Nanoprotease with peroxidase-like activity, preparation method and application thereof

Abstract

The application discloses a nano-enzyme with peroxidase-like activity and a preparation method and application thereof. The nano-enzyme has characteristic peaks of Bi2O3 in an XRD pattern; and the nano-enzyme has characteristic peaks of Fe, C, Bi and O in an XPS full spectrum. The preparation method of the nano-enzyme comprises the following steps: (1) preparing FeBiMOFs by a solvothermal method; and (2) performing thermal activation treatment on the FeBiMOFs, so that the nano-enzyme is obtained. The preparation method is simple and easy to control. The heteroatom-doped Bi2O3-based nano-enzyme has great potential when used as a peroxidase mimic to detect organic phosphorus pesticides, and the application of Bi2O3 in the sensing field is expanded. The heteroatom-doped Bi2O3-based nano-enzyme is formed by accumulation of nanoparticles with a particle size of less than 10 nm, has a large specific surface area and multiple active sites for synergistic action. When applied to a colorimetric analysis method to detect AChE and organic phosphorus pesticides (OPs) such as DDVP, the nano-enzyme has the advantages of low detection limit, wide linear range and strong anti-interference ability, and has strong practicability.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials and peroxidase-like activity, and in particular to nanozymes with peroxidase-like activity, and preparation methods and applications thereof. Background Art

[0002] Nanozymes are a class of nanomaterials with intrinsic enzyme-like catalytic properties. Compared with natural enzymes, nanozymes have many excellent properties, including a wider range of operating conditions (such as temperature, pH, salt concentration), flexible design of structure and composition, and adjustable catalytic activity. In addition, nanozymes are easy to generate, transport and store. To date, many nanomaterials, such as carbon-based materials, metals, metal oxides, transition metal compounds and metal-organic frameworks (MOFs), have been shown to have intrinsic enzyme-like activity. Among them, metal oxides have become promising nanozymes due to their low cost, high stability, and diverse and tunable chemical properties.

[0003] In 2007, Gao et al. first reported that Fe3O4 NPs have intrinsic enzyme catalytic properties. Since then, many metal oxides such as Fe2O3, Mn3O4, Co3O4, ZnO, NiO, CuO, VOx, as well as CoFe2O4, MnFe2O4, MnCo2O4, NiCo2O4 have also been reported to have enzyme-like activities and have been used in many fields. Bismuth (Bi) is well-known for its environmentally friendly and harmless "green metal". As an important bismuth compound and semiconductor, bismuth oxide (Bi2O3) has received widespread attention and application in green catalysis, biomedicine, cosmetics and other fields. Bismuth-based oxides represented by Bi2O3 are well-known oxygen ion conductor solid electrolytes. Bi ions with 6s lone electron pairs can increase the overall oxygen vacancy mobility, thereby enabling the oxygen reduction (ORR) process to proceed at a rate of 4e - Based on this, bismuth-based oxides may have the effect of promoting peroxidase activity. However, there are currently no reports on the construction of nanozymes using bismuth-based oxides.

[0004] In recent years, many methods have been used to modulate and enhance the catalytic activity of nanozymes, and doping is one of the most proven and effective methods. After the introduction of heteroatoms into the nanozyme structure, these doped heteroatoms can promote the formation of catalytic centers, increase conductivity or carrier density, and / or generate additional active sites. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a nanozyme with peroxidase-like activity, a preparation method and an application thereof. The nanozyme is a heteroatom-doped Bi2O3-based nanocomposite material with excellent enzyme catalytic activity, a simple preparation method and a wide range of applications.

[0006] In order to achieve the above objectives, the present invention first provides a nanozyme with peroxidase-like activity, and the technical solution is as follows:

[0007] A nanozyme with peroxidase-like activity, wherein the XRD pattern of the nanozyme has a characteristic peak of Bi2O3; the full XPS spectrum of the nanozyme has characteristic peaks of Fe, C, Bi, and O.

[0008] As a further improvement of the nanozyme of the present invention, the nanozyme is in a three-dimensional flower shape and is composed of stacked nanoparticles with a particle size of ≤10 nm.

[0009] In order to achieve the above-mentioned object, the present invention further provides a method for preparing nanozymes, and the technical solution is as follows:

[0010] The preparation method of nanozyme comprises the following steps: (1) preparing FeBi MOFs by a solvent thermal method; and (2) performing a thermal activation treatment on the FeBi MOFs to obtain the nanozyme.

[0011] As a further improvement to the preparation method of the present invention, the preparation of FeBi MOFs specifically includes the following steps: obtaining a reaction solution comprising SDS, H2BDC, DMF, ethanol, a soluble bismuth salt, and a soluble iron salt; charging the reaction solution into a reactor and subjecting it to heat treatment to generate a solid-liquid mixture containing FeBi MOFs; and subjecting the solid-liquid mixture to solid-liquid separation, washing, and drying to obtain FeBi MOFs. Preferably, SDS and H2BDC are first dissolved in a mixed solvent of DMF and ethanol, and then the soluble bismuth salt and the soluble iron salt are added and dissolved to obtain a reaction solution; the volume ratio of DMF to ethanol is (2-4):1; the concentration ratio of SDS, H2BDC, and the soluble salt in the reaction solution is (1.5-2.5):(1.5-2.5):1; the molar ratio of the soluble iron salt to the soluble salt is 15-30%; the heat treatment temperature is 110-130°C and the heat treatment time is 10-14 hours; and the mixture is washed with DMF and ethanol.

[0012] As a further improvement to the preparation method of the present invention, the thermal activation temperature is 350-400° C., and the thermal activation time is 1.5-3 hours.

[0013] In order to achieve the above object, the present invention further provides a method for detecting the concentration of AChE and a method for detecting the concentration of DDVP, and the technical solutions are as follows:

[0014] The method for detecting AChE concentration includes the following steps: adding ACh, CHOx, a chromogenic substrate and a catalyst to a test liquid, wherein the catalyst is the above-mentioned nanozyme or a nanozyme prepared by the above-mentioned preparation method; then testing the absorbance of the liquid generated by the reaction; substituting the absorbance into a linear equation of absorbance and AChE concentration to obtain the AChE concentration in the test liquid.

[0015] As a further improvement of the method for detecting AChE concentration of the present invention, the test solution, ACh, and CHOx are incubated, quenched after the incubation is completed, and then a color-developing substrate and a catalyst are added for incubation.

[0016] The method for detecting DDVP concentration includes the following steps: adding AChE, ACh, CHOx, a chromogenic substrate, and a catalyst to a test liquid, wherein the catalyst is the above-mentioned nanozyme or a nanozyme prepared by the above-mentioned preparation method; then testing the absorbance of the liquid generated by the reaction; substituting the absorbance into a linear equation between the activity inhibition rate of AChE and the DDVP concentration to obtain the DDVP concentration in the test liquid.

[0017] As a further improvement of the DDVP concentration detection method of the present invention, the test solution is incubated with AChE for a period of time, and then ACh and CHOx are added for incubation. After the incubation is completed, quenching is performed, and then a chromogenic substrate and a catalyst are added for incubation.

[0018] This invention utilizes a simple, easily controllable preparation process to produce a heteroatom-doped Bi2O3-based nanozyme with excellent peroxidase-like activity. This nanozyme demonstrates great potential as a peroxidase mimic for the detection of organophosphorus pesticides, expanding the application of Bi2O3 in sensing. This heteroatom-doped Bi2O3-based nanozyme, composed of nanoparticles ≤10 nm in size, exhibits a large surface area and multiple synergistic active sites. When used in colorimetric assays for the detection of AChE and organophosphorus pesticides (OPs) such as DDVP, it demonstrates low detection limits, a wide linear range, and strong anti-interference capabilities, demonstrating its exceptional practicality.

[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings that constitute a part of the present invention are used to assist in understanding the present invention. The contents provided in the drawings and their related descriptions in the present invention can be used to explain the present invention, but do not constitute improper limitations on the present invention.

[0021] Figure 1UV-visible absorption spectra of the oxidation of three chromogenic substrates catalyzed by Fe / C / Bi2O3 and H2O2.

[0022] Figure 2 The relative activity of Fe / C / Bi2O3 and H2O2 in catalyzing TMB oxidation increases with M 3+ / SDS change curve.

[0023] Figure 3 The relative activity of Fe / C / Bi2O3 and H2O2 in TMB oxidation is affected by the Fe 3+ / (Bi 3+ +Fe 3+ ) change curve.

[0024] Figure 4 The curve showing the relative activity of Fe / C / Bi2O3 and H2O2 catalyzing TMB oxidation as a function of thermal activation temperature.

[0025] Figure 5 The UV-visible spectrum of TMB oxidation catalyzed by Fe / C / Bi2O3 and H2O2 changes with the concentration of Fe / C / Bi2O3.

[0026] Figure 6 Relative activities of different batches of Fe / C / Bi2O3 and H2O2 in catalyzing TMB oxidation.

[0027] Figure 7 Relative activities of Fe / C / Bi2O3 and H2O2 for TMB oxidation catalyzed by different storage times.

[0028] Figure 8 The absorbance curve of TMB oxidation in the presence / absence of Fe, Bi, H2O2 and Fe / C / Bi2O3 changes with time.

[0029] Figure 9 UV-visible absorption spectra of TMB oxidation catalyzed by Fe / C / Bi2O3 in the presence of different H2O2 concentrations.

[0030] Figure 10 This is the curve of absorbance changing with ACh concentration when Fe / C / Bi2O3 detects AChE.

[0031] Figure 11 This is the curve of △A changing with CHOx concentration when Fe / C / Bi2O3 detects AChE.

[0032] Figure 12 This is the curve of the absorbance change with the first incubation time when Fe / C / Bi2O3 was used to detect AChE.

[0033] Figure 13This is the curve of the UV-visible spectrum of Fe / C / Bi2O3 detecting AChE as the AChE concentration changes.

[0034] Figure 14 This is the linear calibration graph of absorbance and AChE concentration when Fe / C / Bi2O3 is used to detect AChE.

[0035] Figure 15 This is the curve of the UV-visible spectrum of Fe / C / Bi2O3 detecting DDVP as the DDVP concentration changes.

[0036] Figure 16 This is the linear calibration diagram of IR and DDVP concentration when Fe / C / Bi2O3 is used to detect DDVP.

[0037] Figure 17 These are the anti-interference test results for Fe / C / Bi2O3 in detecting DDVP.

[0038] Figure 18 XRD patterns of Bi MOFs and FeBi MOFs.

[0039] Figure 19 Infrared spectra of H2BDC, Bi MOFs and FeBi MOFs.

[0040] Figure 20 Microscopic morphologies of FeBi MOFs and Fe / C / Bi2O3.

[0041] Figure 21 XRD patterns of FeBi MOFs and Fe / C / Bi2O3 prepared at different thermal activation temperatures.

[0042] Figure 22 This is the XRD pattern of Fe / C / Bi2O3 prepared at 350℃.

[0043] Figure 23 The infrared spectra of FeBi MOFs and Fe / C / Bi2O3 prepared at different thermal activation temperatures.

[0044] Figure 24 Raman spectra of FeBi MOFs and Fe / C / Bi2O3 prepared at different thermal activation temperatures.

[0045] Figure 25 This is the full XPS spectrum of Fe / C / Bi2O3.

[0046] Figure 26 High-resolution fine spectra of Bi 4f, Fe 2p, O 1s, and C 1s in Fe / C / Bi2O3. DETAILED DESCRIPTION

[0047] The present invention is described clearly and completely below with reference to the accompanying drawings. A person skilled in the art will be able to implement the present invention based on these descriptions. Before describing the present invention with reference to the accompanying drawings, it should be noted that:

[0048] The technical solutions and technical features provided in each part of the present invention, including the following description, may be combined with each other unless there is any conflict.

[0049] In addition, the embodiments of the present invention described below are generally only part of the embodiments of the present invention, rather than all of the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making any creative efforts should fall within the scope of protection of the present invention.

[0050] Regarding the terms and units in the present invention: The terms "include", "have" and any variations thereof in the description of the present invention are intended to cover non-exclusive inclusions.

[0051] The specific embodiments of the nanozyme having peroxidase-like activity and the preparation method thereof of the present invention are as follows:

[0052] The method for preparing the nanozyme having peroxidase-like activity comprises the following steps:

[0053] (1) Preparation of FeBi MOFs by solvothermal method;

[0054] First, sodium dodecyl sulfate (SDS) and terephthalic acid (H2BDC) were dissolved in a mixed solvent of N,N-dimethylformamide (DMF) and ethanol, with the volume ratio of DMF to ethanol being 3:1 and the concentration ratio of SDS to H2BDC being 1:1. Bi(NO3)3·5H2O and Fe(NO3)3·9H2O were then added and dissolved to form a light yellow solution, thereby obtaining a reaction solution. The mixture was stirred for 30 minutes, and then placed in an autoclave for a solvothermal reaction at a temperature of 120°C for 12 hours, thereby generating a solid-liquid mixture containing FeBi MOFs. The solid-liquid mixture was then subjected to solid-liquid separation, washed with DMF and ethanol three times, and dried at 60°C overnight to obtain FeBi MOFs.

[0055] The concentration ratio of soluble salt (i.e., the sum of Bi(NO3)3·5H2O and Fe(NO3)3·9H2O) and SDS in the reaction solution (expressed as M 3+ / SDS) is 1: (1.5-2.5); the molar ratio of Fe(NO3)3·9H2O to soluble salts (expressed as Fe 3+ / (Bi3+ +Fe 3+ )) is 15-30%.

[0056] (2) FeBi MOFs are thermally activated to obtain nanozymes.

[0057] The dried FeBi MOFs were placed in a quartz boat, heated to 350-400°C at a rate of 3°C / min in a tube furnace, kept warm for 2 hours, and then naturally cooled to room temperature to obtain nanozymes, represented by Fe / C / Bi2O3.

[0058] In order to illustrate the beneficial effects of the present invention, two control examples were set up, which differ from the above-mentioned specific embodiments only in the raw materials; in one control example, Fe(NO3)3·9H2O was used to replace the two soluble salts, and the thermally activated product was expressed as C / FeOx; in the other control example, Bi(NO3)3·5H2O was used to replace the two soluble salts, the thermally activated precursor was expressed as BiMOFs, and the thermally activated product was expressed as C / Bi2O3.

[0059] (1) First, the peroxidase-like properties of Fe / C / Bi2O3 were tested using a typical chromogenic substrate and H2O2 colorimetric reaction system. The selected chromogenic substrates were 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS). The resulting reaction system was incubated at 37°C for 15 minutes and the absorbance at 652 nm was measured. The absorbance was measured using an Ocean Optics USB4000 spectrophotometer.

[0060] Figure 1 UV-Vis absorption spectra of the oxidation of three chromogenic substrates catalyzed by Fe / C / Bi2O3 and H2O2. The reaction system consisted of Fe / C / Bi2O3 (20 mg / L), H2O2 (0.1 mM), chromogenic substrate (0.2 mM), and HAc-NaAc buffer (pH 3.5, 0.2 M).

[0061] like Figure 1 As shown, Fe / C / Bi2O3 can catalyze the oxidation of TMB, OPD and ABTS in the presence of H2O2, producing corresponding UV-visible absorption spectra and characteristic colors, exhibiting peroxidase-like activity.

[0062] Figure 2 The relative activity of Fe / C / Bi2O3 and H2O2 in catalyzing TMB oxidation increases with M 3+ / SDS change curve. Figure 3 The relative activity of Fe / C / Bi2O3 and H2O2 in TMB oxidation is affected by the Fe 3+ / (Bi 3+ +Fe 3+ ) change curve. Figure 4 The relative activity of Fe / C / Bi2O3 and H2O2 for TMB oxidation catalyzed by Fe / C / Bi2O3 and H2O2 varies with thermal activation temperature. The reaction system consisted of Fe / C / Bi2O3 (20 mg / L), H2O2 (0.1 mM), TMB (0.2 mM), and HAc-NaAc buffer (pH 3.5, 0.2 M). Relative activity is the ratio of absorbance to the maximum absorbance in the same assay.

[0063] like Figure 2-4 As shown, when M 3+ / SDS is 1: (1.5~2.5), Fe 3+ / (Bi 3+ +Fe 3+ ) ranges from 15% to 30% and the thermal activation temperature is 350°C, achieving the highest relative activity, indicating that Fe / C / Bi2O3 achieves maximum catalytic activity at this temperature. The following experiments (unless otherwise specified) utilize Fe / C / Bi2O3 prepared using the optimal process parameters.

[0064] Figure 5 The UV-Vis spectra of TMB oxidation catalyzed by Fe / C / Bi2O3 and H2O2 vary with Fe / C / Bi2O3 concentration. The reaction system consists of Fe / C / Bi2O3 (0-60 mg / L), H2O2 (0.1 mM), TMB (0.2 mM), and HAc-NaAc buffer (pH 3.5, 0.2 M).

[0065] like Figure 5 As shown, under the experimental conditions, the absorbance of TMB oxidation catalyzed by Fe / C / Bi2O3 and H2O2 increased with the increase of Fe / C / Bi2O3 concentration.

[0066] Figure 6 Relative activities of different batches of Fe / C / Bi2O3 and H2O2 in catalyzing TMB oxidation. Figure 7 Relative activities of Fe / C / Bi2O3 and H2O2 for TMB oxidation catalyzed by different storage times.

[0067] like Figure 6-7 As shown, there was no significant change in Fe / C / Bi2O3 after 90 days of storage or between six different batches, demonstrating good long-term storage stability and reproducibility.

[0068] Figure 8The absorbance curves for TMB oxidation in the presence and absence of Fe, Bi, H₂O₂, and Fe / C / Bi₂O₃ are plotted as a function of time. The reaction system consisted of the catalyst (0 or 20 mg / L), H₂O₂ (0 or 0.1 mM), TMB (0.2 mM), and HAc-NaAc buffer (pH 3.5, 0.2 M). The catalysts used were Fe / C / Bi₂O₃, C / FeOx, and C / Bi₂O₃.

[0069] like Figure 8 As shown, the absorbance at 652 nm of the reaction system composed of Fe / C / Bi2O3+TMB+H2O2 is significantly higher than that of TMB+H2O2 and TMB+Fe / C / Bi2O3 in a time-dependent manner, demonstrating the intrinsic peroxidase-like activity of Fe / C / Bi2O3. The absorbance changes at 652 nm of the thermally activated products without Fe (i.e., C / Bi2O3) and without Bi (i.e., C / FeOx) are negligible, indicating that Fe doping and the crystal structure of the thermally activated precursor play a key role in enhancing the peroxidase-like activity of Fe / C / Bi2O3.

[0070] Figure 9 The UV-Vis absorption spectra of TMB oxidation catalyzed by Fe / C / Bi2O3 in the presence of different H2O2 concentrations were shown. The reaction system consisted of Fe / C / Bi2O3 (20 mg / L), H2O2 (0-0.6 mM), TMB, and HAc-NaAc buffer.

[0071] like Figure 9 As shown, the absorbance of TMB oxidation catalyzed by Fe / C / Bi2O3 and H2O2 increased with the increase of H2O2 concentration.

[0072] (2) Then, Fe / C / Bi2O3 prepared with the optimal process parameters was used to detect acetylcholinesterase (AChE), as follows:

[0073] The detection principle is as follows: AChE can enzymatically hydrolyze acetylcholine chloride (ACh) to produce choline, choline oxidase (CHOx) hydrolyzes choline to form H2O2, Fe / C / Bi2O3 catalyzes H2O2 to produce highly active ·OH, and ·OH oxidizes TMB to produce blue oxTMB (a single-electron oxidation product of TMB). The absorbance of the reaction system containing oxTMB can be used to reflect the AChE concentration in the test solution.

[0074] The assay method involves adding ACh and CHOx to a test solution containing AChE. The reaction is initially incubated at 37°C, followed by quenching. Fe / C / Bi2O3 and TMB are then added and incubated for 15 minutes. The absorbance of the resulting reaction system containing oxTMB is then measured. To stabilize the reaction system, Tris-HCl buffer (pH 7.4) is used. The reaction is then quenched with acetate buffer (HAc-NaAc, pH 3.5).

[0075] The test results are as follows:

[0076] Figure 10 This is the curve of absorbance changing with ACh concentration when Fe / C / Bi2O3 detects AChE. Figure 11 This is the curve of the change of △A (the difference between the absorbance of the reaction system containing AChE and the absorbance without AChE) when Fe / C / Bi2O3 was used to detect AChE as a function of CHOx concentration. Figure 12 The absorbance curve for the detection of AChE by Fe / C / Bi2O3 as a function of the first incubation time is shown. The reaction system, with a volume of 1000 μL, consisted of 10 μL of test solution (AChE concentration: 20 mU / mL), 184 μL of Tris-HCl buffer, 10 μL of ACh solution (10 mM), 16 μL of CHOx solution (2.5 U / mL), 740 μL of HAc-NaAc buffer, 20 μL of Fe / C / Bi2O3 dispersion (1 mg / mL), and 20 μL of TMB solution (10 mM).

[0077] like Figure 10-12 As shown in the figure, the optimal conditions for Fe / C / Bi2O3 to detect AChE are to make the ACh concentration in the reaction system 100 μM, the CHOx concentration 40 mU / mL, and incubate for 40 min.

[0078] Figure 13 This is the curve of the UV-visible spectrum of Fe / C / Bi2O3 detecting AChE as the AChE concentration changes. Figure 14 The linear calibration plot for the detection of AChE by Fe / C / Bi2O3 versus AChE concentration is shown. The reaction volume was 1000 μL, consisting of 10 μL of test solution (AChE concentration ranged from 0 to 10 mU / mL), 184 μL of Tris-HCl buffer, 10 μL of ACh solution (10 mM), 16 μL of CHOx solution (2.5 U / mL), 740 μL of HAc-NaAc buffer, 20 μL of Fe / C / Bi2O3 dispersion (1 mg / mL), and 20 μL of TMB solution (10 mM).

[0079] like Figure 13-14As shown, the suitable linear range of Fe / C / Bi2O3 for detecting AChE is 0.002-1 mU / mL (concentration in reaction system), which meets the regression equation y=0.2540 x +0.3512, R 2 =0.9957, indicating that the colorimetric method based on Fe / C / Bi2O3 has great potential in the determination of AChE concentration.

[0080] (3) Further, Fe / C / Bi2O3 prepared by using the optimal process parameters was used to detect DDVP (dichlorvos), and the detection was as follows:

[0081] The detection principle is as follows: on the basis of the above detection principle of AChE, since DDVP as a typical OP can effectively inhibit the activity of AChE, and finally inhibit the generation of oxTMB, therefore, the absorbance of the reaction system containing oxTMB can also be detected to reflect the concentration of DDVP in the solution to be detected.

[0082] The detection method is as follows: AChE is added to the solution to be detected containing DDVP, and incubated at 37°C for 30 min, then ACh and CHOx are added for further incubation, and after incubation for 40 min, quenching is carried out, then Fe / C / Bi2O3 and TMB are added for further incubation for 15 min, and the absorbance of the reaction system containing oxTMB is tested. In order to make the reaction system more stable, Tris-HCl buffer (pH 7.4) is used. HAc-NaAc buffer (pH 3.5) is used for quenching.

[0083] The detection results are as follows:

[0084] Figure 15 The curve of the ultraviolet-visible spectrum of Fe / C / Bi2O3 for detecting DDVP with the change of the concentration of DDVP. Figure 16The linear calibration plot of the IR versus DDVP concentration for Fe / C / Bi2O3 detection was shown. The experimental reaction system was 1000 μL and consisted of 10 μL of test solution (DDVP concentration ranged from 0 to 100 mg / L), 10 μL of AChE solution (100 mU / mL), 174 μL of Tris-HCl buffer, 10 μL of ACh solution (10 mM), 16 μL of CHOx solution (2.5 U / mL), 740 μL of HAc-NaAc buffer, 20 μL of Fe / C / Bi2O3 dispersion (1 mg / mL), and 20 μL of TMB solution (10 mM). The DDVP concentration in the reaction system ranged from 0 to 1 mg / L. IR is the AChE activity inhibition rate, IR=(A1−A) / (A1−A0)×100, where A0 is the absorbance of the reaction system without the addition of AChE and DDVP, A1 is the absorbance of the reaction system without the addition of DDVP, and A is the absorbance of the experimental group.

[0085] like Figure 15-16 As shown in the figure, there are two linear relationships between IR and DDVP concentration (concentration in the reaction system). When the DDVP concentration is between 0.60 and 10 μg / L, y1=2.57x+2.66, R 2 =0.9926, when the DDVP concentration is between 10 and 100 μg / L, y2=0.62x+21.03, R 2 =0.9985. The detection limit was 0.6 μg / L, indicating that the low-concentration AChE and CHOx enzymatic cascade reaction system constructed by Fe / C / Bi2O3 of the present invention can achieve ultrasensitive analysis of DDVP.

[0086] Figure 17 The results of the anti-interference test of Fe / C / Bi2O3 for detecting DDVP. The volume of the blank group's reaction system was 1000 μL, consisting of 10 μL of test solution (DDVP concentration was 0.1 mg / L), 10 μL of AChE solution (100 mU / mL), 174 μL of Tris-HCl buffer, 10 μL of ACh solution (10 mM), 16 μL of CHOx solution (2.5 U / mL), 740 μL of HAc-NaAc buffer, 20 μL of Fe / C / Bi2O3 dispersion (1 mg / mL) and 20 μL of TMB solution (10 mM). The reaction system of the experimental group was the same as that of the blank group, with the addition of KCl at a 100-fold DDVP concentration. + 、Na + 、Al 3+ 、Fe 3+ 、Fe 2+ 、Mn 2+ 、NO3 - or Cl -, 20 times the concentration of DDVP Ca 2+ Mg 2+ 、Zn 2 + 、SO4 2- Br - 、F - , I - or HCO3 - , 5 times the concentration of DDVP CO3 2- .

[0087] like Figure 17 As shown, the enzymatic cascade reaction system formed by the present invention for detecting DDVP concentration exhibits good anti-interference ability to different anions and cations.

[0088] The standard addition method was used to measure DDVP levels in Fuhe River water and tap water. After pre-treating actual water samples with a 0.22 μm filter membrane, DDVP was spiked at an accurate concentration. DDVP concentrations and recoveries were calculated using a standard curve. As shown in Table 1, the DDVP concentrations measured in the water samples matched the spiked values ​​well, with recoveries ranging from 91.3% to 104.5%. These results demonstrate that the Fe / C / Bi2O3 system presented in this invention also exhibits good accuracy in measuring DDVP concentrations in actual water samples.

[0089] Table 1

[0090]

[0091] The raw materials, thermally activated precursors, and products were characterized using a field emission electron scanning microscope (JSM 7800F Prime, JEOL Ltd.), a transmission electron microscope (JEOL JEM-F 200 (HR), JEOL Ltd.), an X-ray diffractometer (MiniFlex 600, Rigaku Corporation), an X-ray photoelectron spectroscopy analyzer (Thermo Scientific K-Alpha, Thermo Fisher Scientific), an infrared spectrometer (Specturm 2, PerkinElmer Instruments), and a confocal Raman spectrometer (LabRAM HREvolution, HORIBA Group, Japan). All thermally activated precursors and products, unless otherwise noted, were prepared using the optimized process parameters described above.

[0092] Figure 18 XRD patterns of Bi MOFs and FeBi MOFs. Figure 19 Infrared spectra of H2BDC, Bi MOFs and FeBi MOFs.

[0093] like Figure 18As shown in Figure 2, the XRD patterns of Bi MOFs and FeBi MOFs show characteristic diffraction peaks at 9.3°, 10.1°, 16.54° and 20.34°, indicating the formation of highly crystalline MOF structures. Figure 19 As shown, compared with the infrared spectrum of H2BDC, the characteristic absorption peak of carboxylic acid in BiMOFs (-COOH, 1693 cm −1 ) obviously decays, while the carboxyl group (-COO-) at 1367 cm −1 and 1567cm −1 New symmetric and asymmetric vibration absorption peaks appear at the FeBi MOFs and Bi MOFs have similar XRD patterns and infrared spectra, indicating that the addition of a small amount of Fe 3+ The ions do not affect the structure of Bi MOFs.

[0094] Figure 20 Figure 3 is a microscopic morphology of FeBi MOFs and Fe / C / Bi2O3. (a) and (b) are low-magnification and high-magnification SEM images of FeBi MOFs, (c) and (d) are low-magnification and high-magnification SEM images of Fe / C / Bi2O3, and (e) and (f) are low-magnification and high-magnification TEM images of Fe / C / Bi2O3, respectively.

[0095] like Figure 20 As shown, FeBi MOFs are composed of smooth nanosheets stacked into a three-dimensional flower-like structure. Fe / C / Bi2O3, similar in shape to FeBi MOFs, also exhibits a three-dimensional flower-like structure, but lacks the smooth surface of nanosheets and instead consists of stacked nanoparticles ≤10 nm. While FeBi MOFs have a smooth surface, Fe / C / Bi2O3 exhibits numerous pores, confirming the formation of a porous structure during thermal activation. Elemental mapping analysis confirmed that Bi, Fe, C, and O are uniformly distributed throughout the Fe / C / Bi2O3 catalyst.

[0096] Figure 21 XRD patterns of FeBi MOFs and Fe / C / Bi2O3 prepared at different thermal activation temperatures. Figure 22 This is the XRD pattern of Fe / C / Bi2O3 prepared at 350℃. Figure 23 The infrared spectra of FeBi MOFs and Fe / C / Bi2O3 prepared at different thermal activation temperatures. Figure 24 Raman spectra of FeBi MOFs and Fe / C / Bi2O3 prepared at different thermal activation temperatures.

[0097] like Figure 21-22As shown, with increasing calcination temperature, the characteristic diffraction peaks of the precursor gradually decay and eventually disappear, followed by the appearance of numerous new characteristic peaks. The characteristic peaks of the Fe / C / Bi2O3 obtained at 250°C are essentially consistent with those of FeBiMOFs but significantly lower, indicating that the MOFs' crystal structure is destroyed by partial oxidation to form Bi2O3. The characteristic peaks of the Fe / C / Bi2O3 obtained at 350°C, although weak, align with those of γ-Bi2O3 (JCPDS No. 74-1375), confirming the presence of Bi2O3 crystals within the Fe / C / Bi2O3. The Fe / C / Bi2O3 obtained at 450°C exhibits strong diffraction peaks consistent with those of β-Bi2O3 (JCPDS No. 78-1793), indicating the formation of a pure β-Bi2O3 phase.

[0098] like Figure 23 As shown in the figure, with the increase of thermal activation temperature, a peak at 835 cm −1 The δ(OH) band of M-OH-M at 744 cm −1 The ν(CH) bond of benzene gradually disappears. Figure 24 As shown in Figure 2, the typical Raman signal of the 1,4-phthalate organic bridge in FeBi MOFs gradually disappears, which also confirms that the precursor is gradually oxidized. From the infrared and Raman spectra, it can be seen that there are still some carboxyl groups (-COO-) derived from organic ligands in Fe / C / Bi2O3, which are effective binding sites for substrates, especially H2O2.

[0099] Figure 25 This is the full XPS spectrum of Fe / C / Bi2O3. Figure 26 High-resolution fine spectra of Bi 4f, Fe 2p, O 1s, and C 1s in Fe / C / Bi2O3.

[0100] like Figure 25 As shown in Figure 3, the full XPS spectrum of Fe / C / Bi2O3 has characteristic peaks of Fe, C, Bi, and O, indicating that Fe / C / Bi2O3 is mainly composed of Bi, Fe, C, and O elements.

[0101] Bi 4f spectrum at 160.0 eV (Bi 4f 7 / 2 ) and 165.31eV (Bi 4f 5 / 2 ) shows two main peaks with a spin-orbit splitting of 5.31 eV, which corresponds to Bi 3+ 157.88eV(Bi 4f 7 / 2 ) and 162.68eV(Bi 4f 5 / 2 ) has a spin-orbit splitting of 4.8 eV, which is similar to that of Bi 0The peaks are consistent. The Fe 2p spectrum has two peaks at 711.08 and 725.38 eV, corresponding to Fe 3+ 2p 3 / 2 and 2p 1 / 2 states of the species. Meanwhile, the two low intensity peaks at 717.58 and 731.28 eV are typical Fe 3+ satellite peaks. The O 1s spectrum has three peaks at 529.8, 531.8 and 536.8 eV, corresponding to lattice oxygen (Bi-O), surface oxygen (Bi-OH) and adsorbed oxygen, respectively. The C 1s spectrum contains three peaks at 282.5, 284.8 and 288.0 eV, which can be attributed to C* (a low energy carbon species), C-C / C=C and carbon atom bonded to oxygen atom.

[0102] The above description of the present application has been provided. A person of ordinary skill in the art will be able to implement the present application based on the above description. All other embodiments obtained by a person of ordinary skill in the art without creative labor based on the above description of the present application shall fall within the scope of protection of the present application.

Claims

1. A nanozyme having peroxidase-like activity, characterized in that: The nanozyme's XRD pattern has a characteristic peak of γ-Bi2O3; the nanozyme's full XPS spectrum has characteristic peaks of Fe, C, Bi, and O; The nanozyme preparation method includes the following steps: obtaining a reaction solution comprising SDS, H2BDC, DMF, ethanol, a soluble bismuth salt, and a soluble iron salt; The reaction solution is placed in a reactor for heat treatment to generate a solid-liquid mixture containing FeBi MOFs; After solid-liquid separation, washing and drying of the solid-liquid mixture, FeBi MOFs are obtained; The FeBi MOFs were thermally activated at 350°C to obtain nanozymes, which were composed of nanoparticles with a size of ≤10 nm. Among them, FeBi MOFs are composed of smooth nanosheets stacked into a three-dimensional flower shape.

2. The method for preparing the nanozyme according to claim 1, wherein: The following steps are involved: (1) Preparation of FeBi MOFs by solvothermal method; (2) The FeBi MOFs were thermally activated at 350°C to obtain a nanozyme; the nanozyme had a characteristic peak of γ-Bi2O3 in its XRD spectrum; and the nanozyme was composed of nanoparticles with a particle size of ≤10 nm. The preparation of FeBi MOFs specifically includes the following steps: obtaining a reaction solution comprising SDS, H2BDC, DMF, ethanol, a soluble bismuth salt, and a soluble iron salt; The reaction solution is placed in a reactor for heat treatment to generate a solid-liquid mixture containing FeBi MOFs; After solid-liquid separation, washing and drying of the solid-liquid mixture, FeBi MOFs are obtained; FeBi MOFs are stacked into a three-dimensional flower shape by smooth nanosheets.

3. The preparation method according to claim 2, wherein: First, SDS and H2BDC are dissolved in a mixed solvent of DMF and ethanol, and then a soluble bismuth salt and a soluble iron salt are added and dissolved to obtain a reaction solution; the volume ratio of DMF and ethanol is (2-4):1; the concentration ratio of SDS, H2BDC and soluble salt in the reaction solution is (1.5-2.5):(1.5-2.5):1; the molar ratio of soluble iron salt to soluble salt is 15-30%; the heat treatment temperature is 110-130°C, and the heat treatment time is 10-14 hours; and DMF and ethanol are used for washing.

4. The preparation method according to claim 2, wherein: The thermal activation time is 1.5 to 3 hours.

5. A method for detecting AChE concentration, characterized in that: Including steps: Adding ACh, CHOx, a chromogenic substrate and a catalyst to the test solution, wherein the catalyst is the nanozyme according to claim 1 or the nanozyme prepared by the preparation method according to any one of claims 2 to 4; Then test the absorbance of the liquid generated by the reaction; Substituting the absorbance into the linear equation of absorbance and AChE concentration, the AChE concentration in the test solution is obtained.

6. The detection method according to claim 5, wherein: The test solution, ACh, and CHOx are incubated, and after the incubation is completed, the solution is quenched, and then a color development substrate and a catalyst are added for incubation.

7. A method for detecting DDVP concentration, characterized in that: Including steps: AChE, ACh, CHOx, a chromogenic substrate, and a catalyst are added to the test solution, wherein the catalyst is the nanozyme according to claim 1 or the nanozyme prepared by the preparation method according to any one of claims 2 to 4; Then test the absorbance of the liquid generated by the reaction; Substitute the absorbance into the linear equation of AChE activity inhibition rate and DDVP concentration to obtain the DDVP concentration in the test solution.

8. The detection method according to claim 7, wherein: The test solution and AChE are incubated for a period of time, and then ACh and CHOx are added for incubation. After the incubation is completed, the solution is quenched, and then a color-developing substrate and a catalyst are added for incubation.

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