Polymer nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses a polymer nano-enzyme as well as a preparation method and application thereof. The nano-enzyme comprises a nano-enzyme core modified by a surface modifier and a dendritic polymer coupled with the surface modifier. The preparation method of the polymer nano-enzyme comprises the following steps: preparing a nano-enzyme precursor, and then washing the nano-enzyme precursor to obtain a first intermediate; the nano-enzyme precursor is a nano-enzyme core modified by a surface modifier; preparing a first intermediate dispersion liquid, and adding an activator dispersion liquid and a stabilizer dispersion liquid into the first intermediate dispersion liquid to obtain an activation system; the invention also discloses an application of the polymer nano-enzyme. The polymer nano-enzyme provided by the invention can be used as an enzyme label substitute to be widely applied to enzyme-linked immunosorbent assay, immunohistochemistry, immunochromatography, chemiluminescence and other biochemical detection methods, and signal amplification with high sensitivity, wide pH and high temperature tolerance is realized.

Description

Technical Field

[0001] This invention relates to the field of functional nanomaterials technology. Specifically, it relates to a polymeric nanozyme, its preparation method, and its applications. Background Technology

[0002] Nanozymes are a class of nanomaterials with catalytic activities similar to those of natural enzymes. Due to their simple synthesis, high stability, and low cost, they have attracted widespread attention in recent years in fields such as biosensoring, environmental monitoring, and biomedicine. However, there is still room for improvement in the catalytic activity of nanozymes, especially in complex systems where their activity is easily affected by environmental factors, limiting their practical applications.

[0003] Polyamide-amine (PAMAM) dendritic polymers, with their highly branched three-dimensional structure and abundant surface functional groups, have been widely used in drug delivery, gene transfection, and nanomaterial modification. PAMAM's unique structural properties make it a potential regulator of the catalytic activity of nanozymes. However, research on the role of PAMAM in enhancing the catalytic activity of nanozymes is relatively limited, and no systematic report on the mechanisms by which PAMAM enhances nanozyme activity has been found. Therefore, how to effectively utilize the structural properties of PAMAM to improve the catalytic performance of nanozymes and provide new insights for the functionalization and application expansion of nanozymes remains a current research hotspot and challenge. Summary of the Invention

[0004] Therefore, the technical problem to be solved by this invention is to provide a polymeric nanozyme, its preparation method, and its applications. This nanozyme can be widely used as an enzyme label substitute in enzyme-linked immunosorbent assays (ELISA), immunohistochemistry, immunochromatography, chemiluminescence, and other biochemical detection methods, achieving signal amplification with high sensitivity, wide pH range, and high temperature tolerance.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0006] A polymeric nanozyme includes a nanozyme core modified with a surface modifier and a dendritic polymer coupled with the surface modifier.

[0007] The core of the nanozyme is composed of nanoparticles made of gold, platinum, iron tetroxide, or ruthenium.

[0008] The surface modifier is a mixture of one or two or more carboxyl-containing polymers, wherein each carboxyl-containing polymer contains at least one carboxyl functional group;

[0009] The dendritic polymer is a polyamide-amine dendritic polymer, and the generation of the polyamide-amine dendritic polymer is 4.0 to 7.0 generations.

[0010] The algebra of PAMAM refers to the number of branching layers that the molecule expands layer by layer from the core through chemical synthesis. The type of PAMAM core does not affect the preparation of polymer nanozymes.

[0011] The above-mentioned polymer nanozyme has an average particle size of 1-20 nm for the nanozyme core; an average molecular weight of 1500-30000 Da for the surface modifier; and a mass ratio of the nanozyme core modified by the surface modifier to the dendritic polymer of (50-5000):1.

[0012] When the molecular weight of the surface modifier is too small, its electrostatic or steric hindrance effects in solution are weak, which may cause the prepared nanozyme precursor to aggregate in solution. When the molecular weight of the surface modifier is too high, the viscosity of the solvothermal reaction system is large, resulting in uneven local concentration and temperature, leading to anisotropic growth of nanoparticles. The morphology and particle size of the nanoparticles vary greatly, making it difficult to obtain particles with the same morphology and uniform particle size.

[0013] Preferably, the nanozyme core is platinum nanoparticles, the surface modifier is polyacrylic acid, the average relative molecular mass of the polyacrylic acid is 2000 Da, and the generation number of the polyamide-amine dendritic polymer is 5.0; in the polymer nanozyme, the mass ratio of the nanozyme core modified by the surface modifier to the dendritic polymer is 1000:1.

[0014] The surface modifiers of the aforementioned polymer nanozymes are one or a mixture of two or more of the following: polyacrylic acid, polymethacrylic acid, polymaleic acid, polylactic acid-glycolic acid copolymer-carboxyl group, polyethylene glycol-carboxylated derivative, polyaspartic acid, and polyglutamic acid.

[0015] A method for preparing a polymeric nanozyme includes the following steps:

[0016] S1. Prepare nanozyme precursor, then wash nanozyme precursor to obtain first intermediate; nanozyme precursor is nanozyme core modified with surface modifier.

[0017] S2. Prepare the first intermediate dispersion. Add the activator dispersion and the stabilizer dispersion to the first intermediate dispersion to obtain the activation system. Stir the activation system, centrifuge, retain the precipitate, and wash the precipitate under ultrasonic conditions to obtain the second intermediate.

[0018] S3. Prepare a second intermediate dispersion, add a dendritic polymer dispersion dropwise to the second intermediate dispersion to obtain a coupling system; stir the coupling system to carry out a coupling reaction to obtain a third intermediate dispersion.

[0019] S4. Centrifuge the dispersion of the third intermediate, retain the precipitate, and resuspend the precipitate in the sealing solution for sealing incubation;

[0020] S5. After the closed incubation is completed, centrifuge and retain the precipitate to obtain the above-mentioned polymer nanozyme.

[0021] In the above-mentioned method for preparing polymeric nanozymes, the preparation method of the nanozyme precursor in step S1 is as follows:

[0022] The metal hydrate and surface modifier were dissolved in ethylene glycol or water, and anhydrous sodium acetate was added to adjust the pH to obtain a mixed dispersion. The mixed dispersion was subjected to a solvothermal reaction under heating conditions. After the reaction was completed, the obtained solid product was washed to obtain the nanozyme precursor.

[0023] The metal hydrate is chloroplatinic acid hexahydrate, ferric chloride hexahydrate, tetrachloroauric acid tetrahydrate, or ruthenium trichloride hydrate; the mass ratio of the metal hydrate to the surface modifier is 1:(2.5-10); when the metal hydrate is chloroplatinic acid hexahydrate, tetrachloroauric acid tetrahydrate, or ruthenium trichloride hydrate, the solvent is ethylene glycol; when the metal hydrate is ferric chloride hexahydrate, the solvent is water.

[0024] When the amount of surface modifier is too small, the active sites of the nanozyme core are overexposed and easily bind nonspecifically to impurities or proteins; when the amount of surface modifier is too large, the active sites of the nanozyme core are "buried" in the surface modifier shell and have difficulty contacting the substrate.

[0025] Preferably, when using chloroplatinic acid hexahydrate, tetrachloroauric acid tetrahydrate, or ruthenium trichloride hydrate to prepare nanozyme precursors, chloroplatinic acid hexahydrate, tetrachloroauric acid tetrahydrate, or ruthenium trichloride hydrate and a surface modifier are dissolved together in ethylene glycol, anhydrous sodium acetate is added, and the pH is adjusted to 9-13 to obtain a mixed dispersion. The mass fraction of the metal hydrate in the mixed dispersion is 2-5 wt%. The solution is reacted at 160-180°C for 5-10 h, and the resulting product is obtained after washing with water or ethanol and centrifugation.

[0026] Preferably, when using ferric chloride hexahydrate to prepare nanozyme precursors, ferric chloride hexahydrate and the surface modifier are dissolved together in water, anhydrous sodium acetate is added, and the pH is adjusted to alkaline to obtain a mixed dispersion; the solution is placed at 120°C for 10 hours to react, and the resulting product is obtained after washing with pure water and centrifuging.

[0027] In the above-described method for preparing polymeric nanozymes, in step S1, the obtained nanozyme precursor is dispersed in a 2-morpholine ethanesulfonic acid solution to prepare a nanozyme precursor dispersion. The nanozyme precursor dispersion is then centrifuged, the precipitate is retained, and the precipitate is washed with 2-morpholine ethanesulfonic acid solution under ultrasonic conditions to obtain a first intermediate. The pH of the 2-morpholine ethanesulfonic acid solution is 4-7, and the concentration is 0.01-0.05M. In the nanozyme precursor dispersion, the mass concentration of the nanozyme precursor is 0.1-5.0 mg / mL, preferably 0.8-1.2 mg / mL.

[0028] First, the nanozyme precursor is prepared into a nanozyme precursor dispersion to prevent the nanozyme precursor from agglomerating and causing incomplete washing and ineffective removal of impurities. After centrifuging the nanozyme precursor dispersion, the precipitate is washed and ultrasonically treated during washing. The purpose is to replace the original nanozyme preservation solution with the solvent required for the reaction.

[0029] Preferably, after centrifuging the nanozyme precursor dispersion, the precipitate is ultrasonically washed twice at a ratio of 5 mL / mg of 2-morpholine ethanesulfonic acid solution volume to precipitate mass, with an ultrasonic power of 240 W and an ultrasonic time of 5 min.

[0030] In the above-described method for preparing polymeric nanozymes, in step S2, when preparing the first intermediate dispersion, the dispersion medium used is a 2-morpholine ethanesulfonic acid solution with a pH of 4-7 and a concentration of 0.01-0.05M; after dispersing the first intermediate in the 2-morpholine ethanesulfonic acid solution, it is subjected to ultrasonic treatment to obtain the first intermediate dispersion; the ultrasonic time is 30-60 min, and the ultrasonic power is 240-300 W; in the first intermediate dispersion, the mass concentration of the first intermediate is 0.1-5.0 mg / mL, preferably 0.8-1.2 mg / mL;

[0031] The activator dispersion is a dispersion of 1-ethyl-(3-dimethylaminopropyl)carbodiimide with a mass concentration of 15-40 mg / mL, and the stabilizer dispersion is a dispersion of N-hydroxysuccinimide with a mass concentration of 40-80 mg / mL.

[0032] The volume ratio of the first intermediate dispersion, the activator dispersion, and the stabilizer dispersion is controlled so that the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and the first intermediate in the activation system is 1:(1-5):(10-20); the activation time of the system is 20-40 min.

[0033] In the above-described method for preparing polymeric nanozymes, in step S3, when preparing the second intermediate dispersion, the dispersion medium used is a 2-morpholine ethanesulfonic acid solution with a pH of 4-7 and a concentration of 0.01-0.05M. After dispersing the second intermediate in the 2-morpholine ethanesulfonic acid solution, it is subjected to ultrasonic treatment to obtain the second intermediate dispersion. The ultrasonic time is 5-30 min, and the ultrasonic power is 240-300 W. In the second intermediate dispersion, the mass concentration of the second intermediate is 0.1-5.0 mg / mL, preferably 0.8-1.2 mg / mL. When preparing the first intermediate dispersion, the longer ultrasonic time is beneficial to the full dispersion of the first intermediate and to maintain a good dispersion state of the first intermediate in the activation system. Since the first intermediate has been fully dispersed, when preparing the second intermediate dispersion, there is no need to perform long ultrasonic treatment again; only 5-30 min of ultrasonic treatment is required for full dispersion.

[0034] In the dendritic polymer dispersion, the concentration of the dendritic polymer is 5 wt%–10 wt%; in the coupling system, the mass ratio of the second intermediate to the dendritic polymer is (50–5000):1; the coupling reaction time is 0.5–5 h. The amount of dendritic polymer added will affect the activity of the final polymeric nanozyme. When the amount of dendritic polymer added is too large, it will cause the dendritic polymer and the nanozyme (second intermediate) to form an excessively dense network structure, which will block the active sites of the nanozyme core in the second intermediate and have an adverse effect on the activity of the final polymeric nanozyme.

[0035] During the process of the nanozyme precursor being successively transformed into the first intermediate and the second intermediate, the nanozyme precursor, the first intermediate, and the second intermediate are considered to have the same mass; therefore, the mass ratio of the dendritic polymer to the second intermediate is actually equal to the mass ratio of the dendritic polymer to the nanozyme precursor (that is, the nanozyme core modified by the surface modifier).

[0036] Preferably, the mass concentrations of the nanozyme precursor in the nanozyme precursor dispersion, the first intermediate in the first intermediate dispersion, and the second intermediate in the second intermediate dispersion are all the same.

[0037] Preferably, the nanozyme precursor dispersion has a mass concentration of 1.0 mg / mL. Furthermore, during the preparation of the polymeric nanozyme, the mass concentrations of the first intermediate in the first intermediate dispersion and the second intermediate in the second intermediate dispersion are both 1.0 mg / mL. At this concentration, the dispersibility of the nanozyme precursor and the stability of various reaction solutions during preparation are optimal. If the concentration is too low, the efficiency of various reactions during preparation is too low; if the concentration is too high, the nanozyme precursor cannot be effectively dispersed.

[0038] In the above-mentioned method for preparing polymer nanozymes, in step S4, the blocking solution used is a glycine solution, and the amount of glycine in the blocking solution is more than 5 times the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the activation system of step S2; the blocking incubation time is 10-16 h.

[0039] An application of a polymeric nanozyme, using the aforementioned polymeric nanozyme as a catalyst, in any of the following detection methods:

[0040] a) Enzyme-linked immunosorbent assay (ELISA);

[0041] b) Immunohistochemistry;

[0042] c) Immunochromatography;

[0043] d) Chemiluminescence analysis.

[0044] The technical solution of the present invention achieves the following beneficial technical effects:

[0045] 1. In this invention, a stable and highly active catalytic nanozyme is prepared by coupling a dendritic polymer with a surface-modified nanozyme core. Compared with ordinary nanozymes, this polymeric nanozyme exhibits significantly improved dispersibility and stability in aqueous phase and maintains high catalytic activity over a wide temperature and pH range. Specifically, in this invention, platinum, gold, iron tetroxide, or ruthenium nanoparticles are coated with PAA (polyacrylic acid) or other carboxyl-based surface modifiers to obtain a nanozyme precursor. The nanozyme precursor is then coupled with a PAMAM dendritic polymer. Each PAMAM molecule binds multiple nanozyme precursor particles at multiple points, resulting in good dispersibility of the polymeric nanozyme in the liquid phase and full exposure of active sites. Furthermore, PAMAM prevents non-specific adsorption between the polymeric nanozyme and the substrate, enhancing the selectivity of the polymeric nanozyme for the substrate. Experiments show that the polymer nanozyme prepared in this invention has a specific enzyme activity exceeding 300 U / mg and exhibits good sensitivity in ELISA, greatly expanding the application prospects of nanozymes in the field of analytical detection.

[0046] 2. This invention innovatively couples PAMAM dendritic polymers (generations 4.0 to 7.0) with a carboxyl-modified nanozyme core in a high-throughput manner, allowing each PAMAM molecule to bind multiple nanoparticles. This multi-point coupling not only maintains the monodisperse state of the nanoparticles but also results in a highly dense catalytic site, producing a cascade catalytic amplification effect. Furthermore, the polynanozymes obtained through PAMAM treatment exhibit a more compact spherical structure, higher relative density, and more exposed hydrophilic groups compared to natural enzymes. They are fully compatible with the phosphate buffer system of existing ELISA kits and can replace natural enzymes such as HRP without altering standard detection procedures, serving as enzyme labels in various biochemical detection methods, including ELISA. The polymeric nanozyme of this invention, when used to prepare enzyme-labeled antibodies, exhibits a compact structure that reduces fluid resistance, thereby improving membrane penetration and color deposition efficiency. It also shortens the ELISA reaction time to 30 minutes, half the conventional reaction time. Furthermore, it reduces the background signal in ELISA detection by 60%–80%, improves the signal-to-noise ratio by more than 5 times, and increases sensitivity by 1–2 orders of magnitude compared to traditional HRP-ELISA, reaching the pg / mL level. Moreover, compared to the expensive HRP, the production cost of the polymeric nanozyme of this invention can be reduced by approximately 70%, and it can be stored and transported at room temperature, significantly reducing cold chain logistics costs.

[0047] 3. PAMAM exhibits significant advantages in enhancing the chemiluminescence performance of nanozymes. Firstly, its highly branched three-dimensional structure and abundant surface functional groups (such as amino groups) can efficiently load nanozymes and luminescent substrates, significantly increasing local reactant concentrations and accelerating catalytic reactions. Secondly, PAMAM's regular dendritic structure stabilizes the active sites of nanozymes, preventing aggregation and deactivation, while simultaneously optimizing electron transfer efficiency through confinement effects, thus enhancing catalytic activity. Furthermore, surface-modifiable amino groups can synergistically interact with luminescent systems (such as luminol-H₂O₂), amplifying the chemiluminescence signal through electron transfer or energy transfer mechanisms.

[0048] 4. This invention not only solves the key problems of poor stability, high cost and limited sensitivity of natural enzymes in traditional ELISA detection, but also provides an innovative solution for developing a new generation of highly sensitive, highly stable and low-cost immunoassay reagents, which has important application value in the fields of early disease diagnosis, precision medicine and pathological detection. Attached Figure Description

[0049] Figure 1 This is a transmission electron microscope image of the polymer nanozyme prepared in Example 1 of the present invention;

[0050] Figure 2 This is a transmission electron microscope image of the polymer nanozyme prepared in Example 2 of the present invention;

[0051] Figure 3 This is a transmission electron microscope image of the polymer nanozyme prepared in Example 3 of the present invention;

[0052] Figure 4 This is a transmission electron microscope image of the polymer nanozyme prepared in Example 4 of the present invention;

[0053] Figure 5 This is a graph showing the specific enzyme activity of the polymer nanozyme prepared in Example 1 of the present invention;

[0054] Figure 6 This is a graph showing the specific enzyme activity of the polymer nanozyme prepared in Example 2 of the present invention;

[0055] Figure 7 This is a graph showing the specific enzyme activity of the polymer nanozyme prepared in Example 3 of the present invention;

[0056] Figure 8 This is a graph showing the specific enzyme activity of the polymer nanozyme prepared in Comparative Example 1 of the present invention.

[0057] Figure 9 This is a graph showing the specific enzyme activity of the polymer nanozyme prepared in Comparative Example 2 of this invention.

[0058] Figure 10 This is a graph showing the specific enzyme activity of the polymer nanozyme prepared in Comparative Example 3 of this invention.

[0059] Figure 11 This is a graph showing the specific enzyme activity of the polymer nanozyme prepared in Example 4 of the present invention. Detailed Implementation

[0060] To make the objectives, technical solutions, and technical effects of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0061] The following detailed description of a polymer nanozyme, its preparation method, and its application, through specific embodiments, illustrates the present invention.

[0062] Example 1

[0063] In this embodiment, a nanozyme precursor is prepared. The nanozyme precursor is a complex of platinum nanoparticles and polyacrylic acid (PAA), denoted as Pt@PAA.

[0064] The preparation is carried out according to the following method:

[0065] 0.4 g of H₂PtCl₆·6H₂O and 4.0 g of PAA were dissolved in 10 mL of ethylene glycol under ultrasonic assistance. Then, 0.5 g of anhydrous sodium acetate was added to adjust the pH of the system to 9. After dissolution, a mixed dispersion was obtained. This dispersion was transferred to a reaction vessel for reaction at 180℃ for 10 h. After the reaction, the mixture was washed three times with pure water to remove residual PAA and sodium acetate. After each wash, the mixture was centrifuged at 15000 rpm for 10 min. After three washes and centrifugations, the resulting precipitate was Pt@PAA. The precipitate was resuspended in 10 mL of pure water by ultrasonication to obtain the Pt@PAA dispersion.

[0066] During the preparation process, under hydrothermal conditions at 180℃, ethylene glycol (EG) acts as both a solvent and a reducing agent, reducing the Pt in the solution. 4+ (From H2PtCl6·6H2O) reduced to Pt 0 Atom, Pt 0 Atoms nucleate and grow under the coordination of the carboxyl groups of PAA, eventually forming platinum nanoparticles with a diameter of nanometers (i.e., nanozyme cores), which are then coated with PAA (PAA as a surface modifier). These PAA-coated platinum nanoparticles are the nanozyme cores modified with the surface modifier.

[0067] In this embodiment, the mass fraction of Pt in the H2PtCl6·6H2O used should be greater than or equal to 37.5% to ensure the purity of the reagent; the average molecular weight of the polyacrylic acid used is 2000 Daltons (MW~2000). Polyacrylic acid can inhibit the aggregation of platinum nanoparticles. When the molecular weight of polyacrylic acid is small, its electrostatic or steric hindrance effect in solution is weak, which may cause the prepared Pt@PAA to aggregate in solution; while when the molecular weight of polyacrylic acid is too high, the viscosity of the solvothermal reaction system (i.e., the solution containing H2PtCl6·6H2O, PAA and anhydrous sodium acetate) is large, resulting in uneven local concentration and temperature, leading to anisotropic growth of nanoparticles. The morphology and particle size of the nanoparticles vary greatly, making it difficult to obtain particles with the same morphology and uniform particle size.

[0068] Platinum nanoparticles possess activities such as peroxidase. In other embodiments, depending on the desired enzyme activity, the nanozyme core used in the preparation of the nanozyme precursor can be changed, that is, the platinum nanoparticles can be replaced with other nanozymes, such as gold, Fe3O4, or ruthenium nanoparticles. For any nanoparticle used as the nanozyme core, its average particle size should be between 3 and 20 nm (when using the solvothermal method, the particle size of the nanozyme core can be kept within this range by controlling the concentration of the reaction solution, the reaction temperature, and the reaction time); otherwise, the final polymer nanozyme may be too large, affecting the hydrodynamic properties of the polymer nanozyme. If gold nanoparticles are used instead, the nanozyme precursor can be prepared according to the above-described Pt@PAA preparation method, replacing H2PtCl6·6H2O with HAuCl4·4H2O, maintaining a weakly alkaline to neutral pH, and adjusting the reaction temperature and time according to the actual preparation situation.

[0069] Furthermore, when the nanozyme core is replaced, PAA can be replaced with other surface modifiers containing carboxyl groups, such as polymethacrylic acid, polymaleic acid, polylactic-glycolic acid copolymer-carboxyl (PLGA-COOH), polyethylene glycol-carboxylated derivatives (COOH-PEG-COOH), polyaspartic acid, and polyglutamic acid. Different nanozymes and different surface modifiers can be combined in combination, and it is not limited to a certain nanozyme being modified only by a certain surface modifier. Moreover, two or more surface modifiers can be used to modify a nanozyme core. The average molecular weight of the surface modifier is preferably in the range of 1500 to 30000 Da. After these surface modifiers are combined with the nanozyme core, they do not affect the catalytic activity of the nanozyme core itself, and the surface modifiers still have sites that can be coupled with dendritic polymers.

[0070] Furthermore, the aforementioned Pt@PAA is coupled with a dendritic polymer to prepare a polymeric nanozyme. The dendritic polymer used in this embodiment is PAMAM, i.e., polyamidoamine dendritic polymer [Poly(amidoamine), PAMAM], specifically a 4.0 generation PAMAM with an ethylenediamine core, dissolved in methanol at a concentration of 10 wt%.

[0071] The polymeric nanozyme prepared in this embodiment is denoted as Pt@PAA@PAMAM. The preparation method of Pt@PAA@PAMAM includes the following steps:

[0072] 1. Preparation of the first intermediate

[0073] The above-mentioned nanozyme precursor (Pt@PAA) was dispersed in a 0.05M MES (2-morpholinoethanesulfonic acid) solution at pH 6.0 to prepare a 1 mg / mL dispersion, which is the nanozyme precursor dispersion. 4 mL of the nanozyme precursor dispersion was centrifuged at 15000 rpm for 30 min, the supernatant was discarded, and the precipitate was retained. The precipitate was ultrasonically washed twice with a 0.05M MES solution at pH 6.0. During each ultrasonic wash, the volume ratio of the MES solution to the mass of the precipitate was 5 mL / mg. Ultrasonic treatment was performed simultaneously during the soaking and washing process at a power of 240 W for 5 min. After ultrasonication, solid-liquid separation was performed by centrifugation; the solid phase is the first intermediate.

[0074] 2. Preparation of the first intermediate dispersion

[0075] Add 2 mL of MES solution (pH 6.0, concentration 0.05 M) to the first intermediate and treat with ultrasound at 300 W for 1 hour. During ultrasound treatment, place the ultrasound probe below the surface of the MES solution containing the first intermediate. After ultrasound treatment, rinse the ultrasound probe with 2 mL of MES to obtain the first intermediate dispersion. In this step, the first intermediate dispersion is actually prepared by dispersing it in 4 mL of MES solution, resulting in a concentration of 1.0 mg / mL for the first intermediate.

[0076] 3. Preparation of the second intermediate

[0077] 20 μL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) dispersion (activator dispersion) and 20 μL of N-hydroxysuccinimide (NHS) dispersion (stabilizer dispersion) were simultaneously added to the first intermediate dispersion to obtain the activation system. The concentration of the EDC dispersion was 20 mg / mL, and the concentration of the NHS dispersion was 40 mg / mL. EDC acts as the activator, forming an intermediate with the carboxyl group. This intermediate is unstable under acidic conditions and easily hydrolyzes to a carboxyl group. Therefore, NHS acts as the stabilizer, forming an NHS ester with this intermediate, making it less prone to hydrolysis in a short time. Furthermore, the NHS ester releases NHS after forming an amide bond with the amino group, which can be removed by washing. After adding the EDC and NHS dispersions, the activation system was stirred for 30 min to complete the activation.

[0078] After activation, the activated system was centrifuged at 15,000 rpm for 10 min, the precipitate was retained and washed under ultrasonic conditions (the washing method was the same as that used when preparing the first intermediate). After washing, the second intermediate was obtained.

[0079] 4. Preparation of the second intermediate dispersion

[0080] The second intermediate was resuspended in 4 mL of MES solution (the same MES solution used to prepare the dispersion of the first intermediate) and dispersed under ultrasonic conditions for 5 min. The ultrasonic power was 240 W. The concentration of the second intermediate in the resulting dispersion was 1 mg / mL.

[0081] 5. PAMAM Coupling

[0082] 0.7 μL of 10 wt% PAMAM dispersion was slowly added dropwise to the second intermediate dispersion while stirring. After the addition was complete, a coupling system was obtained. The coupling system was stirred for another 4 hours to carry out the coupling reaction. During the coupling reaction, the amino groups on the PAMAM in the coupling system were coupled with the activated carboxyl groups on the second intermediate. Each dendritic PAMAM was bound to multiple second intermediates (i.e., multiple nanozymes). After the coupling reaction was completed, the third intermediate dispersion was obtained.

[0083] PAMAM improves the dispersibility and stability of nanozymes in dispersion systems, allowing more catalytically active sites of the nanozymes to be exposed and contact the substrates within the dispersion. Simultaneously, the compact dendritic structure of PAMAM ensures a small hydrodynamic volume for the final polymeric nanozymes, which enhances membrane permeability and substrate selectivity (PAMAM reduces non-specific adsorption between proteins and nanozymes, preventing competition between proteins and substrates for nanozymes). Furthermore, the addition of PAMAM results in a large number of amino groups on the surface of the prepared polymeric nanozymes. These amino groups protonate at physiological pH, transforming into positively charged -NH groups. 3+ It is easier for negatively charged cell membranes to bind together, thus improving the biocompatibility of polymer nanozymes; and the internal cavity and surface branches of PAMAM can form a "molecular sieve" effect, allowing only substances of specific size or shape to approach the active center of the nanozyme, and further making the prepared polymer nanozymes suitable for preparing enzyme-labeled antibodies with good targeting, thereby significantly improving the application performance of polymer nanozymes.

[0084] 6. Closed incubation

[0085] The dispersion of the third intermediate was centrifuged at 5000 rpm for 5 min, the precipitate was retained, and resuspended in the blocking solution for overnight incubation (12 h). The blocking solution used in this example was a glycine solution with a concentration of 16 mg / mL and a volume of 4 mL. Compared to the amount of carboxyl groups activated by EDC and NHS during the preparation of the second intermediate, the amount of glycine in this step should be at least five times the excess, i.e., the amount of glycine in this step should be at least five times the amount of EDC in the activation system during the preparation of the second intermediate, to ensure complete blocking.

[0086] After the closed incubation was completed, the sample was centrifuged at 5000 rpm, and the precipitate was the prepared polynanozyme Pt@PAA@PAMAM. The prepared polynanozyme could be resuspended in 4 mL of pure water to detect the solid content and enzyme activity. Tests showed that the polynanozyme prepared in this example maintained stable catalytic activity within the pH range of 3–11.

[0087] Example 2

[0088] In this embodiment, the preparation of the polymer nanozyme Fe3O4@HPMA@PAMAM was carried out.

[0089] First, a nanozyme precursor was prepared. In this embodiment, the nanozyme precursor was a complex of Fe3O4 nanoparticles and polymaleic acid (HPMA), which was denoted as Fe3O4@HPMA.

[0090] The nanozyme precursor Fe3O4@HPMA was prepared according to the following method:

[0091] Weigh 1.3 g of FeCl3·6H2O and 6.6 mL of polymaleic acid solution (50 wt% polymaleic acid aqueous solution produced by Shanghai Maclean Biochemical Technology Co., Ltd., catalog number P823909), and dissolve them together in 20 mL of pure water. Then add 3.6 g of anhydrous sodium acetate (to adjust the pH to alkaline). After dissolution, a mixed dispersion is obtained. The mixed dispersion is transferred to a hydrothermal reactor for reaction at 120℃ for 10 h. After the reaction, wash three times with pure water, centrifuging at 15000 rpm for 10 min each time to remove residual HPMA and sodium acetate. Finally, resuspend the centrifuged precipitate in 10 mL of pure water by ultrasonication to obtain a nanozyme precursor (Fe3O4@PMA) dispersion.

[0092] The method for preparing Fe3O4@PMA as Fe3O4@HPMA@PAMAM is the same as in Example 1.

[0093] Example 3

[0094] In this embodiment, the polynanozyme Ru@PAA@PAMAM was prepared.

[0095] First, a nanozyme precursor was prepared. In this example, the nanozyme precursor was a complex of Ru nanoparticles and polyacrylic acid (PAA), which was denoted as Ru@PAA.

[0096] The nanozyme precursor Ru@PAA was prepared according to the following method:

[0097] 0.4 g RuCl3·nH2O and 2.6 g PAA were dissolved in 10 mL of ethylene glycol under ultrasonic assistance. Then, 1.5 g of anhydrous sodium acetate was added to adjust the pH of the system to 13. After dissolution, a mixed dispersion was obtained. This dispersion was transferred to a hydrothermal reactor for reaction at 160℃ for 5 h. After the reaction, the mixture was washed twice with ethanol, centrifuged at 10000 rpm for 10 min each time to remove residual ethylene glycol and PAA. The mixture was then washed once with pure water to remove sodium acetate. Finally, the centrifuged precipitate was resuspended in 10 mL of pure water by ultrasonication to obtain a nanozyme precursor (Ru@PAA) dispersion.

[0098] In this embodiment, the RuCl3·nH2O used is a product of Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity ≥99.95% and catalog number R109234. The PAA used is the same as that used in Example 1.

[0099] The method for preparing Ru@PAA as Ru@PAA@PAMAM is the same as in Example 1.

[0100] Comparative Example 1

[0101] The polymeric nanozyme prepared in this comparative example is Pt@PAA@PAMAM. The preparation method of Pt@PAA@PAMAM in this comparative example is basically the same as that of Pt@PAA@PAMAM in Example 1, the only difference being that in this comparative example, the amount of PAMAM added was changed in the PAMAM coupling step; specifically, 1.4 μL of a 10 wt% PAMAM dispersion was added dropwise to the second intermediate dispersion. All other conditions were the same as in Example 1.

[0102] Comparative Example 2

[0103] The polymeric nanozyme prepared in this comparative example is Fe3O4@PMA@PAMAM. The preparation method of Fe3O4@PMA@PAMAM in this comparative example is basically the same as that in Example 2, except that in the PAMAM coupling step, the amount of PAMAM added was changed; specifically, 1.4 μL of a 10 wt% PAMAM dispersion was added dropwise to the second intermediate dispersion. All other conditions were the same as in Example 2.

[0104] Comparative Example 3

[0105] The polymeric nanozyme prepared in this comparative example is Ru@PAA@PAMAM. The preparation method of Ru@PAA@PAMAM in this comparative example is basically the same as that of Ru@PAA@PAMAM in Example 3, except that the amount of PAMAM added was changed in the PAMAM coupling step; specifically, 1.4 μL of a 10 wt% PAMAM dispersion was added dropwise to the second intermediate dispersion. All other conditions were the same as in Example 3.

[0106] Example 4

[0107] The polymer nanozyme prepared in this embodiment is Pt@PAA@PAMAM. The preparation method of Pt@PAA@PAMAM in this embodiment is basically the same as that of Pt@PAA@PAMAM in Example 1, except that in this embodiment, the generation of PAMAM used is changed in the PAMAM coupling step. 1.4 μL of 5wt% 5.0 generation PAMAM dispersion (5.0 generation PAMAM with ethylenediamine core, dissolved in methanol at a concentration of 5wt%) is added dropwise to the second intermediate dispersion. All other conditions are the same as in Example 1.

[0108] The structures of Pt@PAA@PAMAM (generation 4.0), Fe3O4@PMA@PAMAM, Ru@PAA@PAMAM, and Pt@PAA@PAMAM (generation 5.0) prepared in Examples 1, 2, 3, and 4 were characterized using transmission electron microscopy. All four polymer nanozymes were dispersed in pure water (at the same concentration used in Example 1 for detecting solid content and enzyme activity). Transmission electron micrographs are shown below. Figures 1-4 As shown. Measurements showed that the average particle size of Pt@PAA@PAMAM prepared in Example 1 was 5.3 nm, the average particle size of Fe3O4@PMA@PAMAM prepared in Example 2 was 5.7 nm, the average particle size of Ru@PAA@PAMAM prepared in Example 3 was 4.1 nm, and the average particle size of Pt@PAA@PAMAM prepared in Example 4 was consistent with that of Example 2.

[0109] The above results indicate that the polymer nanozymes prepared in Examples 1-4 have smaller particle sizes, resulting in relatively smaller hydrodynamic volumes. The polymer nanozymes exhibit strong membrane permeability and substrate selectivity.

[0110] like Figure 1 , Figure 2 and Figure 3 Electron micrographs of the polymer nanozymes prepared in Examples 1, 2, and 3, respectively. Figures 1-3It can be seen that Pt@PAA@PAMAM (generation 4.0) and Ru@PAA@PAMAM have good dispersibility, clear particle outlines, and obvious individual aggregates, while Fe3O4@PMA@PAMAM shows more severe agglomeration. Figure 4 Electron micrograph of Pt@PAA@PAMAM (generation 5.0) prepared in Example 4. Figure 4 and Figure 1 Comparative studies show that, compared to the preparation of Pt@PAA@PAMAM using 4.0 generation PAMAM, the preparation of Pt@PAA@PAMAM using 5.0 generation PAMAM improves the dispersibility and alleviates particle overlap and aggregation. However, the average particle size of Pt@PAA@PAMAM (5.0 generation) is slightly larger than that of Pt@PAA@PAMAM (4.0 generation), and differences in hydrodynamic volume and membrane permeability also exist between polymer nanozymes with different particle sizes. In actual production and use, different generations of PAMAM can be selected to prepare polymer nanozymes according to specific needs.

[0111] Furthermore, the specific enzyme activity of the polymeric nanozymes prepared in each embodiment and comparative example was measured. The measurement method was based on standard GB / T 37966-2019, "Method for Measurement of Peroxidase Activity of Iron Oxide Nanoparticles in Nanotechnology". The results of the specific enzyme activity determination are as follows: Figures 5 to 11 As shown in Table 1.

[0112] A comparison of the enzyme activity assay results of Examples 1-3 and Comparative Examples 1-3 shows that the amount of PAMAM added affects the activity of the final polymeric nanozyme when the second intermediate is coupled with PAMAM. When the amount of PAMAM added is too large, it leads to the formation of an excessively dense network structure between the dendritic polymer and the nanozyme (second intermediate), obscuring the active sites of the nanozyme core in the second intermediate and adversely affecting the activity of the final polymeric nanozyme.

[0113] Table 1

[0114]

[0115] Example 5

[0116] This embodiment relates to the application of Pt@PAA@PAMAM. As shown in Table 1, the Pt@PAA@PAMAM prepared in Example 4 has a higher specific enzyme activity, and... Figure 4 It is evident that the Pt@PAA@PAMAM prepared in Example 4 exhibits good dispersibility. In this example, the Pt@PAA@PAMAM prepared in Example 4 was applied to the ELISA validation experiment for primary and secondary antibody affinity.

[0117] The rabbit IgG (primary antibody) and goat anti-rabbit IgG (secondary antibody) used in this embodiment were purchased from Wuhan Aokebotai Biotechnology Co., Ltd., with concentrations of 25.0 mg / mL and 5.9 mg / mL, respectively; the Pt@PAA@PAMAM suspension concentration was 3.0 mg / mL. The chromogenic solution was freshly prepared by volume ratio of 10 mg / mL TMB: 0.2 M sodium acetate buffer (pH 3.6): 30% H2O2 = 20:1:2.

[0118] Conduct the experiment as follows:

[0119] (1) Enzyme-labeled plate coated with primary antibody (rabbit IgG)

[0120] Different concentrations of rabbit IgG (2000 ng / mL, 1000 ng / mL, 500 ng / mL, 250 ng / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, and 0.5 ng / mL) were added to the wells at a rate of 100 μL / well. After sealing, the plates were incubated at 37°C for 2 hours to allow the antibodies to passively adsorb onto the bottom and sidewalls of the wells. The liquid was then discarded, and PBST buffer (containing 0.01 M PBS and 0.05% Tween-20) was added to each well at a rate of 300 μL / well. The plates were washed three times to remove unadsorbed or weakly adsorbed antibodies, and then the bottom of the plates was patted dry.

[0121] Bovine serum albumin (BSA) was dissolved in PBST to prepare a blocking buffer containing 2 wt% BSA. 100 μL of the blocking buffer was added to each well, and the plates were sealed and incubated at 37°C for 2 hours to block non-specific adsorption sites on the plate surface. The liquid was then discarded, and 300 μL of PBST buffer was added to each well. After washing three times, the plates were dried and stored at 2–8°C.

[0122] (2) Pt@PAA@PAMAM secondary antibody (goat anti-rabbit IgG) labeling

[0123] The concentration of the polynanozyme Pt@PAA@PAMAM suspension used in this step was 3.0 mg / mL. 100 μL of the polynanozyme Pt@PAA@PAMAM suspension was diluted to 600 μL with 0.05 M MES (pH 6.0) solution to obtain an enzyme solution with a concentration of 0.5 mg / mL. The enzyme solution was then sonicated at 240 W for 2 min to promote the dispersion of Pt@PAA@PAMAM. After sonication, the solution was centrifuged at 10000 rpm for 10 min, and the supernatant was discarded to obtain MES-washed Pt@PAA@PAMAM.

[0124] The MES-washed Pt@PAA@PAMAM was added to a pre-prepared 0.5 mg / mL EDC / NHS mixed solution (the concentration of both EDC and NHS in this mixed solution was 0.5 mg / mL), and the volume of the EDC / NHS mixed solution used was 600 μL. Then, the mixture was reacted in the dark at room temperature for 30 min. After the reaction, the mixture was centrifuged at 10000 rpm for 5 min, the supernatant was discarded, and the Pt@PAA@PAMAM was washed twice with 0.05 M MES solution to obtain activated Pt@PAA@PAMAM. The activated Pt@PAA@PAMAM was resuspended in 600 μL of 0.05 M MES (pH 6.0) solution to obtain an activated Pt@PAA@PAMAM suspension with a theoretical concentration of 0.5 mg / mL.

[0125] Take 200 μL of activated Pt@PAA@PAMAM suspension, add 200 μL of 0.1 mg / mL goat anti-rabbit IgG antibody, sonicate at 240 W for 2 min to disperse, react on a mixer at 2–8 °C for 16 hours, centrifuge at 10,000 rpm for 5 min, discard the supernatant, and wash twice with 200 μL of 0.05 M Tris solution. After washing, centrifuge and discard the supernatant to obtain labeled Pt@PAA@PAMAM.

[0126] Add 100 μL of 10 wt% BSA solution to the labeled Pt@PAA@PAMAM, and sonicate at 240 W for 2 min under ice bath conditions (2℃~10℃). After confirming that all particulate matter has disappeared, block the reaction on a mixer at 2~8℃ for 3 h. After the blocking reaction is completed, centrifuge at 10000 rpm for 5 min, discard the supernatant, add 100 μL of 1 wt% BSA solution to the precipitate, and resuspend to obtain the Pt@PAA@PAMAM-labeled goat anti-rabbit IgG stock solution. The concentration of Pt@PAA@PAMAM-labeled goat anti-rabbit IgG in this stock solution is 1 mg / mL.

[0127] (3) Detection limit test

[0128] Following the steps in “(1) Coating of primary antibody (rabbit IgG) on ELISA plate”, different concentrations of rabbit IgG were coated onto the ELISA plate, and a blank control was set up at the same time: the coating wells were kept blank, no primary antibody was added, only PBST buffer was added for blocking, and washing and blocking with 2wt% BSA were completed in sequence. The plate was stored at 2-8℃ for testing.

[0129] Dilute the above Pt@PAA@PAMAM-labeled goat anti-rabbit IgG stock solution 500-fold, and add 100 μL / well to each well. Seal the wells and incubate at 37°C for 0.5 hours. After incubation, wash three times with 300 μL / well of PBST buffer and blot dry. Add 100 μL / well of chromogenic solution, incubate at room temperature in the dark for 5 minutes, and then stop the reaction with 2M H2SO4. Measure the absorbance at 450 nm using a microplate reader. Measure each sample (each primary antibody concentration) three times.

[0130] The relevant measurement data are shown in Table 2. The experimental results show that the detection limit can reach 0.5 ng / mL (i.e., 500 pg / mL), at which point the cutoff value is >3.

[0131] Table 2

[0132]

[0133] In this embodiment, after adding Pt@PAA@PAMAM (concentration 1 mg / mL, diluted 500 times) labeled with goat anti-rabbit IgG to the enzyme label wells, the colorimetric reaction can be carried out after incubation at 37°C for 30 min. Compared with traditional HRP-labeled secondary antibodies, which often require incubation of 60 min or longer in ELISA, the polymeric nanozyme-antibody complex prepared by this invention significantly shortens the reaction time. On this basis, due to the shortened reaction time, the risk of non-specific adsorption (caused by long incubation) is significantly reduced, which is beneficial to improving the detection accuracy.

[0134] Example 6

[0135] In this embodiment, the catalytic activity of the Pt@PAA@PAMAM prepared in Example 4, and the nanozyme precursor Pt@PAA used in the preparation of the Pt@PAA@PAMAM, was detected by chemiluminescence method to verify the catalytic performance of the polymer nanozyme.

[0136] In this embodiment, the hydrogen peroxide solution and luminol / enhancing agent were purchased from Thermo Fisher Scientific, catalog number 37070, and the instrument used was a Cosma SMART 6500 chemiluminescence analyzer.

[0137] Perform the test as follows:

[0138] 1. Take the Pt@PAA and Pt@PAA@PAMAM prepared in Example 4, and prepare dispersions with mass concentrations of 1000, 100, 10, and 1 ng / mL respectively using 0.1M PBS buffer. Place the chemiluminescent substrate A (hydrogen peroxide solution) and substrate B (luminol / enhancer) into chambers B and C of the analyzer's reagent rack, respectively.

[0139] 2. Take 10 μL of nanozyme precursor dispersion (Pt@PAA dispersion) or polymeric nanozyme dispersion (Pt@PAA@PAMAM dispersion) as a sample and add it to the sample well of the analyzer;

[0140] 3. Add 50 μL of substrate A and 50 μL of substrate B to the sample well of the analyzer in sequence;

[0141] 4. Reaction and detection: After 2 minutes of reaction, start the analyzer to automatically acquire signals and record the chemiluminescence intensity (RLU value).

[0142] At the same time, a blank control (background) was set up, using 10 μL of 0.1M PBS solution as the sample (instead of nanozyme dispersion or nanozyme precursor dispersion).

[0143] The test results are shown in Table 3. As can be seen from Table 3, at the same concentration, the signal intensity of Pt@PAA@PAMAM is significantly higher than that of Pt@PAA@. Specifically, at a concentration of 100 ng / mL, the chemiluminescence signal intensity of the polymeric nanozyme dispersion (Pt@PAA@PAMAM dispersion) is approximately 265 times stronger than that of the nanozyme precursor dispersion (Pt@PAA dispersion). This indicates that the polymeric nanozyme constructed through PAMAM polymerization has a significant luminescence enhancement effect. This luminescence enhancement effect can not only be used for real-time monitoring of the nanozyme's catalytic process but also expand its applications in fields such as bioimaging and disease diagnosis, providing new ideas and technical support for the development of multifunctional integrated nanoplatforms.

[0144] Table 3

[0145] Concentration (ng / mL) Pt@PAA Pt@PAA@PAMAM Enhancement factor 1000 20903 4560606 217 100 10644 2832606 265 10 3734 43930 11 1 1311 4510 2 Background 237 248

Claims

1. A polymeric nanozyme, characterized in that, This includes a nanozyme core modified with a surface modifier, and a dendritic polymer coupled with the surface modifier; The core of the nanozyme is composed of nanoparticles made of gold, platinum, iron tetroxide, or ruthenium. The surface modifier is a mixture of one or two or more carboxyl-containing polymers, wherein each carboxyl-containing polymer contains at least one carboxyl functional group; The dendritic polymer is a polyamide-amine dendritic polymer, and the generation of the polyamide-amine dendritic polymer is 4.0 to 7.0 generations.

2. The polymer nanozyme according to claim 1, characterized in that, The average particle size of the nanozyme core is 1–20 nm; the average molecular weight of the surface modifier is 1500–30000 Da. In the polymer nanozyme, the mass ratio of the nanozyme core modified with the surface modifier to the dendritic polymer is (50-5000):

1.

3. The polymer nanozyme according to claim 1, characterized in that, The surface modifier is one or a mixture of two or more of the following: polyacrylic acid, polymethacrylic acid, polymaleic acid, polylactic acid-glycolic acid copolymer-carboxyl group, polyethylene glycol-carboxylated derivative, polyaspartic acid, and polyglutamic acid.

4. A method for preparing a polymeric nanozyme, characterized in that, Includes the following steps: S1. Prepare nanozyme precursor, then wash nanozyme precursor to obtain first intermediate; The nanozyme precursor is a nanozyme core modified with a surface modifier; S2. Prepare the first intermediate dispersion. Add the activator dispersion and the stabilizer dispersion to the first intermediate dispersion to obtain the activation system. Stir the activation system, centrifuge, retain the precipitate, and wash the precipitate under ultrasonic conditions to obtain the second intermediate. S3. Prepare a second intermediate dispersion, and add a dendritic polymer dispersion dropwise to the second intermediate dispersion to obtain a coupling system; The stirring coupling system undergoes a coupling reaction to obtain a third intermediate dispersion; S4. Centrifuge the dispersion of the third intermediate, retain the precipitate, and resuspend the precipitate in the sealing solution for sealing incubation; S5. After the closed incubation is completed, centrifuge and retain the precipitate to obtain the polynanozyme as described in any one of claims 1-3.

5. The method for preparing polymeric nanozymes according to claim 4, characterized in that, In step S1, the preparation method of the nanozyme precursor is as follows: Metal hydrate and surface modifier are dissolved in solvent, and anhydrous sodium acetate is added to adjust the pH to 9-13 to obtain a mixed dispersion. The mixed dispersion is subjected to a solvothermal reaction at 160-180℃ for 5-10 hours. After the reaction is completed, the obtained solid product is washed to obtain the nanozyme precursor. The metal hydrate is chloroplatinic acid hexahydrate, ferric chloride hexahydrate, tetrachloroauric acid tetrahydrate, or ruthenium trichloride hydrate; when the metal hydrate is chloroplatinic acid hexahydrate, tetrachloroauric acid tetrahydrate, or ruthenium trichloride hydrate, the solvent is ethylene glycol; when the metal hydrate is ferric chloride hexahydrate, the solvent is water; the mass ratio of the metal hydrate to the surface modifier is 1:(2.5–10); the mass fraction of the metal hydrate in the mixed dispersion is 2–5 wt%.

6. The method for preparing polymeric nanozymes according to claim 4, characterized in that, In step S1, the method for preparing the first intermediate is as follows: The prepared nanozyme precursor was dispersed in a 2-morpholine ethanesulfonic acid solution to prepare a nanozyme precursor dispersion. The nanozyme precursor dispersion was then centrifuged, and the precipitate was retained and washed with 2-morpholine ethanesulfonic acid solution under ultrasonic conditions to obtain the first intermediate. The pH of the 2-morpholine ethanesulfonic acid solution used was 4-7, and the concentration was 0.01-0.05 M. The mass concentration of the nanozyme precursor in the nanozyme precursor dispersion was 0.1-5.0 mg / mL.

7. The method for preparing polymeric nanozymes according to claim 5, characterized in that, In step S2, when preparing the first intermediate dispersion, the dispersion medium used is a 2-morpholine ethanesulfonic acid solution with a pH of 4-7 and a concentration of 0.01-0.05M. After dispersing the first intermediate in the 2-morpholine ethanesulfonic acid solution, it is subjected to ultrasonic treatment to obtain the first intermediate dispersion. The ultrasonic time is 30-60 min, and the ultrasonic power is 240-300 W. The mass concentration of the first intermediate in the first intermediate dispersion is 0.1-5.0 mg / mL. The activator dispersion is a dispersion of 1-ethyl-(3-dimethylaminopropyl)carbodiimide with a mass concentration of 15-40 mg / mL; the stabilizer dispersion is a dispersion of N-hydroxysuccinimide with a mass concentration of 40-80 mg / mL. The volume ratio of the first intermediate dispersion, the activator dispersion, and the stabilizer dispersion is controlled so that the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and the first intermediate in the activation system is 1:(1-5):(10-20); the activation time of the system is 20-40 min.

8. The method for preparing polymeric nanozymes according to claim 6, characterized in that, In step S3, when preparing the second intermediate dispersion, the dispersion medium used is a 2-morpholine ethanesulfonic acid solution with a pH of 4-7 and a concentration of 0.01-0.05M; The second intermediate was dispersed in a 2-morpholine ethanesulfonic acid solution and then subjected to ultrasonic treatment to obtain a dispersion of the second intermediate. The ultrasonic time was 5–30 min and the ultrasonic power was 240–300 W. The mass concentration of the second intermediate in the dispersion was 0.1–5.0 mg / mL. In the dendritic polymer dispersion, the concentration of the dendritic polymer is 5wt% to 10wt%; in the coupling system, the mass ratio of the second intermediate to the dendritic polymer is (50 to 5000): 1; and the coupling reaction time is 0.5 to 5 h.

9. The method for preparing polymeric nanozymes according to claim 7, characterized in that, In step S4, the blocking solution used is a glycine solution, and the amount of glycine in the blocking solution is more than 5 times the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the activation system of step S2; the blocking incubation time is 10-16 hours.

10. An application of a polymeric nanozyme, characterized in that, Using the polymer nanozyme as described in any one of claims 1-3 as a catalyst, apply it to any of the following detection methods: a) Enzyme-linked immunosorbent assay (ELISA); b) Immunohistochemistry; c) Immunochromatography; d) Chemiluminescence analysis.

Images