specifically discloses an iron-based nanozyme based on polydopamine, a preparation method thereof, and applications thereof. Background Art

[0002] As an important intermediate in the nitrogen cycle, nitrite widely exists in natural water bodies and foods. In the environment, nitrite is mainly produced by microbial nitrification, and human activities such as industrial wastewater discharge and agricultural fertilizer application further exacerbate its accumulation in water bodies. Excessive nitrite not only destroys the ecological balance of water bodies, but also causes health problems such as methemoglobinemia and carcinogenic risks in the human body through the food chain. Therefore, the development of highly sensitive and reliable nitrite detection technology is crucial for environmental protection and food safety.

[0003] At present, due to its simple operation and low cost, the colorimetric method has become the mainstream method for nitrite detection. The traditional colorimetric method reflects the concentration of the target substance through a single-wavelength signal (such as 652 nm), but it is easily interfered by the environment (such as impurity light absorption and reagent fluctuations) during low-concentration detection, resulting in insufficient sensitivity and accuracy. Although the ratio colorimetric method can partially offset the interference through the ratio of dual-wavelength absorbances (such as A 450 / A 652 ), its core still relies on highly efficient and stable catalytic materials to improve the detection performance.

[0004] In recent years, due to their catalytic activity comparable to that of natural enzymes and their controllability, nanozymes have become a research hotspot in the detection field. Among them, iron-based nanozymes have attracted much attention due to their high catalytic efficiency and low cost, but their practical applications still face two major bottlenecks:

[0005] 1. Low iron atom utilization rate: Ferrous ions are easily oxidized and inactivated, resulting in a decrease in catalytic activity.

[0006] 2. Poor stability: Traditional iron-based materials are prone to aggregation in complex environments, reducing the reliability of long-term use. Summary of the Invention

[0007] To solve the above problems, an iron-based nanozyme based on polydopamine, a preparation method thereof, and applications thereof use polydopamine (PDA) microspheres as functional carriers to chelate ferrous ions through their rich catechol / amine groups, and in-situ construct composite nanozymes (PDA@Fe). The polydopamine microspheres can not only chelate ferrous ions and inhibit oxidation, but also their uniform nanostructure can improve the dispersibility of the material, thereby significantly enhancing the catalytic stability and detection sensitivity. This design provides a new idea for the development of highly efficient, convenient, and stable nitrite detection technology.

[0008] The present invention includes the following technical solutions:

[0009] A poly-dopamine-based iron nanozyme, which is formed by compounding poly-dopamine microspheres and ferrous ions through a chelation reaction. The particle size of the poly-dopamine microspheres is 150-300 nm, and the nanozyme has peroxidase-like catalytic activity and can catalyze the color reaction of 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2).

[0010] Furthermore, for the above poly-dopamine-based iron nanozyme, the preparation method of the poly-dopamine microspheres includes the following steps:

[0011] a. Add dopamine hydrochloride to a mixed solution of ammonia water, water and ethanol with a volume ratio of 2:9-6:9, and stir and react for 24-48 h;

[0012] b. Centrifuge and separate the product, wash and freeze-dry to obtain poly-dopamine microspheres with uniform particle size.

[0013] Furthermore, for the above poly-dopamine-based iron nanozyme, in step a, the dosage of dopamine hydrochloride is 0.4-0.7 g, the dosage of ammonia water is 1-4 mL, the reaction temperature is 20-25 °C, and the stirring rate is 150-300 rpm.

[0014] Furthermore, for the above poly-dopamine-based iron nanozyme, in the chelation reaction, the mass ratio of poly-dopamine microspheres to ferrous chloride is 1:3-1:10, the reaction is carried out in an ethanol solution, the reaction time is 4-8 h, and the reaction temperature is 20-25 °C.

[0015] The present invention also discloses a preparation method of the above iron nanozyme, including the following steps:

[0016] a. Prepare poly-dopamine microspheres;

[0017] b. Mix the poly-dopamine microspheres and ferrous chloride in an ethanol solution, stir and react under a nitrogen atmosphere, centrifuge and wash, and then freeze-dry to obtain the iron nanozyme.

[0018] Furthermore, for the above preparation method of the iron nanozyme, in step b, the dosage of poly-dopamine microspheres is 10-30 mg, the dosage of ferrous chloride is 10-300 mg, the volume of the ethanol solution is 10-50 mL, and the stirring rate is 150-300 rpm.

[0019] The present invention also discloses a nitrite detection method based on the above iron nanozyme, including the following steps:

[0020] a. Mix the iron nanozyme, TMB, H2O2 and buffer solution, and add the sample to be tested;

[0021] b. Determine the absorbance ratio (A450 / A652) at 450 nm and 652 nm by ultraviolet-visible spectrophotometer, and quantitatively analyze the nitrite concentration according to the standard curve.

[0022] Further, in the above detection method, the buffer solution is acetic acid-sodium acetate buffer solution with pH 2.0 - 6.0, the concentration of iron-based nanozyme is 5 - 30 μg / mL, the concentration of H2O2 is 0.05 - 0.50 mM, and the concentration of TMB is 0.1 - 1.0 mM.

[0023] The present invention also discloses the application of the above iron-based nanozyme in environmental monitoring, food safety or biomedical detection, which is characterized in that the application is based on the peroxidase-like catalytic activity of the nanozyme and the sensitivity to nitrite.

[0024] Further, in the above application, the detection limit of nitrite is 1.0 μM, the linear range is 5 - 100 μM, and the detection time is 10 - 30 min.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. High catalytic activity and stability

[0027] Chelate ferrous ions through polydopamine (PDA) microspheres, significantly improving the utilization rate of iron atoms and avoiding oxidation inactivation. Experiments show that the catalytic activity of PDA@Fe nanozyme for H2O2 and TMB is better than that of traditional iron-based materials (K m value is as low as 0.124 mM, V max reaches 1.88×10 -8 M / s), and the enzyme activity still remains above 98% after storage at 4°C for 3 months.

[0028] 2. High sensitivity and anti-interference ability

[0029] Based on the ratio colorimetry of the dual-wavelength absorbance ratio (A 450 / A 652 ), effectively eliminate environmental interference. The detection limit is as low as 1.0 μM, and the linear range is wide (5 - 100 μM), which is suitable for the accurate detection of trace nitrite.

[0030] 3. Excellent material homogeneity and dispersibility

[0031] Optimize the preparation process (such as 0.5 g of dopamine, 3 mL of ammonia water, and the volume ratio of ethanol / water is 3:9) to obtain PDA@Fe microspheres with uniform particle size (150 - 300 nm) and good dispersibility (PDI < 0.1), ensuring high repeatability of the catalytic reaction.

[0032] 4. Strong environmental adaptability

[0033] The detection system is stable in the pH range of 2.0 - 6.0 and can be compatible with complex sample matrices (such as water bodies and food extracts), without the need for complex pretreatment.

[0034] 5. Low cost and simple operation

[0035] PDA microspheres are synthesized by a one-step self-polymerization method, combined with a chelation reaction at room temperature. The process is simple and does not require expensive equipment. The detection process only takes 20 minutes, significantly reducing the time and economic costs.

[0036] 6. Broad application prospects

[0037] This nanozyme can be integrated into a portable detection device and is applicable to environmental monitoring (such as industrial wastewater), food safety (such as nitrite screening in pickled products), and biomedical diagnosis fields, promoting the development of on-site rapid detection technology. Description of the drawings

[0038] Figure 1 Infrared structural analysis of PDA microspheres;

[0039] Figure 2 Effect of different masses of dopamine on the particle size of PDA microspheres;

[0040] Figure 3 Effect of different volumes of ammonia water on the particle size of PDA microspheres;

[0041] Figure 4 Effect of different volume ratios of ethanol to water on the particle size of PDA microspheres;

[0042] Figure 5 Transmission electron microscope photograph of polydopamine microspheres;

[0043] Figure 6 Transmission electron microscope photograph of PDA@Fe nano-microspheres;

[0044] Figure 7 Elemental analysis of PDA@Fe nano-microspheres;

[0045] Figure 8 Ultraviolet absorption spectra of enzyme activity corresponding to buffer solutions with different pH values;

[0046] Figure 9 Ultraviolet absorption spectra of enzyme activity corresponding to different enzyme concentrations;

[0047] Figure 10 Ultraviolet absorption of enzyme activity corresponding to different H2O2 concentrations: (a) Ultraviolet absorption spectra of catalytic activity of PDA@Fe under different concentrations of hydrogen peroxide; (b) Absorbance at the maximum absorption wavelength (652 nm) of catalytic activity of PDA@Fe under different concentrations of hydrogen peroxide;

[0048] Figure 11 UV absorption of enzyme activity corresponding to different concentrations of TMB: (a) UV absorption spectrum of the catalytic activity of PDA@Fe under different concentrations of TMB; (b) Absorbance at the maximum absorption wavelength (652 nm) of the catalytic activity of PDA@Fe under different concentrations of TMB.

[0049] Figure 12 Enzymatic kinetics of PDA@Fe for H2O2: (a) Curve of velocity change with varying H2O2 concentration at a TMB concentration of 0.8 mM; (b) Double-reciprocal Lineweaver-Burk plot based on the Michaelis-Menten equation.

[0050] Figure 13 Enzymatic kinetics of PDA@Fe for TMB: (a) Curve of velocity change with varying TMB concentration at an H2O2 concentration of 0.45 mM; (b) Double-reciprocal Lineweaver-Burk plot based on the Michaelis-Menten equation.

[0051] Figure 14 Peroxidase stability of PDA@Fe nanospheres

[0052] Figure 15 Linear relationship diagram between different nitrite concentrations and the absorbance ratio (A 450 / A 652 ) of the detection system. Specific implementation manners

[0053] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0054] Example 1

[0055] Iron-based nanozyme based on polydopamine, preparation method and application

[0056] (1) Preparation of polydopamine microspheres

[0057] At room temperature of 25 °C, measure 1-4 mL of ammonia water (NH₃·H₂O), 100-200 mL of a mixed solution of water and ethanol with a volume ratio of 2:9-6:9, add them to a 250 mL three-necked flask, and stir at a speed of 150-300 rpm for 20-60 min to make them fully mixed; then weigh 0.4-0.7 g of dopamine hydrochloride and dissolve it in 5-15 mL of water, inject it into the above mixed solution, and continue to stir and react at a speed of 200 rpm for 24-48 h. After the reaction is completed, centrifuge at a speed of 8000-12000 rpm for 5-15 min, discard the supernatant, add an appropriate amount of water for washing and centrifugation, and repeat 2-3 times to obtain the final product. Collect the product in the centrifuge tube, freeze-dry to obtain a black powder, and store the obtained sample powder in a 4 °C refrigerator for later use.

[0058] (2) Preparation of PDA@Fe nanospheres

[0059] At room temperature of 25 °C, weigh 10-30 mg of polydopamine microspheres and 10-300 mg of ferrous chloride powder, add them to a 100 mL three-necked flask containing 10-50 mL of ethanol solution, stir in an N₂ atmosphere at a speed of 150-300 rpm to make them fully dissolve and mix, and then react at room temperature for 4-8 h. After the reaction is completed, centrifuge at a speed of 5000-12000 rpm for 8-15 min, discard the supernatant, add an appropriate amount of water for washing and centrifugation, and repeat 3 times to remove ethanol and unreacted ferrous chloride to obtain the final chelation product. After freeze-drying, a black powder is obtained, and the obtained sample powder is stored in a 4 °C refrigerator for later use.

[0060] (3) Optimization of peroxidase catalytic performance of PDA@Fe microspheres

[0061] Mix 0.1-0.3 mL of PDA@Fe microsphere dispersion (150-900 μg / mL), 0.1-0.3 mL of TMB solution (1-30 mM), 0.1-0.3 mL of H₂O₂ solution (1.5-15 mM) and 2.1-2.7 mL of buffer solution with different pH values (2.0-6.0) evenly, let it stand and incubate at room temperature for 10-20 min, then perform spectral scanning in the range of 400-800 nm through an ultraviolet-visible spectrophotometer, observe the ultraviolet absorbance at 652 nm, and analyze the effects of buffer solutions with different pH values, different PDA@Fe concentrations, and different concentrations of H₂O₂ solution and TMB solution on the peroxidase catalytic performance.

[0062] (4) Peroxidase kinetics of PDA@Fe microspheres

[0063] Mix the HAc-NaAc buffer solution (pH 2.0 - 6.0), TMB (0.1 - 0.8 mM), H2O2 (0.05 - 0.40 mM) and PDA@Fe (5 - 30 μg / mL) evenly, and record the UV-visible absorption spectrum at 652 nm every 30 s to track the reaction kinetics. Use the Lineweaver-Burk equation: V = V max ×[S] / (K m +[S]) to calculate the Michaelis-Menten constant (K m ), where V max is the maximum reaction rate; V corresponds to the initial rate; [S] is the concentration of the substrate; calculate this constant to evaluate the affinity of the nanozyme for the substrate.

[0064] (5) Stability analysis

[0065] To analyze the storage stability of the freeze-dried powder of PDA@Fe, measure the peroxidase activity stored in the refrigerator at 0 - 4 °C for 0 - 3 months respectively.

[0066] (6) Detection of nitrite

[0067] Detect the concentration of nitrite by monitoring the absorbance at the maximum values of two different characteristic wavelengths (λ = 652 and λ = 450) in the detection system. First, evenly mix the HAc-NaAc buffer solution (pH 2 - 6), TMB (0.1 - 1.0 mM), H2O2 (0.1 - 0.5 mM) and PDA@Fe microspheres (5 - 30 μg / mL), then add sodium nitrite solutions with different concentrations (5 - 100 μM) to the mixture respectively, and continue to react at room temperature for 10 - 30 min. Subsequently, measure the UV-visible spectrum using a spectrophotometer in the range of 400 - 750 nm. By measuring the absorbance values at 652 nm and 450 nm, plot the relationship diagram between the absorbance ratio (A 450 / A 652 ) and the concentration of sodium nitrite.

[0068] Test examples

[0069] 1. Preparation research of polydopamine microspheres (PDA)

[0070] Use Fourier transform infrared spectroscopy (FT-IR) to measure and analyze the structure of PDA nano-microspheres ( Figure 1 ). The broad peak at 3000 - 3500 cm -1 indicates the presence of O-H / N-H, and 3100 cm -1 is the absorption peak of C-H stretching vibration, mainly caused by the aromatic C-H vibration on the benzene ring. 1650 cm -1The absorption peak may be attributed to the vibration of quinone-type C=O or conjugated C=N, further confirming the oxidative polymerization of dopamine. The absorption peak at 800 cm -1 is assigned to the out-of-plane bending vibration of C-H in the ortho-substituted benzene ring. The positions and intensities of these infrared characteristic absorption peaks confirm the structural characteristics of the polydopamine microspheres. Meanwhile, the influence of different dopamine masses on the particle size of PDA microspheres was also tested, and the results are shown in Table 1.

[0071] Table 1 Influence of different dopamine masses on the particle size of PDA microspheres

[0072]

[0073] a The volume of ammonia water used was 3 mL, and the volume ratio of ethanol to water was 3:9;

[0074] b The polydispersity index is used to characterize the particle size distribution.

[0075] As shown in Table 1 and Figure 2 indicated, when the dosage of dopamine increased from 0.4 g to 0.7 g, the particle size of the microspheres increased from 155.51 nm to 757.30 nm, and the polydispersity index (PDI) increased. The above results show that the more dopamine is used, the larger the particle size of the polydopamine microspheres and the worse the dispersibility. When the dopamine dosage is 0.5 g, the particle size of the polydopamine microspheres is appropriate, and the uniformity and dispersibility are good. Therefore, a dopamine dosage of 0.5 g was selected in the subsequent research.

[0076] Subsequently, the influence of different ammonia water dosages on the particle size of dopamine microspheres was studied, and the results are shown in Table 2.

[0077] Table 2 Influence of different ammonia water dosages on the particle size of dopamine microspheres

[0078]

[0079] a The mass of dopamine used was 0.5 g, and the volume ratio of ethanol to water was 3:9;

[0080] b The polydispersity index is used to characterize the particle size distribution.

[0081] As shown in Table 2 and Figure 3 indicated, the more ammonia water is used, the higher the pH of the solution and the smaller the particle size of the polydopamine microspheres. Among them, ammonia water, as a catalyst for the solvent containing dopamine, can further improve the efficiency of synthesizing PDA nanoparticles in the water-ethanol mixture. Considering that smaller and more uniform particle sizes can increase peroxidase activity, 3 mL was selected as the optimal ammonia water dosage.

[0082] Subsequently, the effect of the volume ratio of ethanol to water on the particle size of the microspheres was studied, and the results are shown in Table 3.

[0083] Table 3 Effect of the volume ratio of ethanol to water on the particle size of the microspheres

[0084]

[0085] a The mass of dopamine used was 0.5 g, and the volume of ammonia water was 3 mL;

[0086] b The polydispersity index is used to characterize the particle size distribution.

[0087] Table 3 and Figure 4 show that when the volume ratio changes from 2:9 to 5:9, the particle size of the polydopamine microspheres changes little, only increasing slightly. However, when the volume ratio reaches 6:9, the particle size of the polydopamine microspheres increases significantly. Considering that smaller and more uniform particle sizes can increase peroxidase activity, the optimal volume ratio of ethanol to water was selected as 3:9.

[0088] As Figure 5 shown, the particle size of the polydopamine prepared under the electron microscope was about 150 nm, showing a spherical shape as a whole, without large agglomeration, and the particle size was relatively uniform, with good overall dispersibility. In summary, considering that the activity of peroxidase is related to the particle size, uniformity, and dispersibility, the optimal reaction conditions were selected as 0.5 g of dopamine, 3 mL of ammonia water, and a volume ratio of ethanol to ammonia water of 3:9.

[0089] 2. The preparation of PDA@Fe nanomicrospheres was studied.

[0090] Table 4 Zeta potential and particle size of PDA@Fe microspheres with different mass ratios of FeCl2 to PDA microspheres

[0091]

[0092] a The mass of dopamine used was 0.5 g, and the volume of ammonia water was 3 mL;

[0093] b The polydispersity index is used to characterize the particle size distribution.

[0094] As shown in Table 4, when the mass ratio of FeCl2 to PDA microspheres changes from 0:1 to 10:1, its zeta potential changes from -13.5 mV to 13.2 mV, and the particle size changes from 202.15 nm to 310.75 nm; from the above results, it can be seen that with the 2+ increase in the amount of Fe used, both the particle size and zeta potential of the PDA@Fe microspheres increase, because Fe 2+Chelated with the hydroxyl groups on the surface of PDA microspheres, introducing positive charges, increasing their potential and particle size. Considering the particle size, dispersibility of the microspheres, and the content of Fe 2+ , we selected the PDA@Fe microspheres prepared with a mass ratio of FeCl2 to PDA microspheres of 7:1 as the object of subsequent research.

[0095] As Figure 6 shown, the particle size of PDA@Fe microspheres under the electron microscope is about 250 nm, showing a spherical shape overall, with relatively uniform particle size, no large agglomeration phenomenon, and Fe 2+ attachment on the edge, indicating that Fe 2+ was successfully chelated onto the surface of PDA microspheres.

[0096] The XPS analysis results showed ( Figure 7 ), in addition to the obvious characteristic peaks of C, N, and O elements in the spectrum, characteristic peaks of Fe 2+ also appeared at 711.49 and 727.13 eV, and the peak signal intensity was strong, indicating that Fe 2+ was successfully chelated with PDA microspheres.

[0097] 3. Optimization of the peroxidase catalytic performance of PDA@Fe microspheres

[0098] As Figure 8 shown, buffer variable groups with different pH values (pH = 2.0, 3.0, 4.0, 5.0, 6.0) were prepared. Through ultraviolet spectrum analysis, the results showed that the ultraviolet absorbance at 652 nm was the largest at pH 3.0. Therefore, the acetic acid-sodium acetate buffer solution with pH 3.0 was selected as the buffer solution for subsequent experiments.

[0099] As Figure 9 shown, for PDA@Fe catalytic microspheres with different concentrations (5, 10, 15, 20, 30 μg / mL), the absorbance at 652 nm increased with the increase in the concentration of PDA@Fe. Therefore, the concentration of PDA@Fe was selected as 30 μg / mL for subsequent enzyme kinetic experiments.

[0100] It can be seen from Figure 10 that in the range of 0.05 - 0.45 mM H2O2 concentration, with the increase in the concentration of H2O2, the absorbance at 652 nm increased and showed a good linear relationship (A = 0.83505C + 0.13931, R 2 = 0.98). Therefore, 0.45 mM was selected as the optimal H2O2 concentration.

[0101] As Figure 11As shown, in the catalytic system, as the concentration of TMB increases, the absorbance also gradually rises, indicating that the more the content of TMB, the more the amount of TMB oxide generated under the enzymatic catalysis of PDA@Fe. When the TMB concentration increases from 0.05 mM to 0.8 mM, the ultraviolet absorbance at 652 nm within the same incubation time increases with the increase of TMB concentration, and there is a good linear relationship in the range of 0.05 - 0.6 mM.

[0102] 4. Peroxidase-catalyzed kinetic study of PDA@Fe microspheres

[0103] The results of enzymatic kinetics showed that the enzymatic kinetic analysis of PDA@Fe on H2O2 ( Figure 12 (a) and (b)) follows the typical Michaelis-Menten model. Therefore, the catalytic activity of PDA@Fe nanospheres on H2O2 was evaluated by the Michaelis-Menten model. Through calculation, the apparent K m value of PDA@Fe for H2O2 is 0.124 mM, and the V max value is 1.88×10 -8 M / s, indicating that PDA@Fe has good affinity for the substrate H2O2.

[0104] The results of enzymatic kinetics showed that the enzymatic kinetic analysis of PDA@Fe on TMB ( Figure 13 (a) and (b)) follows the typical Michaelis-Menten model. Therefore, the catalytic activity of the nanospheres (PDA@Fe) on TMB was evaluated by the Michaelis-Menten model. Through calculation, the apparent K m value of PDA@Fe for TMB is 1.39 mM, and the V max value is 1.69×10 -8 M / s, indicating that PDA@Fe has good affinity for the substrate TMB. Therefore, PDA@Fe has excellent peroxidase-like catalytic activity.

[0105] 5. Stability study of PDA@Fe nanospheres

[0106] Good stability is crucial for the application of nanozymes. Therefore, we tracked the peroxidase activity of PDA@Fe nanospheres with a mass ratio of FeCl2 to PDA microspheres of 7:1 for 1 week. The results showed that almost no activity change was observed ( Figure 14 ), and even on the 7th day, the relative activity was still close to 100%, indicating that PDA@Fe nanospheres have excellent stability.

[0107] 6. Detection and analysis of nitrite

[0108] The addition of nitrite consumes blue oxTMB, resulting in a decrease in the 652 nm signal; with the increase in nitrite concentration, the formation of diazotized oxTMB leads to a significant increase in absorbance at 450 nm. As Figure 15 shown, in the range of 0.005 - 0.1 mM, the A 450 / A 652 ratio shows a linear growth relationship with nitrite concentration (A 450 / A 652 = 14.71C + 0.2259, R 2 = 0.9977). This method has a low detection limit (1.0 μM), indicating that this method has high sensitivity for the quantitative detection of nitrite.

[0109] Example 2

[0110] This example is the best example of the present invention

[0111] Polydopamine-based iron-based nanozyme and its preparation method and application.

[0112] (1) Preparation of polydopamine microspheres

[0113] At room temperature of 25 °C, measure 3 mL of ammonia water (NH3·H2O) and 130 mL of a mixed solution of water and ethanol with a volume ratio of 3:9, add them to a 250 mL three-necked flask, and stir at 200 rpm for 30 min to fully mix them; then weigh 0.5 g of dopamine hydrochloride and dissolve it in 10 mL of water, inject it into the above alkaline water-ethanol mixed solution, and continue to stir and react at 200 rpm for 30 h after the solution turns completely black. After the reaction, centrifuge at 10000 rpm for 10 min, discard the supernatant, add an appropriate amount of water for washing and centrifugation, and repeat 3 times to obtain the final product. Collect the product in the centrifuge tube, freeze-dry to obtain a black powder, and store the obtained sample powder in a 4 °C refrigerator for standby.

[0114] (2) Preparation of PDA@Fe nanospheres

[0115] At room temperature of 25 °C, weigh 20 mg of polydopamine microspheres and 140 mg of FeCl2 powder, add them to a 50 mL three-necked flask containing 20 mL of ethanol solution, stir in an N2 atmosphere at 200 rpm to fully dissolve and mix them, and then react at room temperature for 6 h. After the reaction, centrifuge at 10000 rpm for 10 min, discard the supernatant, add an appropriate amount of water for washing and centrifugation, and repeat 3 times to remove ethanol and unreacted FeCl2 to obtain the final chelation product. After freeze-drying, obtain a black powder, and store the obtained sample powder in a 4 °C refrigerator for standby.

[0116] (3) Optimization of the peroxidase catalytic performance of PDA@Fe microspheres

[0117] ① Effect of buffer solutions with different pH values on catalytic activity

[0118] Mix 0.1 mL of PDA@Fe microsphere dispersion (900 μg / mL), 0.1 mL of TMB solution (30 mM), 0.1 mL of H2O2 solution (13.5 mM), and 2.7 mL of buffer solutions with different pH values (2.0 - 6.0) evenly. After standing and incubating at room temperature for 15 min, perform spectral scanning in the range of 400 - 800 nm using a UV-visible spectrophotometer, and observe the UV absorbance at 652 nm.

[0119] ② Effect of PDA@Fe microspheres with different concentrations on catalytic activity

[0120] Prepare variable groups of PDA@Fe microsphere dispersions with different concentrations (0.15 mg / mL, 0.3 mg / mL, 0.45 mg / mL, 0.6 mg / mL, 0.9 mg / mL). Mix 0.1 mL of PDA@Fe microsphere dispersions with different concentrations, 0.1 mL of 30 mM TMB solution, 0.1 mL of 13.5 mM H2O2, and 2.7 mL of acetate - sodium acetate buffer (pH 3.0) evenly. After standing at room temperature for 15 min, perform spectral scanning in the range of 400 - 800 nm using a UV-visible spectrophotometer, and observe the UV absorbance at 652 nm.

[0121] ③ Effect of H2O2 with different concentrations on enzyme catalytic activity

[0122] Prepare H2O2 solutions with different concentrations of 1.5 mM, 3 mM, 4.5 mM, 6 mM, 7.5 mM, 9 mM, 12 mM, and 13.5 mM respectively. Mix 0.1 mL of 900 μg / mL PDA@Fe microsphere dispersion, 0.1 mL of 30 mM TMB solution, 0.1 mL of H2O2 solutions with different concentrations, and 2.7 mL of acetate - sodium acetate buffer (pH 3.0) evenly. After standing at room temperature for 15 min, perform spectral scanning in the range of 400 - 800 nm using a UV-visible spectrophotometer, and observe the UV absorbance at 652 nm.

[0123] ④ Effect of TMB with different concentrations on enzyme catalytic activity

[0124] Prepare TMB solutions with different concentrations of 1.5 mM, 3 mM, 6 mM, 9 mM, 12 mM, 15 mM, 18 mM, and 24 mM respectively. Mix 0.1 mL of 900 μg / mL PDA@Fe microsphere dispersion, 0.1 mL of TMB solutions with different concentrations, 0.1 mL of 13.5 mM H2O2, and 2.7 mL of acetic acid-sodium acetate buffer (pH 3.0) evenly. After standing at room temperature for 15 min, perform spectral scanning in the range of 400 - 800 nm using a UV-visible spectrophotometer to observe the UV absorbance at 652 nm.

[0125] (4) Peroxidase-catalyzed kinetics of PDA@Fe microspheres

[0126] Mix HAc-NaAc buffer solution (pH = 3.0), TMB (0.1 - 0.8 mM), H2O2 (0.05 - 0.45 mM), and PDA@Fe (30 μg / mL) evenly, and record the UV-visible absorption spectrum at 652 nm every 30 s to track the reaction kinetics. Use the Lineweaver-Burk equation: V = V max ×[S] / (K m +[S]) to calculate the Michaelis-Menten constant (K m ), where V max is the maximum reaction rate; V corresponds to the initial rate; [S] is the concentration of the substrate; K m is the Michaelis-Menten constant, and this constant is obtained by calculation to evaluate the affinity of the nanozyme for the substrate.

[0127] (5) Stability analysis

[0128] To analyze the storage stability of the freeze-dried powder of PDA@Fe, measure the peroxidase activity stored in the refrigerator at 4°C for 0 - 3 months respectively.

[0129] (6) Detection of nitrite

[0130] Detect the concentration of nitrite by monitoring the absorbance at the maximum values of two different characteristic wavelengths (λ = 652 and λ = 450) in the detection system. First, mix HAc-NaAc buffer solution (pH = 3), TMB (0.8 mM), H2O2 (0.45 mM), and Fe / PDA microspheres (30 μg / mL) evenly, and then add sodium nitrite solutions with different concentrations (5 - 100 μM) to the mixture respectively and continue to react at room temperature for 20 min. Subsequently, measure the UV-visible spectrum in the range of 400 - 800 nm using a spectrophotometer. By measuring the absorbance values at 652 nm and 450 nm, plot the absorbance ratio (A 450 / A652 ) Relationship diagram with the concentration of sodium nitrite.

[0131] As can be seen from the above examples, through optimizing the preparation conditions, the present invention has successfully synthesized polydopamine microspheres with uniform particle size and excellent dispersibility, and combined with the Fe 2+ chelation process to prepare PDA@Fe nanozymes with controllable surface charge, high iron atom utilization rate and excellent peroxidase activity. The nanozymes exhibit catalytic activities significantly superior to traditional iron-based materials, have extremely strong affinities for H2O2 and TMB, high catalytic efficiency and a wide reaction linear range. Based on the ratio colorimetry method, trace detection of nitrite has been achieved, with high detection sensitivity and strong anti-interference ability. At the same time, the PDA@Fe nanozymes have the characteristics of excellent long-term stability, solving the problem that traditional iron-based nanozymes are easily oxidized and inactivated. In addition, the preparation process is simple and low-cost, suitable for large-scale production, and the detection process is fast and convenient. Therefore, the nanozymes can be widely applied in the fields of environmental monitoring, food safety and biomedical diagnosis, providing efficient and reliable solutions for on-site rapid analysis, and having significant industrial application prospects.

[0132] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Therefore, based on the innovative concept of the present invention, the changes and modifications made to the embodiments described herein, or the equivalent structural or equivalent process transformations made using the content of the specification of the present invention, directly or indirectly applying the above technical solutions to other related technical fields, are all included in the protection scope of the present invention patent.

Claims

1. A polydopamine-based iron-based nanozyme, characterized in that, The nanozyme is composed of polydopamine microspheres and ferrous ions through chelation reaction. The particle size of the polydopamine microspheres is 150 - 300 nm, and the nanozyme has peroxidase-like catalytic activity and can catalyze the color reaction of 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2).

2. The iron-based nanozyme according to claim 1, wherein The preparation method of the polydopamine microspheres includes the following steps: a. Add dopamine hydrochloride to a mixed solution of ammonia water, water and ethanol with a volume ratio of 2:9 - 6:9, and stir and react for 24 - 48 h; b. Centrifuge and separate the product, wash and freeze-dry to obtain polydopamine microspheres with uniform particle size.

3. The iron-based nanozyme according to claim 2, characterized in that, In step a, the dosage of dopamine hydrochloride is 0.4 - 0.7 g, the dosage of ammonia water is 1 - 4 mL, the reaction temperature is 20 - 25 °C, and the stirring rate is 150 - 300 rpm.

4. The iron-based nanozyme according to claim 1, characterized in that, In the chelation reaction, the mass ratio of polydopamine microspheres to ferrous chloride is 1:3 to 1:

10. The reaction is carried out in an ethanol solution, the reaction time is 4 - 8 h, and the reaction temperature is 20 - 25 °C.

5. A method for preparing the iron-based nanozyme according to any one of claims 1-4, characterized in that, It includes the following steps: a. Prepare polydopamine microspheres; b. Mix the polydopamine microspheres and ferrous chloride in an ethanol solution, stir and react under a nitrogen atmosphere, centrifuge, wash and then freeze-dry to obtain the iron-based nanozyme.

6. The preparation method according to claim 5, characterized in that, In step b, the dosage of polydopamine microspheres is 10 - 30 mg, the dosage of ferrous chloride is 10 - 300 mg, the volume of the ethanol solution is 10 - 50 mL, and the stirring rate is 150 - 300 rpm.

7. A method for detecting nitrite based on the iron-based nanozyme described in claim 1, characterized in that, It includes the following steps: a. Mix the iron-based nanozyme, TMB, H2O2 and buffer solution, and add the sample to be tested; b. Measure the absorbance ratio (A450 / A652) at 450 nm and 652 nm by ultraviolet-visible spectrophotometer, and quantitatively analyze the nitrite concentration according to the standard curve.

8. The detection method according to claim 7, wherein The buffer solution is acetic acid-sodium acetate buffer solution with pH 2.0 - 6.

0. The concentration of the iron-based nanozyme is 5 - 30 μg / mL, the concentration of H2O2 is 0.05 - 0.45 mM, and the concentration of TMB is 0.1 - 1.0 mM.

9. Use of the iron-based nanozyme according to claim 1 in environmental monitoring, food safety or biomedical detection, characterized in that, The application is based on the peroxidase-like catalytic activity of the nanozyme and its sensitivity to nitrite.

10. The application according to claim 9, characterized in that, The detection limit of the nitrite is 1.0 μM, the linear range is 5 - 100 μM, and the detection time is 10 - 30 min.

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