High-entropy alloy nano-enzyme, biosensor as well as preparation method and application of high-entropy alloy nano-enzyme and biosensor

Abstract

The invention discloses a high-entropy alloy nano-enzyme PDA-HEA with peroxidase-like activity, a biosensor based on the PDA-HEA as well as a preparation method and application of the high-entropy alloy nano-enzyme PDA-HEA. The preparation method of the high-entropy alloy nano-enzyme PDA-HEA comprises the following steps: S1, preparing a PtRuFeCoNi HEA nanowire by a low-temperature oil phase method, and S2, synthesizing PDA-HEA: adding the PtRuFeCoNi HEA nanowire into a solvent B for dispersing, adding dopamine and a Tris-HCl solution into the solvent B, stirring until the color of the solution becomes black, and centrifuging to obtain a precipitate, namely PDA-HEA. The high-entropy alloy nano enzyme PDA-HEA or a biosensor based on PDA-HEA can be applied to detection of acetylcholin esterase, amino-acid ester pesticides and organophosphorus pesticides, and is convenient to operate and high in detection sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry, and in particular to a high entropy alloy nanozyme, a biosensor, and a preparation method and application thereof. Background Art

[0002] Carbamate drugs are a class of neurotoxins that inhibit the activity of acetylcholinesterase, preventing the neurotransmitter acetylcholine from being decomposed, leading to neuromuscular conduction disorders. Methomyl is a highly toxic oxime carbamate pesticide. In addition to common dosage forms, it can also be used as a compound agent and is widely used. It controls a variety of pests by inhibiting acetylcholinesterase (AChE). Due to the widespread use of pesticides, the monitoring of pesticide residues in agricultural products and environmental matrices is undoubtedly the key to ensuring food safety and ecological health.

[0003] Many laboratory-based techniques have been developed to detect carbamate pesticides, including gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography, liquid chromatography-tandem mass spectrometry, etc. Although these methods are quite mature in terms of technology and have high accuracy and sensitivity, there are still certain limitations in the analysis of biological samples of carbamate pesticides, such as expensive instruments and equipment, complex and time-consuming sample pretreatment, and the need for professional laboratory personnel to complete the operation, which greatly limits their on-site detection and use. Therefore, in order to further simplify the sample analysis steps, improve the selectivity and sensitivity of the method, and realize the visualization and intelligent detection of biological sample detection, it is urgent to develop a simple, fast and instant new method to detect trace carbamate pesticide residues in fruits, vegetables and environmental samples.

[0004] Nanozymes are a class of enzyme mimics that have both nanomaterial properties and catalytic functions. They are able to mimic the functions of natural enzymes such as peroxidases and oxidases, and have many unique advantages, including low cost, high stability, easy modification, adjustable catalytic activity, and suitability for large-scale production. The unique physicochemical properties of nanomaterials provide important opportunities for regulating their catalytic properties. Therefore, nanomaterials are widely used in biochemical sensing, disease diagnosis and treatment, and environmental protection. At present, various types of nanocatalyst materials have been developed, including metal-based nanozymes, carbon-based nanozymes, metal oxide / sulfide nanomaterials, and nanocomposites. Although nanozymes have made great progress, problems such as limited catalytic activity, complex catalytic mechanism, and biocompatibility have become bottlenecks restricting their development. Therefore, it is necessary to try to develop new nanozymes, design strategies to regulate their activity, and conduct in-depth research on their catalytic mechanisms. It is generally believed that the performance of nanozymes is often closely related to the structural properties and electron distribution of their surfaces. Therefore, improving catalytic activity through surface modification engineering or modifying different ligands is an effective way to optimize and regulate catalysts.

[0005] High-entropy alloys (HEAs) are single-phase solid solution alloys with simple crystal structures composed of five or more equal or approximately equal metals, with the atomic ratio of each element being 5-35%. In recent years, they have received extensive attention. Unlike traditional bimetallic and trimetallic alloys, HEAs have remarkable mechanical properties. Therefore, the research on HEAs is mainly focused on the study of metal structural materials. The subtle interaction between the internal geometric structure and electronic structure of HEAs gives them unique characteristics. By rationally adjusting the preparation conditions to control the material composition and geometric structure, HEAs have broad application prospects and provide an effective means to improve their catalytic performance. There are many different methods for synthesizing HEAs. Different methods have different synthesis difficulties, and the catalytic activity, dispersibility and morphology of the synthesized HEAs are also different. At the same time, the catalytic activity and dispersibility are affected by the type and proportion of metals. The difference in atomic size of different elements in HEAs leads to obvious lattice distortion, which increases the energy barrier for atomic diffusion and is conducive to the formation and stability of HEAs. In addition, the cocktail effect of HEAs regulates the adsorption of reactants and intermediates on active sites through the synergistic effect of multiple elements. A large number of studies have used DFT calculations to explain the mechanism of HEAs' superior catalytic performance, which mainly involves the interaction of d orbitals of various metals in HEAs, creating more electron transfer channels and active centers for catalytic reactions, making them exhibit excellent electrocatalytic performance. At present, there are few studies on the enzyme activity of HEAs, and there are no reports on the selection of surface modification to further improve the enzyme activity based on the catalytic activity given by the intrinsic structure of HEAs. Therefore, the development of HEAs with more efficient enzyme activity can not only better exert their catalytic performance, but also provide impetus for the development of high entropy nanozymes (HEAzymes) and promote the exploration of new fields of nanoenzymology. Summary of the invention

[0006] The purpose of the present invention is to provide a high entropy alloy nanozyme, a biosensor and a preparation method and application thereof in view of the above problems.

[0007] In order to achieve its purpose, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a method for preparing a high entropy alloy nanozyme PDA-HEA having peroxidase-like activity, comprising the following steps:

[0009] S1. Preparation of PtRuFeCoNi HEA nanowires by low-temperature oil phase method

[0010] Take metal precursor raw materials of Pt, Ru, Fe, Co, Ni, a structure directing agent, and a reducing agent, add them to solvent A, mix them thoroughly, heat the solution to 70-90° C. and keep it for 4-9 minutes under stirring conditions, then heat the solution to 200-240° C. and keep it for 80-100 minutes, cool it, collect the precipitate by centrifugation, wash it with a detergent, collect the precipitate, and dry it to obtain PtRuFeCoNi HEA nanowires;

[0011] The metal precursor is an acetylacetonate metal complex;

[0012] The structure directing agent is selected from hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium chloride, and dodecyltrimethylammonium chloride;

[0013] The reducing agent includes Mo(CO)6 and glucose;

[0014] S2. Synthesis of PDA-HEA

[0015] The PtRuFeCoNi HEA nanowires prepared in step S1 are dispersed in solvent B and mixed thoroughly. Dopamine and Tris-HCl solution are added thereto and stirred (magnetic stirring 500-1000 rpm, preferably 800 rpm) until the color of the solution turns black. Centrifuge and the resulting precipitate is the high entropy alloy nanozyme PDA-HEA.

[0016] Preferably, in the preparation method, the mass ratio of each raw material is: metal precursor of Pt: metal precursor of Ru: metal precursor of Fe: metal precursor of Co: metal precursor of Ni: structure directing agent: Mo(CO)6: glucose is 6-10:8-12:4-8:4-8:4-8:50-70:60-72:8-12, and the mass volume ratio of the structure directing agent to the solvent A is 50-70 mg:3-5 mL;

[0017] In step S1, each raw material is added to solvent A and then ultrasonically mixed to obtain uniform mixture. The ultrasonic conditions are: power 100-400 W, time 1.5-2.5 h;

[0018] The solvent A is oleylamine;

[0019] The detergent is a mixture of ethanol and cyclohexane, preferably the detergent is a mixture of ethanol and cyclohexane in a volume ratio of 7-11:1;

[0020] The solvent B is ethanol.

[0021] Preferably, in step S2, the raw materials are mixed according to the ratio of PtRuFeCoNi HEA nanowires, solvent B, dopamine, and 1M Tris-HCl solution of 1.5-2.5 mg: 6-12 mL: 0.2-0.4 mg: 0.8-1.2 mL (preferably 2 mg: 9 mL: 0.3 mg: 1 mL);

[0022] After adding PtRuFeCoNi HEA nanowires to solvent B, ultrasonic treatment is used to fully mix them. The ultrasonic conditions are: power 100-400W, time 20-40min; after all the raw materials are mixed, stir for 5-7h until the color of the solution turns black, centrifuge, and the resulting precipitate is the high entropy alloy nanozyme PDA-HEA;

[0023] Preferably, the metal precursor raw materials are (Pt(a-cac)2), (Ru(acac)3), (Fe(acac)3), (Co(a-cac)3) and (Ni(acac)2), and the structure directing agent is hexadecyltrimethylammonium bromide.

[0024] The second aspect of the present invention provides a high entropy alloy nanozyme PDA-HEA with peroxidase activity, which is prepared by any of the preparation methods described above.

[0025] The third aspect of the present invention provides a biosensor based on PDA-HEA, which is prepared by loading the above-mentioned high entropy alloy nanozyme PDA-HEA onto a hydrogel; preferably, the hydrogel is a sodium alginate hydrogel or a hyaluronic acid silicone gel.

[0026] The fourth aspect of the present invention provides a method for preparing the above-mentioned PDA-HEA-based biosensor, comprising the following steps:

[0027] S1, taking a hydrogel monomer, a cross-linking agent, the high entropy alloy nanozyme PDA-HEA described in claim 4 and water, mixing them evenly to obtain a hydrogel mixed solution, wherein the hydrogel monomer is selected from sodium alginate or sodium hyaluronate; the cross-linking agent of the sodium alginate hydrogel is CaCl2; the raw materials of the hyaluronic acid silicone gel include sodium hyaluronate and medical silicone;

[0028] Preferably, the hydrogel monomer is selected from sodium alginate, the cross-linking agent is CaCl2, and the components in the hydrogel mixed solution are proportioned according to the following mass ratio: the mass ratio of hydrogel monomer: cross-linking agent: PDA-HEA: water is 6-10: 0.10-0.14: 0.010-0.014: 1 (preferably 8: 0.12: 0.012: 1);

[0029] Preferably, the hydrogel monomer is first added into water, heated to dissolve, and then a cross-linking agent is added, mixed, and then PDA-HEA is added and mixed to obtain a hydrogel mixed solution;

[0030] S2. Add the prepared mixed solution into a mold, and let it stand at 4-10° C. to solidify and form the biosensor in a hydrogel state.

[0031] The fifth aspect of the present invention provides the use of the above-mentioned high-entropy alloy nanozyme PDA-HEA or the above-mentioned PDA-HEA-based biosensor in the detection of acetylcholinesterase, amino acid ester pesticides, and organophosphorus pesticides; the amino acid ester pesticides include methomyl, isoprocarb, aldicarb, and carbofuran.

[0032] The application method of the above-mentioned high entropy alloy nanozyme PDA-HEA for detecting acetylcholinesterase comprises the following steps:

[0033] S1. Mix and incubate acetylcholinesterase solutions of known different concentrations with acetylthiocholine solutions, add NaAc buffer, PDA-HEA, TMB solution and H2O2 solution to the mixed solution after incubation to form an ATCh-TMB-H2O2 detection system, and measure the absorption spectrum after sufficient reaction (preferably measuring the absorption spectrum at 652 nm);

[0034] S2. Obtain the absorbances corresponding to acetylcholinesterase solutions of different concentrations respectively, and use the concentration of acetylcholinesterase as the horizontal axis and the change in absorbance as the vertical axis to obtain an absorbance change-concentration standard curve;

[0035] S3. Take the acetylcholinesterase to be tested, detect its absorbance according to the same method, and calculate its concentration by comparing it with the absorbance change-concentration standard curve; wherein, the detection linear range of AChE is 0.1-1.1mU / mL, and the detection limit is 0.064mU / mL;

[0036] Preferably, the PDA-HEA is irradiated with 808 nm near-infrared laser for 4-6 min and then operated according to steps S1-S3;

[0037] The application method of the high entropy alloy nanozyme PDA-HEA for detecting methomyl comprises the following steps:

[0038] S1. Mix an acetylcholinesterase solution of known concentration with an acetylthiocholine solution and incubate them, add different concentrations of methomyl, NaAc buffer, PDA-HEA, TMB solution and H2O2 solution to the mixed solution after incubation to form an ATCh-TMB-H2O2 detection system, and measure the absorption spectrum after sufficient reaction (preferably measuring the absorption spectrum at 652 nm);

[0039] S2, respectively obtain the absorbance corresponding to the different concentrations of methomyl solution, take the concentration of methomyl as the abscissa and the change of absorbance as the ordinate, and obtain the absorbance change-concentration standard curve;

[0040] S3, take the methomyl to be tested, detect its absorbance according to the same method, and calculate its concentration by comparing with the absorbance change-concentration standard curve; the linear range of methomyl detection is 0.4-40, 150-1000ng / mL, and the detection limit is 0.624ng / mL;

[0041] Preferably, the PDA-HEA is irradiated with 808 nm near-infrared laser for 4-6 min and then operated according to steps S1-S3; after 808 nm near-infrared laser irradiation for 4-6 min, the linear range of methomyl detection is 0.1-5, 10-2000 ng / mL, and the detection limit is 0.068 ng / mL.

[0042] The application method of the above-mentioned biosensor for detecting acetylcholinesterase comprises the following steps:

[0043] ATCh and AChE standards of different concentrations are mixed and incubated to obtain a standard incubation solution, ATCh and a sample to be tested are mixed and incubated to obtain a sample incubation solution, and then the standard incubation solution and the sample incubation solution are respectively added dropwise to different detection units of the biosensor according to claim 5, and then H2O2 and TMB are added to react fully, and the gel color change of the detection unit is recorded, and the content of AChE in the sample to be tested is judged and analyzed according to the color change of the detection unit to which the standard incubation solution is added;

[0044] The above-mentioned application method of the biosensor for detecting methomyl comprises the following steps:

[0045] AChE and ATCh are mixed and incubated, and the incubation solution is dripped onto the biosensor according to claim 5, followed by dripping methomyl, H2O2 and TMB, and different concentrations of methomyl standards and samples to be tested are dripped into different detection units of the biosensor, and reacted fully, and the gel color change of the detection unit is recorded, and the content of methomyl in the sample to be tested is judged and analyzed according to the color change of the detection unit to which the methomyl standard is added.

[0046] The linear range of the biosensor for detecting AChE is 0.1-12 mU / mL; the linear range of the biosensor for detecting methomyl is 0.45-2 ng / mL.

[0047] The detection principle of the high entropy alloy nanozyme and biosensor of the present invention is:

[0048] When used for acetylcholinesterase (AChE), if the sample to be tested contains AChE, acetylthiocholine (ATCh) is specifically decomposed into thiocholine (TCh) by AChE. PDA-HEA has peroxidase-like activity. TCh binds to PDA-HEA, and the sulfur atom in TCh coordinates with the metal ions or other functional groups in the PDA-HEA enzyme, changing the spatial structure of the enzyme or the properties of the active site. At the same time, TCh may compete with H2O2 to bind to the active site of the enzyme, thereby inhibiting the decomposition reaction of H2O2 and reducing the generation of ·OH. Therefore, TCh reduces its ability to decompose H2O2 to generate hydroxyl radicals by interfering with the catalytic activity of PDA-HEA peroxidase. Since there is no hydroxyl radical to oxidize the colorless TMB to blue oxTMB, the solution does not produce an absorbance change or the gel does not change color; at the same time, since TCh can reduce hydroxyl radicals, it further cooperates with the phenomenon that TMB does not turn blue. That is, if the sample to be tested contains AChE, the sample will not turn blue; if the sample to be tested does not contain AChE, the sample will turn blue after the reaction.

[0049] When used to detect methomyl, the principle is that carbamate pesticides (for example, methomyl) decompose AChE, so ATCh is retained as a substrate, and no TCh is generated to act on PDA-HEA. Therefore, PDA-HEA can decompose H2O2 into hydroxyl radicals, which oxidize TMB into blue oxTMB, and the sample turns blue. That is, if the sample to be tested contains methomyl, the sample turns blue; if the sample to be tested does not contain methomyl, the sample does not change color.

[0050] The beneficial effects of the present invention are:

[0051] To overcome the problems existing in the prior art, we introduced surface modification engineering on the surface of HEAs and studied the effects of surface modification on their enzyme-like activity. Inspired by the adhesion properties of mussel foot proteins, polydopamine (PDA) coating has emerged as a versatile surface modification strategy. The surface of PDA materials contains abundant catechol, imine and amine groups, which have excellent metal ion chelation function, biocompatibility, hydrophilicity and adhesion properties. This enables them to be firmly attached to different surfaces and provide a stable interface for further functionalization. At the same time, PDA also has strong near-infrared absorption ability and produces photothermal conversion efficiency under 808nm infrared light irradiation. In addition, PDA coating has been shown to enhance the activity of nanozymes by induced accumulation and electron transfer effects. However, there are few reports on the dual strategy of composition adjustment and surface modification to regulate the catalytic performance of nanozymes. The Sabatier principle suggests that a good catalyst should have an appropriate binding strength with the key substrate to balance the coverage of the adsorbed products and improve the adsorption / desorption efficiency, thereby achieving the best catalytic effect. The center position of the d-band of HEAs is closely related to the adsorption strength of the catalyst to the reactants. Based on the Sabatier principle and d-band center theory, high entropy alloy catalysts can be designed in a targeted manner.

[0052] In this study, PtRuFeCoNi HEA NWs with POD-like activity were designed and synthesized, and the surface of HEA NWs was modified with PDA. We studied the changes in its catalytic performance and the effect of photothermal conversion on the enzyme-like activity. Theoretical studies revealed the reasons why PDA modification improves the catalytic performance of HEA. The Sabatier principle explains the increase in HEA enzyme activity after PDA coating, which is to optimize the surface charge distribution, appropriately reduce the d-band center, balance the adsorption of substrates, further improve the adsorption / desorption efficiency, and improve the catalytic performance. As a conceptual application, a PDA-HEA-based biosensor was constructed for the sensitive detection of acetylcholinesterase (AChE) and methomyl. PDA-HEA loaded with sodium alginate (SA) hydrogel can achieve portable visual detection in combination with a smartphone. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1Synthesis and characterization of PtRuFeCoNi HEA: A) Schematic diagram of the HEA formation process; B) HRTEM image of HEA; C) HRTEM image of PDA-HEA; D) XRD pattern; E) HRTEM image of PDA-HEA; the insert shows the atomic arrangement; F) FCC pattern on the

[111] crystal plane axis of PDA-HEA (region 1-region 3); G) HAADF-STEM-EDS spectrum and metal element content obtained by ICP-OES; H) HAADF-STEM-EDS mapping of PDA-HEA; I) FT-IR spectrum.

[0054] Figure 2 Peroxidase-like activity and optimization of HEA and PDA-HEA are shown: A) Schematic diagram of peroxidase-like activity evaluation of PDA-HEA (using OPD, ABTS and TMB); B) UV-visible absorption spectra and visual colors of different color development reactions of PDA-HEA; C) UV-visible absorption spectra and visual colors of FeCoNi, RuFeCoNi and HEA; D) PDA-FeCoNi, PDA-RuFeCoNi and PDA-HEA; E) UV-visible absorption spectra of PDA, HEA and PDA-HEA; F) Time-dependent absorbance spectra of dopamine at different concentrations; G) Time-dependent absorbance spectra of PDA-HEA at different concentrations; H) Peroxidase-like activity and visual color of PDA-HEA at different pH values; Data are presented as mean ± SD (n = 6 independent samples).

[0055] Figure 3The effects of photothermal conversion on the POD-like activity and mechanism of PDA-HEA are shown: A) UV-visible absorption spectra of HEA, PDA-HEA and NIR-PDA-HEA; B) Time-dependent absorption spectra of HEA, PDA-HEA and NIR-PDA-HEA; C) Photothermal conversion performance of PDA-HEA at different concentrations; D) Effect of different temperatures on the POD-like activity of PDA-HEA; Data are presented as mean ± SD (n = 6 independent samples); E) Control group (catalytic system without PDA-HEA), PDA, HEA and PDA-HEA; the legend contains an insert of the infrared thermal images of these four systems; F) UV-visible absorption spectra of the control group (catalytic system without PDA-HEA), PDA, HEA and PDA-HEA under NIR light; the legend contains an insert of the infrared thermal images of these four systems under NIR light; G) UV-visible absorption spectra of PDA-HEA at 50°C and room temperature (under dark conditions); H) Temperature-time heat map of the infrared thermal images of PDA, HEA and PDA-HEA under NIR irradiation, with a time range of 0-5 minutes.

[0056] Figure 4 The peroxidase-like mechanism of HEA, PDA-HEA and NIR-PDA-HEA is shown: A-C) Michaelis-Menten curves of HEA, PDA-HEA and NIR-PDA-HEA at different concentrations of TMB; data are presented as mean ± SD (n = 6 independent samples); D-F) Michaelis-Menten curves of HEA, PDA-HEA and NIR-PDA-HEA at different concentrations of H2O2; data are presented as mean ± SD (n = 6 independent samples) Figure 3 shows the absorbance of ·OH produced by H2O2 in the presence of different scavengers; G) Specific activities of HEA, PDA-HEA and NIR-PDA-HEA; H) ESR spectra of ·OH produced by H2O2; data are presented as mean ± SD (n = 3 independent samples); I) Absorbance in PDA-HEA + TMB system containing different scavengers; for superoxide dismutase (SOD) (ae: 0, 5, 10, 15 and 20 U / L), tryptophan (ae: 0, 0.5, 1, 1.5 and 2 mM) and isopropanol (ae: 0, 0.1, 0.5, 1 and 1.5 mM).

[0057] Figure 5DFT calculations of the electron distribution, structural configuration and theoretical calculations of the POD-like mechanism of HEA and PDA-HEA are shown: A) 3D atomic model of the PDA-HEA crystal structure; B) Different reaction pathways for the decomposition of H2O2 to produce ·OH on HEA and PDA-HEA; C-D) PDOSs and partially enlarged adsorption of H2O2 on HEA; E) Comparison of the d-band centers of single elements and bulk HEA, respectively; F-G) PDOSs and partially enlarged H2O2 adsorbed on PDA-HEA; H) Comparison of the d-band centers of single elements and bulk PDA-HEA; I) Schematic diagram of the Sabatier principle of HEA and PDA-HEA; J) Free energy spectrum of the decomposition of H2O2 on HEA and PDA-HEA along the homolytic pathway.

[0058] Figure 6 The colorimetric detection platform of acetylcholinesterase and methomyl is shown: A) AChE-controlled color development reaction based on TMB+PDA-HEA nanozyme, illustrating the detection mechanism of AChE and methomyl; B) UV-visible absorption spectra of PDA-HEA / TMB / H2O2 system of different compounds to study the feasibility of detecting AChE; C) UV-visible spectra of AChE at different concentrations; D) Relationship between the change of absorbance at 652nm and AChE concentration and its linear relationship; E) Different compounds Figure 1: UV-visible absorption spectrum of PDA-HEA / TMB / H2O2 system of the compound to investigate the feasibility of methomyl detection; F) UV-visible spectra of methomyl at different concentrations; G) Relationship between the change of absorbance at 652nm and the concentration of methomyl and their linear relationship; H) UV-visible spectra of methomyl at different concentrations (under near-infrared light); I) Relationship between the change of absorbance at 652nm and the concentration of methomyl and the corresponding linear relationship (under near-infrared light); J) Interference experiment of methomyl colorimetric detection platform.

[0059] Figure 7The construction of a comprehensive and portable detection platform for hydrogel@PDA-HEA is shown: A) Schematic diagram of the synthesis of hydrogel@PDA-HEA and the construction of a portable methomyl detection platform; B) Digital photos of hydrogel, hydrogel@PDA, and hydrogel@PDA-HEA under white light; C) SEM image of hydrogel@PDA-HEA; the red circle indicates that PDA-HEA is located inside the hydrogel; D) The relationship between B / G and methomyl concentration and the corresponding linear relationship; inside are digital photos of different concentrations of methomyl detected by hydrogel@PDA-HEA; E) The relationship between B / R and AChE concentration and the corresponding linear relationship; inside are digital photos of different concentrations of AChE detected by hydrogel@PDA-HEA; F) Digital photos of hydrogel@PDA-HEA and TMB, OPD, and ABTS in different shapes (strawberry, pear, apple) under white light. DETAILED DESCRIPTION

[0060] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0061] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0062] Example 1

[0063] 1 Reagents and Materials

[0064] Ruthenium acetylacetonate (Ru(acac)3), platinum acetylacetonate (Pt(acac)2), iron acetylacetonate (Fe(acac)3), nickel acetylacetonate (Ni(acac)2), cobalt acetylacetonate (Co(acac)3), oleylamine (OAm), trimethylammonium bromide (CTAB), hexacarbonyl molybdenum Mo(CO)6, glucose, and acetylthiocholine (ATCh) were purchased from Macklin Biochemical Technology (Shanghai) Co., Ltd. 2,2'-aza-bis(3-ethylbenzothiazole-6-sulfonic acid) diammonium salt (ABTS), 3,3',5,5'-tetramethylbenzidine (TMB), and o-phenylenediamine (OPD) were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. Ethanol and H2O2 (30%) were obtained from Chongqing Chuandong Chemical Co., Ltd. Acetylcholinesterase (AChE) and NaAc buffer (pH 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 0.2 M) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Horseradish peroxidase (HRP), glucose oxidase (GOx), neutral protease (NEP), and acid phosphatase (ACP) were obtained from Shanghai SigmaAldrich Trading Co., Ltd. (Shanghai, China). Bovine serum albumin, V component, and heat shock isolate (BSA) were purchased from Shenggong Biotechnology Co., Ltd. (Shanghai). Methomyl (CAS No. 16752-77-5) was purchased from LGC Labor GmbH Bgm (Augsburg, Germany). All reagents were not further purified. All experimental aqueous solutions were prepared with distilled water.

[0065] 2 Methods

[0066] 2.1 Synthesis of FeCoNi NPs, RuFeCoNi NPs and HEANWs

[0067] HEANWs were synthesized according to previous reports with slight modifications as follows:

[0068] CTAB, transition metal organic complex raw material, Mo(CO)6, and glucose were added to a flask containing OAm (4 mL) in sequence. After the mixture was ultrasonicated at room temperature for 2 h (power 100-400 W) to obtain a homogeneous solution, the solution was heated to 80 ° C and maintained for 5 min under magnetic stirring in an oil bath, and then the solution was heated to 220 ° C and maintained for 90 min. The mixture was quickly cooled to room temperature and centrifuged to collect the black product. The product was washed several times with an ethanol / cyclohexane mixture (v / v: 9:1). Finally, the precipitate was freeze-dried and stored for later use. FeCoNi NPs and RuFeCoNi NPs were synthesized under the same conditions and processes, but with different compositions.

[0069] The raw materials for the synthesis of FeCoNi NPs are: CTAB (60 mg), Fe(acac)3 (6 mg), Co(acac)3 (6 mg), Ni(acac)2 (6 mg), Mo(CO)6 (66 mg) and glucose (10 mg).

[0070] The raw materials for the synthesis of RuFeCoNiNPs are: CTAB (60 mg), (Ru(acac)3) (10 mg), Fe(acac)3 (6 mg), Co(acac)3 (6 mg), Ni(acac)2 (6 mg), Mo(CO)6 (66 mg) and glucose (10 mg).

[0071] The raw materials for the synthesis of HEANWs (i.e., PtRuFeCoNi HEA nanoparticles) are: CTAB (60 mg), (Ru(acac)3) (10 mg), (Pt(acac)2) (8 mg), Fe(acac)3 (6 mg), Co(acac)3 (6 mg), Ni(acac)2 (6 mg), Mo(CO)6 (66 mg) and glucose (10 mg).

[0072] 2.2 Synthesis of PDA-FeCoNi NPs, PDA-RuFeCoNi NPs, and PDA-HEANWs:

[0073] The following steps were followed: HEANWs (2 mg) prepared in the previous step were added to 9 mL of ethanol and treated with ultrasound (power 100-400 W) for 30 min; 30 μL of dopamine (10 mg / mL) was added to 1 mL of Tris-HCl solution (1 M, pH 8.5) and mixed, and then added to the ethanol solution of HEANWs and stirred vigorously (800 rpm). The color of the solution slowly changed from light brown to black. After continuous stirring for 6 hours, the residue obtained by centrifugation of the reaction solution was dissolved in ultrapure water to obtain PDA-HEANWs.

[0074] PDA-FeCoNi NPs and PDA-RuFeCoNi NPs were synthesized following the same method.

[0075] 2.3 Characteristics of HEANWs and PDA-HEANWs

[0076] TEM and HRTEM images were characterized by FEI Tecnai G2 F20 TEM equipment (FEI, USA). AC-STEM images and EDS patterns were obtained using JEM-arm300f (JEM, Japan). Powder XRD patterns were recorded on an x-ray diffractometer (D8Advance, Bruker, Germany). XPS data were retrieved using a K-Alpha x-ray photoelectron spectroscopy (XPS) spectrometer from Thermo, USA. The composition of HEANWs was determined using ICP-OES (720ES, Agilent, US). FT-IR data in the wavenumber range of 400-4000 cm-1 were measured using a NicoletIS50 Fourier transform infrared (FT-IR) spectrometer (Thermo, USA). Electron spin resonance (ESR) data were obtained using an electron paramagnetic resonance spectrometer (A300-10 / 12, Bruker, Germany). Zeta potential was evaluated by Zetasizer Nano ZS90 (Malvern, UK). UV-visible absorption spectra were obtained using a UV-visible spectrophotometer (TU-1901, PERSEE, China). The absorbance of all experimental groups was measured at 652 nm using a microplate reader (Multiskan Go, Thermo Fisher Scientific, USA).

[0077] The configurational entropy of a high entropy alloy is defined by the following formula:

[0078]

[0079] ΔS configuration >1.5R is a high entropy material; ΔS configuration 1.0–1.5R is a medium entropy material; ΔS configuration <1.0R is a low entropy material.

[0080] In the present invention, the ΔS of HEANWs configuration =1.85R, classified as a high entropy material.

[0081] 2.4 Enzyme-like activities of HEANWs, PDA-HEANWs, and NIR-PDA-HEANWs

[0082] The POD-like activity of HEA and PDA-HEANWs was detected by TMB colorimetric assay. -1 ) and H2O2 (2 μL, 30%) were added to a pH 4.5 NaAc buffer (2 mL, 0.2 M) containing 5 μg mL -1HEANWs or PDA-HEANWs. The absorbance at 652 nm was measured at room temperature after a 5-min reaction period. NIR-PDA-HEANPs were irradiated with 808 nm near-infrared laser for 5 min. -1 ), different pH values ​​(3.5-7.0) and different power densities (0.2-1.2 W cm -2 ) of PDA-HEANPs to determine the optimal enzyme activity conditions of HEANPs.

[0083] NIR-PDA-HEANWs refers to nanozymes prepared by PDA-HEANPs irradiated with 808nm near-infrared laser for 5min.

[0084] 2.5 Enzyme kinetics of HEANWs, PDA-HEANWs and NIR PDA-HEANWs

[0085] Kinetic measurements were performed in time course mode by monitoring the change in absorbance at 652 nm. Peroxidation reactions were performed using different concentrations of TMB (0.1–2.0 mM) and H2O2 (1.0–10 mM). In each experiment, HEANWs or PDA-HEANWs (5 μg mL -1 ) was added to the reaction solution containing H2O2 and TMB substrate in NaAc buffer (pH 4.5, 0.2M, 2mL). In addition, the NIR-PDA-HEA NWs were irradiated with 808nm near-infrared laser for 5min and the absorbance at 652nm was detected. The Michaelis-menten constant (Km) was determined using the Lineweaver-Burk plot, using the double reciprocal of the Michaelis-menten equation:

[0086] v=v max ×[S] / (K m +[S]),

[0087] Where v represents the initial reaction rate, v max is the maximum reaction rate, [S] is the substrate concentration, K m is the Michaelis constant.

[0088] 2.7 Determination of specific activity

[0089] A series of concentrations of HEANWs or PDA-HEANWs were mixed with H2O2 (2 μL, 30%) and TMB (8 μL, 10 mg mL -1) were mixed in 2 mL of NaAc buffer (pH 4.5, 0.2 M), and the initial reaction rate of the solution was calculated by the absorbance change (Δa / Δt) of TMB. In addition, the absorbance of NIR-PDA-HEANWs was detected after irradiating them with 808 nm near-infrared laser for 5 min. The specific activity was determined by the linear fitting curve of nanozyme concentration and initial reaction rate.

[0090] 2.8ESR Measurement

[0091] Electron spin resonance (ESR) spectroscopy was used to measure the hydroxyl radical (·OH), superoxide anion (·O 2- ) and singlet oxygen ( 1 In brief, the test was conducted under the following conditions, including H2O2, DMPO and HEANWs or PDA-HEANWs. All mixtures were dispersed in NaAc buffer (0.2 M) at pH 4.5. After reacting at room temperature for 10 min, the solution was immediately subjected to ESR analysis. In addition, the same operation was performed after irradiating NIR-PDA-HEANWs with 808 nm near-infrared laser for 5 min.

[0092] 2.9 Colorimetric detection of acetylcholinesterase in solution

[0093] After 5 μL of different concentrations of AChE and 10 μL of acetylthiocholine (10 mM) were incubated at 37°C for 30 min, 1970 μL of NaAc buffer (pH 4.5, 0.2 M), 5 μL of HEA NWs or PDA-HEANWs (2 mg mL -1 ), H2O2 (2 μL, 30%) and 8 μL TMB (10 mmol / L). After reacting at 25°C for 30 min, the absorption spectrum was measured. In addition, the NIR-PDA-HEANWs were irradiated with 808 nm near-infrared laser for 5 min and then the same operation was performed.

[0094] 2.10 Colorimetric detection of methomyl in solution

[0095] After 5 μL of AChE (0.55 mU / mL) and 10 μL of ACh (10 mM) were incubated at 37 °C for 30 min, 5 μL of different concentrations of methomyl, 1965 μL of NaAc buffer (pH 4.5, 0.2 M), 5 μL of HEA NWs or PDA-HEANWs (2 mg mL -1), H2O2 (2μL, 30%) and 8μLTMB (10mmol / L). After reacting at 25℃ for 30min, the absorption spectrum was measured. In addition, the NIR-PDA-HEANWs were irradiated with 808nm near-infrared laser for 5min and then the same operation was performed.

[0096] 2.11 Construction of a portable colorimetric platform for acetylcholinesterase and methomyl hydrogel

[0097] To prepare the hydrogel, 8 mg of sodium alginate (SA) was added to a 5 mL glass bottle containing 1 mL of ultrapure water and stirred in a water bath at 60 ° C for 15 minutes. Under continuous stirring, 20 μL of CaCl2 (6 mg / mL) was added and the mixture was stirred for another 15 minutes to achieve homogeneity. Subsequently, 6 μL of PDA-HEANWs (2 mg mL-1) was added to the mixture. It was then added to a 96-well plate mold (300 μL per well) and refrigerated for 12 hours to form the detection hydrogel Hydrogel@HEzymes@PDA.

[0098] For AChE detection, 5 μL of different concentrations of AChE and 10 μL ACh (10 mM) were incubated at 37 °C for 30 min and then added to Hydrogel@HEzymes@PDA in a 96-well plate. Subsequently, 2 μL H2O2 (30%) and 50 μL TMB (10 mM) were added and reacted for 5 min. The color information was captured using a smartphone camera, and the images were converted into numerical data using ImageJ software, thereby achieving quantitative analysis of AChE.

[0099] Similarly, for the detection of methomyl, 5 μL of AChE (0.55 mU / mL) and 10 μL of ATCh (10 mM) were incubated at 37°C for 30 minutes and then added to Hydrogel@HEzymes@PDA in a 96-well plate. Next, 5 μL of different concentrations of methomyl, 2 μL of H2O2 (30%), and 50 μL of TMB (10 mM) were added and reacted for 5 minutes. The color was recorded with a smartphone camera, and the images were processed with ImageJ software to obtain numerical values, thereby performing quantitative analysis of methomyl.

[0100] We first evaluated the enzyme-like activity of HEA by colorimetry, using 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. In the presence of H2O2, HEA can convert colorless TMB into blue oxidized TMB (oxTMB), and its characteristic absorption peak is monitored at 652nm by UV-visible absorption spectroscopy. In addition, 2,2'-azino (3-ethylbenzothiazole-6-sulfonic acid) diammonium salt (ABTS) and o-phenylenediamine (OPD) were used to verify the peroxidase (POD)-like activity of HEANWs, and the results showed that HEANWs have good substrate versatility. For comparison, we successfully prepared FeCoNi and RuFeCoNi with less metal precursors using the same synthesis method. By comparing the POD-like activities of the three materials, it was found that the catalytic performance gradually increased. Studies have shown that noble metals perform better than general metals, so we speculate that the improvement in enzyme activity is attributed to the doping of noble metals, and the drastic lattice distortion gives HEANWs excellent POD-like activity. In order to illustrate the universality of PDA modification and coating, we further successfully coated PDA on the surface of FeCoNi, RuFeCoNi and HEANWs. It is worth noting that the catalytic performance of PDA-FeCoNi, PDA-RuFeCoNi and PDA-HEA is significantly improved compared with the unmodified materials. Thanks to the excellent hydrophilicity and biocompatibility of PDA, the solubility of PDA-HEA in water is significantly enhanced. To exclude the influence of PDA, we synthesized a pure PDA phase without HEANWs and investigated its POD-like activity. The results show that pure PDA has no catalytic ability, while the catalytic activity of PDA-HEANWs shows a "1+1>2" effect compared with pure HEA and PDA. At the same time, by changing the concentration of added dopamine during the formation of PDA-coated HEANWs, the obtained samples exhibited different POD-like activities. When a higher concentration of dopamine was added, the enzyme-like activity decreased significantly, which may be due to the excessive and heavy PDA coating, which masked the active sites of the nanozyme and affected the catalytic performance. Based on this, the optimal coating concentration was selected.

[0101] 3 Results and analysis

[0102] 3.1 Properties of PtRuFeCoNi HEA nanowires and PDA-HEA nanozymes

[0103] PDA-HEA nanozyme is a kind of HEA nanozyme that adopts dopamine-mediated growth strategy (such as Figure 1 First, a simple low-temperature oil phase method was used to prepare PtRuFeCoNi HEA nanowires ( Figure 1 B. Figure 1C). We used platinum (II) acetylacetonate (Pt(acac)2), ruthenium (III) acetylacetonate (Ru(acac)3), iron (III) acetylacetonate (Fe(acac)3), cobalt (III) acetylacetonate (Co(acac)3) and nickel (II) acetylacetonate (Ni(acac)2) as metal precursors, OAm as solvent, hexadecyltrimethylammonium bromide (CTAB) as structure-directing agent, molybdenum hexacarbonyl (Mo(CO)6) and glucose as reducing agents. Tris-HCL solution and dopamine were then added to anhydrous ethanol to disperse the HEA nanowires. During this process, dopamine (DA) itself was oxidized and aggregated to form PDA, which was then modified on the surface of the HEA nanowires. This is crucial because the surface of PDA contains abundant catechol, imine and amine groups, which give it excellent metal ion chelating properties. The three characteristic peaks in the X-ray diffraction (XRD) pattern of HEA nanowires belong to the face-centered cubic (fcc) structure of FePt (PDF#02-1167) Figure 1 D). The diffraction peak intensity of PDA-HEA is significantly higher than that of HEA, which may be due to the encapsulation of PDA. Compared with the diffraction peaks of pure Pt, Ru, Fe, Co and Ni, the position of the broad diffraction peak has shifted significantly, which proves that these metals have been incorporated into the HEA nanowire structure to form an fcc structure. High-resolution transmission electron microscopy (HRTEM) images of HEA nanowires show an average intercrystalline spacing of 0.223nm ( Figure 1 E), which corresponds to the (111) crystal plane of FePt, which is consistent with the XRD results. At the same time, the integrated intensity of the (111) lattice in the selected area shows that the average value of the lattice spacing determined by fast Fourier transform (FFT) analysis varies from region 1 (0.223nm) to region 2 (0.224nm) to region 4 (0.225nm) (S2), thus proving that the synthesized HEA has lattice distortion. In addition, the selected area electron diffraction (SAED) of the high entropy nanowires further confirmed its typical face-centered cubic structure ( Figure 1 F), which is consistent with the results of XRD and HRTEM. All these data indicate the successful combination of HEA nanowires.

[0104] Energy dispersive X-ray analysis (EDS) images of HEA show that the five elements are evenly distributed throughout the nanowires. ICP results show that the relative atomic ratios of Pt, Ru, Fe, Co, and Ni are 26:20:19:7:28 ( Figure 1 G). Through calculation, its configuration entropy is 1.83R, which belongs to the high entropy level. These results prove that the nanowire meets the conceptual definition of high entropy alloy and belongs to the category of high entropy materials. Obviously, the EDS data of PDA-HEA also shows the existence of coating ( Figure 1H).

[0105] Since DA is rich in catecholamines, and PDA, as an oxidative polymerization product of DA, also retains this structure. The changes in the chemical bonds on the surface of HEA nanowires after PDA encapsulation were analyzed by infrared spectroscopy (FTIR). Figure 1 I). The diffraction peak intensity of PDA-HEA is higher than that of HEA, and out-of-plane distortion of the CH bonds of the aromatic ring occurs, confirming the successful modification of PDA.

[0106] X-ray photoelectron spectroscopy (XPS) was used to observe the metal distribution on the surface of HEA and PDA-HEA. The peaks of Pt 4f7 / 2 and Pt4f5 / 2 were attributed to 71.12eV and 74.46eV, respectively, while the peaks of Ru 3p3 / 2 and Ru 3p1 / 2 were attributed to 463.15eV and 485.02eV, respectively. The peaks of Fe 2p3 / 2 and Fe 2p1 / 2 were attributed to 712.09eV and 724.45eV, respectively, while the peaks of Co2p3 / 2 and Co 2p1 / 2 were attributed to 780.91eV and 796.79eV, respectively. The peaks of Ni 2p3 / 2 and Ni 2p1 / 2 were attributed to 855.75eV and 873.45eV, respectively. The results show that HEA contains five metals, Pt, Ru, Fe, Co and Ni, with different valence states. However, the XPS results of PDA-HEA showed that the peak intensity signals of each metal element decreased significantly, and the signal intensity of the C element increased. In addition, we performed Zeta potential analysis on HEA and PDA-HEA, and the potential changed from positive to negative, further proving that PDA was successfully coated on the surface of HEA (S5).

[0107] 3.2 Peroxidase-like enzyme activity of HEA and PDA-HEA

[0108] We first evaluated the enzymatic activity of HEA using a colorimetric assay, using 3,3',5,5'-tetramethylbenzidine (TMB) as the substrate. In the presence of H2O2, HEA can convert the colorless TMB into a blue OX TMB, and the characteristic absorption peak at 652nm was monitored by UV-visible absorption spectroscopy. In addition, we also used 2,2'-diphenylindoline-3-sulfonic acid ammonium salt (ABTS) and o-phenylenediamine (OPD) to verify the catalase-like activity of HEA, proving that HEA nanozymes have good substrate versatility (S6). For comparison, we also successfully synthesized FeCoNi and RuFeCoNi containing less metal precursors using the same synthesis method. By comparing the catalase-like activity of the three materials, the catalytic performance showed a gradually improving effect. Studies have shown that precious metals outperform ordinary metals ( Figure 2C), we therefore speculate that the improvement in enzyme activity is due to the incorporation of noble metals, and the severe lattice distortion makes HEA have superior catalase-like activity.

[0109] To illustrate the universality of PDA modification and coating, we also successfully coated FeCoNi, RuFeCoNi, and HEA with PDA. Furthermore, the catalytic performance of PDA-FeCoNi, PDA-RuFeCoNi, and PDA-HEA was significantly improved compared to the materials without PDA modification ( Figure 2 D). It is worth noting that the catalytic ability of nanozymes is usually related to their degree of dispersion. Thanks to the excellent hydrophilicity and biocompatibility of PDA, the solubility of PDA-HEA in water is much higher than that of HEA. Therefore, the enhanced dispersion makes it easier for the active sites to bind to the substrate, thereby improving the catalytic performance. In order to exclude the influence of PDA, we synthesized pure phase PDA without HEA and studied its POD-like activity. The results showed that pure PDA has no catalytic ability ( Figure 2 E), while the catalytic activity of PDA-HEA is "1+1>2" than that of pure HEA and PDA. At the same time, by changing the added dopamine concentration during the preparation of PDA-coated HEA nanozymes, samples with obvious catalase (POD)-like activity can be obtained. When the added dopamine concentration is high (0.07mg / mL-0.1mg / mL), the enzyme-like activity is significantly reduced. This may be due to the formation of excessive and thicker PDA coatings, which in turn mask the active sites of the nanozymes and affect the catalytic performance. Therefore, based on this result, the optimal coating concentration (0.03mg / mL) was selected. As the concentration of PDA-HEA increases, the corresponding absorbance also increases. Based on this result, a suitable material concentration was selected. Similarly, in the pH-dependence test, when determining the optimal pH value, we found that only limited OXD-like activity was exhibited compared to POD-like activity. Therefore, the effect of OXD-like activity can be basically ignored.

[0110] 3.3 Photothermal conversion effect of PDA-HEA on POD-like activity and its mechanism

[0111] Due to the molecular structure and physical properties of PDA, when used as a coating, PDA not only enhances the POD-like activity of HEA but also enables it to have photothermal conversion properties. Inspired by this mechanism and the fact that the decomposition of H2O2 is affected by temperature, we investigated the mechanism by which PDA-HEA exhibits its photothermal conversion properties and its influence on its pod-like activity. Figure 3 A It can be directly seen that after PDA modification and NIR treatment, the catalytic activity of HEA gradually increased. In addition, with the extension of irradiation time, the absorbance at 652nm also increased ( Figure 3B), which preliminarily showed that PDA-HEA after near-infrared treatment had enhanced pod-like activity. Subsequently, the effect of different concentrations of catalyst on the photothermal conversion effect was studied, indicating that the photothermal conversion effect is related to the material concentration ( Figure 3 C). In addition, PDA-HEA also exhibited excellent photothermal conversion stability, with no significant change in concentration after four on / off laser irradiation cycles. The irradiation power of the laser was also fine-tuned to maximize its photothermal conversion performance.

[0112] To elucidate the mechanism of NIR irradiation-enhanced catalytic activity, we used a water bath heating method instead of NIR treatment. Under 50 °C water bath treatment, an absorbance increase similar to that of the previous NIR treatment was observed, which is also consistent with the results of temperature optimization ( Figure 3 D and 3G). These results indicate that the increase in pod-like activity after near-infrared treatment is due to the increase in ambient temperature. The absorbance values ​​of different concentrations of PDA in dark and near-infrared environments were compared to analyze the effect of PDA on the photothermal conversion performance of PDA-HEA. We found that the higher the PDA concentration, the greater the corresponding increase in activity. At the same time, the intensity of the photothermal conversion effect of PDA, HEA, and PDA-HEA was compared, and the temperature changes were recorded using a thermal imager ( Figure 3 H). The results show that HEA has only limited photothermal conversion ability, while PDA and PDA-HEA both show good photothermal conversion ability. This also proves that the photothermal conversion effect of PDA-hea mainly comes from the modification of PDA. In addition, we compared the absorbance and temperature changes of the blank and color development systems in the dark and near-infrared treatment after adding PDA, HEA and PDA-HEA ( Figure 3 E and 3F). We found that although PDA itself has photothermal conversion performance, considering the amount of catalyst added and the fact that PDA itself has no pod-like activity, the absorbance and temperature of the entire system remained basically unchanged after NIR treatment. This further shows that only when PDA encapsulates HEA in the reaction system can the most ideal effect be achieved. The key is that NIR treatment not only increases the temperature of the PDA-HEA microenvironment, but also promotes the decomposition of H2O2 and increases the diffusion rate of free radicals, thereby further enhancing the catalytic activity. Finally, we studied the stability of HEA and PDA-HEA and their photothermal conversion capabilities. After being placed at room temperature for 30 days, there was no obvious change in the catalytic ability and photothermal conversion performance of the two catalysts, indicating that both catalysts exhibited good stability.

[0113] 3.4 Kinetics of Nanozymes and Free Radical Analysis

[0114] To evaluate the effects of PDA-coated particles and near-infrared irradiation on the enhancement of PODS-like activity, we varied the TMB ( Figure 4AC) and H2O2( Figure 4 The concentration of DF) was used to determine the enzyme kinetics, and the curve of substrate concentration and initial reaction rate was obtained, which was consistent with the classic Michaelis-menten model, indicating that the pod-like catalytic process followed the Michaelis kinetic law.

[0115]

[0116] According to the calculation of the above formula, the Lineweaver-Burk double reciprocal plot reveals the Michaelis constant (KM) and maximum reaction rate (vmax) of the corresponding substrate, and derives the catalytic constant (Kcat) and catalytic efficiency (Kcat / KM). It is generally believed that the key first step in the catalytic mechanism of nanomaterials simulating pod-like activity is to adsorb H2O2 onto the nanozyme, catalyze the decomposition of H2O2 to release free radicals and catalyze the reaction with the chromogenic substrate, and it is of great significance to construct nanozymes with better affinity for H2O2. Compared with HEA, PDA-HEA has a lower KM and a higher vmax for H2O2, which indicates that the PDA coating can enhance the affinity and catalytic rate of HEA for H2O2. More importantly, compared with the treatment under dark conditions, PDA-HEA irradiated with near-infrared further improves its affinity and catalytic rate for H2O2. In addition, Kcat represents the ability of a single enzyme molecule to catalyze the conversion of substrates into products per unit time. The higher the Kcat value, the stronger its intrinsic catalytic activity. Kcat / Km represents the number of substrate molecules that can be converted per enzyme molecule per second under substrate saturation conditions. The larger the value, the higher the catalytic efficiency of the enzyme. Compared with HEA, the Kcat and Kcat / Km of PDA-HEA for H2O2 increased by 2.74 times and 3.19 times, respectively. Compared with PDA-HEA treated in the dark, near-infrared irradiation further increased Kcat and Kcat / Km by 1.86 times and 2.06 times (Table 1). This shows that PDA modification and near-infrared irradiation have gradually improved the catalytic ability of nanozymes. Similarly, we also compared the relevant kinetic parameters with TMB (Table 2). Compared with HEA, the Kcat and Kcat / Km of PDA-HEA increased by 4.43 times and 1.97 times, respectively. In addition, compared with PDA-HEA under dark conditions, the Kcat and Kcat / Km of PDA-HEA treated with near-infrared increased by 2.79 times and 1.21 times, respectively. The catalytic ability of these nanozymes was further evaluated by specific activity (NIR-treated PDA-HEA: 13.87 U mg-1, PDA-HEA: 7.33 U mg-1, HEA: 1.75 U mg-1) ( Figure 4G). Taken together, these results demonstrate the feasibility of our strategy to modify the HEA surface by PDA and to gradually enhance the enzyme-like activity under the assistance of NIR.

[0117] Table 1 Kinetic parameters of POD-like performance of HEANWs, PDA-HEANWs and NIR-PDA-HEANWs with H2O2

[0118]

[0119] Table 2 Kinetic parameters of POD-like performance of HEANWs, PDA-HEANWs and NIR-PDA-HEANWs with TMB

[0120]

[0121] In order to better analyze the catalytic mechanism of PDA-HEA, the possible types of reactive oxygen species (ROS) generated during H2O2 catalysis were discussed. Generally, ·OH, 1O2 and ·O2- were generated. We used isopropanol, tryptophan and superoxide dismutase (SOD) as scavengers of ·OH, 1O2 and ·O2-, respectively. With the increase of the concentration of the three scavengers, the relative catalytic activity of PDA-HEA gradually decreased, suggesting that there may be ·OH, 1O2 and ·O2- ( Figure 4 I). To verify this experimental result, we further used ESR spectroscopy to analyze the types of free radicals produced during the catalytic process. The results showed that ·OH, 1O2, and ·O2- were produced, and the signal intensity gradually increased with time, which mutually verified the results of the above free radical scavenger experiment. At the same time, without the addition of H2O2, the signals of the three free radicals were almost undetectable, indicating that PDA-HEA had almost no oxd-like activity, which was consistent with the previous pH optimization results ( Figure 4 G).

[0122] 3.5 DFT calculations of HEA and PDA-HEA nanozymes

[0123] From previous experiments, it can be seen that the modification of PDA significantly improves the catalytic performance of HEA. It is worth noting that the catalytic activity of nanozymes is often closely related to their surface properties and the electronic structure of the active site. In order to further understand the reasons for the enhanced pod-like activity of PDA-HEA, we used DFT calculation methods to conduct theoretical studies on HEA and PDA-HEA. According to the relevant characterization results such as ICP and XRD, the HEA structure and reaction site with the lowest energy and the best stability were selected as the theoretical research model, and the PDA-HEA model was designed on this basis. The total density of states (TDOS) shows that both PDA-HEA and HEA show rich electron distribution near the Fermi level (EF). After modification with PDA, the electronic structure of HEA changed, and the electron abundance was higher at the EF.

[0124] Subsequently, we used the partial predicted density of state (PDOS) of HEA and PDA-HEA to analyze the active sites of their overall structures, revealing the synergistic effect between different metals. First, there is a significant overlap between the d orbitals of different metals, indicating that there is a strong inter-bonding interaction between them. Among them, the three transition metals Fe, Co, and Ni show a wide band in the middle, with more orbital overlap, verifying the strong coupling of the three-dimensional orbitals of the transition metals. In addition, the Pt-5d orbital is located near -5 eV, which is the orbital farthest from EF. This indicates that during the catalytic process, the surface Pt plays the role of an electron reservoir and balances the valence state of HEA. It is worth noting that the highest peak of Ru is at -2.2 eV, which is closest to EF. At the same time, Pt also has a sharp peak near EF. The electronic structure of the Pt-Ru bimetallic site enhances the electron density at EF, which is more conducive to the adsorption and transfer of substrates. Therefore, the synergistic complementarity of the three transition metals in HEA and the Pt-Ru bimetallic leads to the lattice distortion and uneven bonding orbitals of HEA. Through electron transfer and orbital hybridization, it provides abundant active sites for its catalytic activity. In addition, PDA-HEA also shows a similar trend of metal element distribution, indicating that the modification of PDA does not affect the internal element binding and synergistic effect of HEA.

[0125] Figure 5E and 5H depict the overall structure of HEA and PDA-HEA and the d-band centers of the five internal metal elements. According to the d-band center theory, for HEA, Ru (spin up, 0.39 eV; spin down, 0.98 eV), the higher the d-band center position, the stronger the adsorption of reactants. This is because the electron cloud is farther away from the electron nucleus, more "diffuse", and more easily reacts with molecules. Crucially, the d-band center of Pt is at a lower energy level (spin up, -0.656 eV; spin down, -0.446 eV) and has a higher electron cloud density, indicating that it is more likely to provide electrons in catalytic reactions and promote electron transfer, which is consistent with its role as an electron reservoir. Based on d-band model, the d-band center of the metal determines the location of the electron donor-acceptor. There is a significant difference in the d-band center of the Pt-Ru dual site, with the Ru center having a higher energy and the Pt center having the lowest energy. This energy difference provides the driving force for the transfer of electrons from Ru to Pt, which is confirmed by experimental electron transfer. The self-complementary induction of the two metals makes the substrate and its intermediates more stably adsorbed. The three transition metals, Fe, Co and Ni, have higher energy in the spin-down state and lower energy in the spin-up state. These transition metals can synergize with Pt-Ru to promote electron transfer and play a catalytic role. After PDA modification, although the change trend of the d-band center of the metal elements in the overall catalyst remains unchanged, the overall structure of PDA-HEA and the d-band centers of the five metals become more negative compared with HEA. This indicates that compared with HEA, the electron density on the surface of PDA-HEA is relatively increased, and it is easier to dissociate and transform the substrate on the basis of stable adsorption. In addition, the more negative d-band center of PDA-HEA changes the donor-acceptor properties between it and the substrate, making it easier to provide / accept electrons, enhance electron transfer, and facilitate the reaction. Therefore, the modification of PDA has no effect on the internal structure of HEA. Instead, it fine-tunes the d-band center of HEA and optimizes the electron cloud density on the surface, further improving its catalytic performance.

[0126] The pod-like activity mechanism of PDA-HEA and HEA was further explored. According to the study, there are two cleavage mechanisms for the catalytic decomposition of H2O2, namely homolysis and heterolysis, such as Figure 5As shown in B, under the catalysis of PDA-HEA and HEA, H2O2 is homolytically and heterolytically decomposed to eventually generate ·OH, which catalyzes the oxidation of TMB to blue. We first compared the TDOS spectra of PDA-HEA and HEA after adsorbing H2O2. PDA-HEA has a higher electron density at EF than HEA. Similar results were found in the TDOS data of PDA-HEA and HEA after decomposing and adsorbing ·OH. These results demonstrate that PDA-HEA has more electronic states available for electron occupation and electronic transitions compared with HEA. In order to evaluate the adsorption capacity of PDA-HEA and HEA for H2O2 in more detail, the PDOS diagram clearly shows that when H2O2 is adsorbed on the surface of PDA-HEA and HEA, the s orbital and p orbital of the adsorbed H2O2 have obvious overlap with the D orbital of the adjacent metal, indicating that both catalysts can stably adsorb H2O2 ( Figure 5 C, D and 5F, G). In addition, the adsorption of the intermediate ·OH on PDA-HEA and HEA also showed similar results.

[0127] According to the dissociation mechanism of H2O2, we calculated the Gibbs free energy changes of each step in the catalytic process, which provided a more intuitive comparison of the catalytic activities of the two catalysts. Figure 5 As shown in Figure 1, the adsorption energies of HEA and PDA-HEA are both negative (HEA, -0.754 eV; PDA-HEA, -0.279 eV), further proving that both can stably adsorb H2O2. According to the Sabatier principle, for an ideal catalyst, the adsorption of reactants on the catalyst surface cannot be too strong or too weak. Excessive adsorption will cause the reactants to adsorb too tightly, which is not conducive to the desorption of the products. In contrast, weak adsorption is not conducive to the adsorption of reactants. PDA modification makes the d-band center more negative, optimizing the adsorption state of reactants and intermediates. It ensures that the adsorption will not be too stable, preventing decomposition and dissociation, nor will it be too weak to effectively catalyze decomposition, thereby achieving the optimal catalytic state ( Figure 5 I) Subsequently, the peroxide bonds in H2O2 are evenly broken to generate two OH( Figure 5J). Crucially, the rate-determining step (RDS) of the catalytic decomposition of H2O2, i.e., the slowest step of the reaction, is the key factor in determining the rate of the catalytic reaction. The superiority of the catalyst is reflected in the reduction of its RDS activation energy, especially the step with the highest energy barrier, the formation of ·OH from the OH* intermediate. The energy barrier spanned by the rate-determining step (RDS) of the homogeneous decomposition of H2O2 catalyzed by PDA-HEA to form ·OH is significantly lower than that of HEA (PDA-HEA, ΔE = 0.741; HEA, ΔE = 2.207), which may be the reason for the faster catalytic rate of PDA-HEA. We also studied the free energy changes of each step in the heterogeneous decomposition of H2O2 catalyzed by the two catalysts. Similarly, for PDA-HEA, the energy barrier required to generate the first ·OH is higher (ΔE = 1.062), which is the rate-determining step (RDS) of the heterogeneous decomposition of H2O2. For HEA, the energy barrier required for the RDS of the heterogeneous decomposition of H2O2 is (ΔE = 1.785). This indicates that PDA-HEA still exhibits good catalytic performance in catalyzing the heterogeneous decomposition of H2O2.

[0128] In general, for HEA, the RDS of heterolytic cleavage is lower than that of homolytic cleavage, indicating that in HEA, the catalytic effect may be achieved by catalyzing the heterolytic cleavage of H2O2 to generate ·OH. For PDA-HEA, the RDS of homolytic cleavage is lower than that of heterolytic cleavage, indicating that for PDA-HEA, the catalytic effect of homolytic cleavage of H2O2 plays a dominant role. Of course, whether it is homolytic or heterolytic, the energy barrier required for PDA-HEA to catalyze the decomposition of RDS of H2O2 is smaller than that of HEA. The relevant results of DFT clarify the relationship between the PDA modification to improve the catalytic performance of HEA and the Sabatier principle, that is, by optimizing the charge distribution on the HEA surface and appropriately reducing the energy of the d-band center to balance the coverage of the products, the adsorption-dissociation efficiency is improved, thereby improving its pod-like activity.

[0129] 3.6 Construction of PDA-HEA-based biosensor

[0130] Acetylcholinesterase (AChE) can terminate the signal transmission of neurotransmitters in the synaptic cleft and is widely used as a biomarker for disease diagnosis and treatment and biosensing. Acetylthiocholine (ATCh) can be specifically decomposed into thiocholine (TCh) by AChE. TCh, as a strong reducing agent, can reduce blue oxTMB to colorless TMB. Based on this principle, in order to explore the potential application of PDA-HEA, we constructed a PDA-HEA-based biosensor for the visual detection of AchE ( Figure 6 A). Figure 6 As shown in B, when ATCh and AChE coexist, the absorbance of the color system at 652nm decreases significantly, and ATCh and AChE alone have little effect on the catalytic activity of PDA-HEA. In order to achieve the best performance of the biosensor, the concentration of acetylcholinesterase was optimized. As the concentration of AChE increases, the absorbance of oxTMB gradually decreases ( Figure 6 C). In the range of AChE content of 0.1 to 1.1 mU / mL, it showed a good linear relationship with the absorbance change (ΔA652), and the detection limit (LOD) was 0.064 mU / mL ( Figure 6 D). Compared with the previously reported AChE detection sensing platform, it exhibits an ultra-low LOD, which also proves the excellent catalytic performance of PDA-HEA from another perspective. Subsequently, the specificity of AChE detection was studied. Horseradish peroxidase (HRP), glucose oxidase (GOx), enkephalinase (NEP), acid phosphatase (ACP), papain, bovine serum albumin (BSA) and other proteins were selected as interfering substances. In the presence / absence of AChE, the effects of these proteins on the absorbance of the colorimetric system were basically negligible, indicating that the sensing platform showed good specificity for AChE detection.

[0131] Compared with amino acid ester pesticides, a large number of organophosphorus pesticide (OPs) detection sensing platforms based on nanozymes have been developed. Amino acid ester pesticides are widely used worldwide for pest control of crops such as fruits and vegetables. They can accumulate in the environment and cause damage to the human central nervous system by inhibiting the activity of acetylcholinesterase (AChE). Therefore, their detection is of great significance. Based on the inhibitory effect of amino acid ester pesticides on acetylcholinesterase, we selected methomyl as a typical carbamate pesticide and detected it using the biosensor constructed in this article. Figure 6 As shown in Figure E, the introduction of pure methomyl has little effect on the change of absorbance, eliminating its own interference. As the concentration of methomyl increases, the activity of AChE decreases, and then the suppressed absorbance recovers ( Figure 6 F). Methomyl showed a good linear relationship in the concentration range of 0.4-40 and 150-1000 ng / mL, and the LOD was 0.624 ng / mL ( Figure 6 G). At the same time, the detection performance of this method for methomyl is better than other methods. Considering the complexity of the actual environment, we studied the specificity of methomyl detection and selected different ions and other types of pesticides as interfering substances ( Figure 6 J). Again, the sensor exhibited considerable selectivity in the presence / absence of these interfering substances.

[0132] In addition, in order to explore the role of photothermal conversion effect in the detection of methomyl, we carried out the same detection process as above under NIR conditions. The linear range of the sensing platform for the detection of methomyl under NIR treatment is 0.1-5, 10-2000 ng / mL, and the LOD is only 0.068 ng / mL, which is 10 times lower than that without NIR irradiation ( Figure 6 H and I). These results all indicate that the photothermal conversion effect improves the sensitivity of methomyl detection by enhancing the catalytic performance of PDA-HEA.

[0133] 3.7 Portable detection platform based on water-in-gel@PDA-HEA combined with smartphone

[0134] Sodium alginate (SA) is a natural polysaccharide derived from seaweed. It is less irritating and less toxic to organisms. SA contains a large number of carboxyl groups. 2+ In the presence of 2+ A cross-linking reaction occurs to form a three-dimensional network hydrogel structure, which fixes the nanozyme and color reagent inside, plays a role in screening size, and ensures the stability of color development. In addition, the high hydrophilicity and hydration capacity of SA make it more sensitive and rapid in responding to target analytes. Based on this, we chose SA hydrogel to encapsulate PDA-HEA (Hydrogel@PDA-HEA) in a 96-well plate and integrate it with a smartphone to build a portable high-throughput detection platform (such as Figure 7 A). Figure 7 B shows the construction and injectability of Hydrogel@PDA-HEA. Color changes can be observed by adding TMB and H2O2, while the addition of PDA-HEA has little effect on the color development, thus demonstrating the feasibility of the Hydrogel@PDA-HEA sensor for colorimetric detection. SEM images of the internal morphology of the SA hydrogels revealed by freeze drying show a favorable porous structure. The SEM images of Hydrogel@PDA-HEA also show a similar porous mesh structure. Upon magnification, black PDA-HEA loading can be observed within the cavity ( Figure 7 C). Thanks to the ultrastructure of PDA-HEA, it is relatively easy to load PDA-HEA without destroying the original organizational structure of the hydrogel, which also lays the foundation for the catalytic performance of Hydrogel@PDA-HEA.

[0135] In a 96-well plate containing Hydrogel@PDA-HEA, TMB and H2O2 were added in sequence, followed by the gradual introduction of ATCh and different concentrations of AChE, and the resulting color signal gradually weakened. The signal was collected by taking photos with a smartphone, and the color information of the photos was analyzed by RGB. Figure 7 As shown in D and E, in the range of 0 to 12 mU / mL, the B / R value showed an excellent linear relationship with the AChE concentration. Similarly, after adding methomyl on the basis of the above, the color development of Hydrogel@PDA-HEA gradually recovered with the increase of methomyl concentration. The B / G value of methomyl showed a good linear relationship in the range of 0 to 2 ng / mL. These results confirm that the visualization biosensing platform constructed based on Hydrogel@PDA-HEA has a certain detection feasibility. Interestingly, thanks to the three-dimensional network structure of the hydrogel and the flexibility of the molecular chain, Hydrogel@PDA-HEA can be cross-linked and cured using different molds to obtain a hydrogel product consistent with the shape of the mold. Considering that methomyl is mainly used in agricultural applications, we cleverly designed Hydrogel@PDA-HEA into the shapes of strawberries, pears and apples, and used TMB, OPD and ABTS as chromogenic substrates, which were successfully applied to the detection of methomyl ( Figure 7 F).

[0136] 4 Conclusion

[0137] In summary, we preliminarily designed and synthesized PtRuFeCoNi HEA nanoparticles. Subsequently, we used a surface modification strategy to modify PDA on the surface of HEA, and further improved the enzyme-like activity through the photothermal conversion effect. DFT results explained the reasons for the improved catalytic performance based on the Sabatier principle and PDA modification. On the basis of the inherent structure of HEA, the coating of PDA caused the d-band center to shift downward, the surface electron abundance to increase, and the electron mobility to enhance, thereby promoting the adsorption-desorption conversion of the substrate and achieving improved catalytic efficiency. In addition, PDA-HEA was used as a biosensor to analyze acetylcholinesterase and methomyl, and a smartphone-assisted hydrogel detection platform was established to achieve rapid visual detection. In summary, this paper introduced the PDA surface modification strategy into the field of high-entropy nanozymes, verified the relationship between the Sabatier principle and the photothermal conversion effect that enhanced POD-like activity, and provided a new perspective for the design and application of complex high-entropy nanozymes.

[0138] Example 2 Specific Application

[0139] In order to verify the feasibility and practicality of PDA-HEANWs in actual samples, the residues of methomyl in pears, apples and strawberries were analyzed. The spike recovery rate was used to evaluate the precision of the detection method. The samples needed to be pretreated before detection as follows: 2 mg of pears were placed in 10 mL of ethanol solution, fully shaken for 5 minutes, and then sonicated for 2 hours. Subsequently, 5 μL of ChE (0.55 mU / mL), 10 μL of TCh (10 mM) and 5 μL of different concentrations of methomyl were incubated at 37 °C for 30 min. Then, 1965 μL of fruit supernatant, 5 μL of PDA-HEANWs (2 mg mL-1), 2 μL of H2O2 (30%) and 8 μL of TMB (10 mM) were added to the mixture. The absorbance spectrum was measured after 30 minutes of reaction at 25 °C. Apples and strawberries were treated as above.

[0140] Table 3. PDA-HEA detection results of methomyl in actual samples

[0141]

[0142] Table 3 a Recovery rate (%) = 100 × ((c 平均检测量 -c0) / c 加标量 ).

[0143] From the results in Table 3, it can be seen that for the actual sample detection of methomyl, the recovery rate is above 95%, and the relative standard deviation is within 3.5%, which proves that the nanozyme detection in the present invention has considerable feasibility and practicality in actual samples, and provides development prospects for portable detection of carbamate pesticides.

Claims

1. A method for preparing a high entropy alloy nanozyme PDA-HEA with peroxidase-like activity, characterized in that: The steps include: S1. Preparation of PtRuFeCoNi HEA nanowires by low-temperature oil phase method Take metal precursor raw materials of Pt, Ru, Fe, Co, Ni, a structure directing agent, and a reducing agent, add them to solvent A, mix them thoroughly, heat the solution to 70-90° C. and keep it for 4-9 minutes under stirring conditions, then heat the solution to 200-240° C. and keep it for 80-100 minutes, cool it, collect the precipitate by centrifugation, wash it with a detergent, collect the precipitate, and dry it to obtain PtRuFeCoNi HEA nanowires; The metal precursor is an acetylacetone metal complex; The structure directing agent is selected from hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium chloride, and dodecyltrimethylammonium chloride; The reducing agent includes Mo(CO)6 and glucose; S2. Synthesis of PDA-HEA The PtRuFeCoNi HEA nanowires prepared in step S1 are dispersed in solvent B and mixed thoroughly. Dopamine and Tris-HCl solution are added thereto and stirred until the color of the solution turns black. The solution is centrifuged to obtain the precipitate, which is the high entropy alloy nanozyme PDA-HEA.

2. The preparation method according to claim 1, characterized in that: The mass ratio of each raw material is: metal precursor of Pt: metal precursor of Ru: metal precursor of Fe: metal precursor of Co: metal precursor of Ni: structure directing agent: Mo(CO)6: glucose is 6-10:8-12:4-8:4-8:4-8:50-70:60-72:8-12, and the mass volume ratio of structure directing agent to solvent A is 50-70 mg:3-5 mL; In step S1, each raw material is added to solvent A and then uniformly mixed by ultrasound, and the ultrasound conditions are: power 100-400W, time 1.5-2.5h; The solvent A is oleylamine; The detergent is a mixture of ethanol and cyclohexane, preferably the detergent is a mixture of ethanol and cyclohexane in a volume ratio of 7-11:1; The solvent B is ethanol.

3. The preparation method according to claim 1, characterized in that: In step S2, the raw materials are mixed according to the ratio of PtRuFeCoNi HEA nanowires, solvent B, dopamine, and 1M Tris-HCl solution of 1.5-2.5 mg: 6-12 mL: 0.2-0.4 mg: 0.8-1.2 mL; After adding PtRuFeCoNi HEA nanowires to solvent B, ultrasonic treatment is used to fully mix them. The ultrasonic conditions are: power 100-400W, time 20-40min; after all the raw materials are mixed, stir for 5-7h until the color of the solution turns black, centrifuge, and the resulting precipitate is the high entropy alloy nanozyme PDA-HEA; Preferably, the metal precursor raw materials are (Pt(a-cac)2), (Ru(acac)3), (Fe(acac)3), (Co(a-cac)3) and (Ni(acac)2), and the structure directing agent is hexadecyltrimethylammonium bromide.

4. A high entropy alloy nanozyme PDA-HEA with peroxidase activity, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 3.

5. A biosensor based on PDA-HEA, characterized in that: The high entropy alloy nanozyme PDA-HEA described in claim 4 is loaded onto a hydrogel to prepare the hydrogel; preferably, the hydrogel is a sodium alginate hydrogel or a hyaluronic acid silicone gel.

6. The method for preparing the PDA-HEA-based biosensor according to claim 5, characterized in that: The steps include: S1, taking a hydrogel monomer, a cross-linking agent, the high entropy alloy nanozyme PDA-HEA described in claim 4 and water, mixing them evenly to obtain a hydrogel mixed solution, wherein the hydrogel monomer is selected from sodium alginate or sodium hyaluronate; the cross-linking agent of the sodium alginate hydrogel is CaCl2; the raw materials of the hyaluronic acid silicone gel include sodium hyaluronate and medical silicone; Preferably, the hydrogel monomer is selected from sodium alginate, the cross-linking agent is CaCl2, and the components in the hydrogel mixed solution are mixed according to the following mass ratios: the mass ratio of hydrogel monomer: cross-linking agent: PDA-HEA: water is 6-10: 0.10-0.14: 0.010-0.014: 1; Preferably, the hydrogel monomer is first added into water, heated to dissolve, and then a cross-linking agent is added, mixed, and then PDA-HEA is added and mixed to obtain a hydrogel mixed solution; S2. Add the prepared mixed solution into a mold, and let it stand at 4-10° C. to solidify and form the biosensor in a hydrogel state.

7. Application of the high entropy alloy nanozyme PDA-HEA according to claim 4 or the PDA-HEA-based biosensor according to claim 5 in detecting acetylcholinesterase, amino acid ester pesticides, and organophosphorus pesticides; the amino acid ester pesticides include methomyl, isoprocarb, aldicarb, and carbofuran.

8. The use according to claim 7, characterized in that: The application method of the high entropy alloy nanozyme PDA-HEA for detecting acetylcholinesterase according to claim 4 comprises the following steps: S1. Mix and incubate acetylcholinesterase solutions of known different concentrations with acetylthiocholine solutions, add NaAc buffer, PDA-HEA, TMB solution and H2O2 solution to the mixed solution after incubation to form an ATCh-TMB-H2O2 detection system, and measure the absorption spectrum after sufficient reaction (preferably measuring the absorption spectrum at 652 nm); S2. Obtain the absorbances corresponding to acetylcholinesterase solutions of different concentrations respectively, and use the concentration of acetylcholinesterase as the horizontal axis and the change in absorbance as the vertical axis to obtain an absorbance change-concentration standard curve; S3, take the acetylcholinesterase to be tested, detect its absorbance according to the same method, and calculate its concentration by comparing with the absorbance change-concentration standard curve; preferably, irradiate PDA-HEA with 808nm near-infrared laser for 4-6min and then operate according to steps S1-S3; The application method of the high entropy alloy nanozyme PDA-HEA for detecting methomyl according to claim 4 comprises the following steps: S1. Mix an acetylcholinesterase solution of known concentration with an acetylthiocholine solution and incubate them, add different concentrations of methomyl, NaAc buffer, PDA-HEA, TMB solution and H2O2 solution to the mixed solution after incubation to form an ATCh-TMB-H2O2 detection system, and measure the absorption spectrum after sufficient reaction (preferably measuring the absorption spectrum at 652 nm); S2, respectively obtain the absorbance corresponding to the different concentrations of methomyl solution, take the concentration of methomyl as the abscissa and the change of absorbance as the ordinate, and obtain the absorbance change-concentration standard curve; S3, take the methomyl to be tested, detect its absorbance according to the same method, and calculate its concentration by comparing with the absorbance change-concentration standard curve; preferably, irradiate PDA-HEA with 808nm near-infrared laser for 4-6min and then operate according to steps S1-S3.

9. The use according to claim 7, characterized in that: The application method of the biosensor for detecting acetylcholinesterase according to claim 5 comprises the following steps: ATCh and AChE standards of different concentrations are mixed and incubated to obtain a standard incubation solution, ATCh and a sample to be tested are mixed and incubated to obtain a sample incubation solution, and then the standard incubation solution and the sample incubation solution are respectively added dropwise to different detection units of the biosensor according to claim 5, and then H2O2 and TMB are added to react fully, and the gel color change of the detection unit is recorded, and the content of AChE in the sample to be tested is judged and analyzed according to the color change of the detection unit to which the standard incubation solution is added; The application method of the biosensor for detecting methomyl according to claim 5 comprises the following steps: AChE and ATCh are mixed and incubated, and the incubation solution is dripped onto the biosensor according to claim 5, followed by dripping methomyl, H2O2 and TMB, and different concentrations of methomyl standards and samples to be tested are dripped into different detection units of the biosensor, and reacted fully, and the gel color change of the detection unit is recorded, and the content of methomyl in the sample to be tested is judged and analyzed according to the color change of the detection unit to which the methomyl standard is added.

10. The use according to claim 9, characterized in that: The linear range of AChE detection is 0.1-12mU / mL; the linear range of methomyl detection is 0.45-2ng / mL.

Images