Metal-nucleosidyl nanoscle hydrogels with dual enzymatic activities, methods of synthesis and uses thereof

Abstract

This invention discloses a metal-nucleoside-based nanozyme hydrogel with dual-enzyme activity, its synthesis method, and its applications. This hydrogel is synthesized by introducing Fe into a guanosine-boric acid hydrogel. 2+ It is formed by embedding gold-adenosine monophosphate (GA) coordination nanoclusters and possesses both glucose oxidase-like and peroxidase-like activities. Its synthesis method includes: preparing gold-adenosine monophosphate (GA) coordination nanoclusters, and preparing Fe-containing... 2+ This invention relates to a guanosine-boric acid sol, and the gelation of the sol by incorporating nanoclusters. This nanoenzyme hydrogel is inexpensive, easy to synthesize, exhibits high enzyme activity at pH ≤ 5, and can adapt to the microenvironment of fruit spoilage and exert antibacterial effects. Films and coatings prepared based on this hydrogel and polyvinyl alcohol can extend the shelf life of strawberries, and the material has good biocompatibility and biodegradability. This invention provides a novel, efficient, safe, and environmentally friendly fruit preservation material.

Description

Technical Field

[0001] This invention belongs to the field of nanoenzyme antibacterial food preservation packaging, specifically relating to a metal-nucleoside nanoenzyme hydrogel with dual enzyme activity and its synthesis method, as well as the application of the nanoenzyme hydrogel in antibacterial and fruit shelf-extending. Background Technology

[0002] Foodborne illnesses are a leading cause of death worldwide, and bacterial contamination is the primary cause of food spoilage and food poisoning. Food packaging and storage, as the final step before consumption, play a crucial role in food preservation. Ideal packaging should not only protect food but also inhibit microbial growth without causing pollution or harming the environment. Traditional petroleum-based plastic packaging is widely used, but its low recycling rate and poor degradability pose serious environmental challenges. Furthermore, microplastics can accumulate through the food chain, posing a potential threat to human health. Therefore, the food packaging industry is gradually shifting towards environmentally friendly and functional alternative materials to meet increasingly stringent environmental regulations.

[0003] Antimicrobial food packaging has garnered widespread attention due to its potential to enhance both food safety and preservation. During storage, food produces acidic substances such as oxalic acid and carbonic acid, and the accumulation of some microbial metabolites can create an acidic microenvironment within the food system. However, most antimicrobial agents (such as antimicrobial peptides and organic acids) become inactive under these conditions. Therefore, developing food packaging materials that maintain antimicrobial properties under acidic conditions while also possessing excellent biocompatibility and biodegradability has become an important direction for sustainable food preservation.

[0004] Nucleoside-based materials, including nucleosides, nucleotides, and nucleotides, are important components of genetic material. Nucleobases and phosphate groups provide numerous binding sites for metal ion coordination, exhibiting excellent metal coordination properties. This coordination form endows metal-nucleoside nanozymes with high catalytic performance and biocompatibility, making them excellent materials for food preservation and antibacterial purposes. Hydrogel films possess good mechanical properties, swelling properties, and biodegradability, effectively inhibiting food spoilage and representing a potential alternative to traditional plastic packaging. Furthermore, their three-dimensional network structure can effectively encapsulate antibacterial agents. Summary of the Invention

[0005] The purpose of this invention is to provide a metal-nucleoside nanoenzyme hydrogel with dual enzyme activity, characterized by high catalytic efficiency, good antibacterial activity, high biocompatibility, and high biodegradability, as well as its preparation method, and to provide new applications for this hydrogel.

[0006] The metal-nucleoside nanoenzyme hydrogel with dual enzyme activity provided by this invention comprises: a three-dimensional network framework formed by cross-linking of guanosine and boronic acid through hydrogen bonds and boronic acid ester bonds; and Fe dispersed and coordinated within the three-dimensional network framework. 2+ ; and gold-adenosine coordination nanoclusters embedded and dispersed in the pores of the three-dimensional network framework; the metal-nucleoside nanoenzyme hydrogel simultaneously possesses glucose oxidase-like (GOD activity) and peroxidase-like (POD) activity.

[0007] Furthermore, the gold-adenosine coordination nanoclusters are coordination nanoclusters formed by the hydrothermal reaction of chloroauric acid and adenosine monophosphate in the presence of sodium citrate.

[0008] The method for synthesizing the metal-nucleoside nanoenzyme hydrogel with dual enzyme activity of the present invention includes the following steps:

[0009] Step 1: Chloroauric acid and adenosine monophosphate are mixed, sodium citrate is added, and the mixture is hydrothermally reacted in a sealed container at 80-120℃ for 10-60 min. After separation and drying, gold-adenosine monophosphate coordination nanoclusters (Au-AMP NCs) are obtained.

[0010] Step 2: Combine guanosine, boric acid, alkaline substances, and Fe 2+ Salts are mixed and heated until dissolved, forming a clear, transparent substance containing Fe. 2+ Guanosine-boric acid sol.

[0011] Step 3: Add the gold-adenosine coordination nanoclusters from Step 1 to the Fe-containing nanoclusters from Step 2. 2+ The metal-nucleoside nanoenzyme hydrogel (Fe-GB@Au hydrogel) was obtained by mixing the guanosine-boric acid sol evenly and then naturally cooling it to room temperature.

[0012] Further, in step 1, the molar ratio of chloroauric acid to adenosine monophosphate and sodium citrate is 3-1:1:20-30; the hydrothermal reaction temperature is 100℃ and the reaction time is 30 min.

[0013] Further, in step 2, the molar ratio of guanosine to boric acid and the alkaline substance is 1:0.0005-0.002:0.0005-0.002, the alkaline substance is KOH, and the Fe... 2+ The salt is ferrous sulfate or ferrous chloride; the Fe-containing salt... 2+ Fe in guanosine-boric acid sol 2+ The concentration is 1–10 mM; the heating temperature is 90–100 °C.

[0014] Furthermore, the amount of gold-adenosine coordination nanoclusters added in step 3 is such that they are present in Fe... 2+The concentration of guanosine-boric acid sol is 50–200 μg·mL. -1 .

[0015] The present invention also provides the application of the aforementioned metal-nucleoside nanoenzyme hydrogel with dual enzyme activity in the preparation of antibacterial materials. Further, the antibacterial material is used to inhibit *Escherichia coli* and / or methicillin-resistant *Staphylococcus aureus*.

[0016] The present invention also provides the application of the metal-nucleoside nanoenzyme hydrogel with dual enzyme activity in the preparation of fruit preservatives or fruit preservative coatings.

[0017] The present invention also provides a biodegradable composite film, which is composed of the aforementioned metal-nucleoside nanoenzyme hydrogel with dual enzyme activity and polyvinyl alcohol. The mass ratio of the hydrogel to polyvinyl alcohol is 0.3-0.4 g of polyvinyl alcohol per 6 mL of hydrogel, and the film is prepared by drying at 60-80°C for 1-2 h.

[0018] The beneficial effects of this invention are as follows:

[0019] This invention utilizes the coordination interaction between a metal and a nucleoside-based material to prepare a Fe-GB@Au hydrogel with dual-enzyme activity. This material exhibits both peroxidase-like and glucose oxidase-like activities, and at pH ≤ 5, it can cascade and catalyze the production of reactive oxygen species from glucose, thus achieving highly efficient antibacterial activity. When this hydrogel is combined with polyvinyl alcohol to form a film, it demonstrates excellent practical application results in fruit preservation, extending the shelf life of strawberries to 8 days. This material also exhibits excellent biodegradability, with a degradation rate of up to 97% within 30 days, significantly superior to traditional non-degradable preservation films. The Fe-GB@Au hydrogel not only achieves highly efficient antibacterial action but also aligns with environmental protection principles due to its rapid degradability. This Fe-GB@Au hydrogel combines high antibacterial efficiency, excellent preservation performance, and environmental friendliness, providing a promising new material for developing functional, safe, and green next-generation food packaging. Attached Figure Description

[0020] Figure 1This is an assay to verify the enzyme activity of Fe-GB@Au hydrogel. (A) shows the UV-Vis absorption spectra of TMB solution under different conditions; (B) shows the absorbance at 652 nm of Fe-GB hydrogel, TMB, and H2O2 mixed solution at different pH values; (C) shows the UV-Vis absorption spectra of ABTS solution under different conditions; (D) shows the absorbance at 420 nm of Au-AMP NCs, glucose, and ABTS mixed solution; (E) and (F) show the absorbance at 420 nm of Fe-GB@Au hydrogel, glucose, and ABTS mixed solution containing different concentrations of Au-AMP NCs and at different pH values, respectively.

[0021] Figure 2 This section describes the antibacterial properties of Fe-GB@Au hydrogel. (A) and (B) show colony plate images and bacterial activity after treatment with different materials for E. coli and MRSA, respectively; (C) shows colony plate images of E. coli and MRSA after treatment with different concentrations of Fe-GB@Au hydrogel; (D)-(E) show the bacterial activity after treatment with different concentrations of Fe-GB@Au hydrogel for E. coli and MRSA; and (F) shows fluorescence imaging of live / dead bacteria after treatment with different materials for E. coli.

[0022] Figure 3 The viability of L929 cells after treatment with different concentrations of Fe-GB@Au hydrogel.

[0023] Figure 4 This is a diagram (A) showing different treatments of strawberries and photographs of strawberries at different times (B).

[0024] Figure 5 It shows the change in the weight loss of strawberries over time.

[0025] Figure 6 This represents the change in weight loss over time of commercially available cling film and Fe-GB@Au film.

[0026] Figure 7 These are the UV-Vis absorption spectra of different M-GB hydrogels mixed with TMB and H2O2 solutions.

[0027] Figure 8 The absorbance at 420 nm is the absorbance of composite hydrogel materials containing GOD, Au@BSA (A) and Au@BSA (B) at different concentrations with a mixture of ABTS and glucose. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0029] Example 1

[0030] Step 1: Mix 1 mL of 10 mM HAuCl4 aqueous solution and 200 μL of 50 mM adenosine monophosphate solution thoroughly, then add 0.5 mL of 0.5 M sodium citrate aqueous solution. Transfer the mixture to a polytetrafluoroethylene hydrothermal reactor and react hydrothermally at 100 °C for 30 min. After the reaction is complete, allow it to cool naturally, add 20 mL of acetone, centrifuge at 10000 r for 5 min, remove the supernatant, and freeze-dry the precipitate under vacuum for 12 h to obtain Au-AMP NCs, which are then stored at 4 °C for later use.

[0031] Step 2: Mix 500 mL of 0.2 M guanosine aqueous solution with 100 μL of 0.5 M boric acid aqueous solution, and after ultrasonic dispersion, add 100 μL of 0.5 M KOH aqueous solution and 20 μL of 0.1 M ferrous chloride aqueous solution sequentially. Heat in a 95°C water bath until the mixed solution becomes clear and transparent, obtaining Fe-containing... 2+ Guanosine-boric acid sol. The Fe-containing... 2+ Fe in guanosine-boric acid sol 2+ The concentration was 2.8 mM.

[0032] Step 3: Disperse 0.2 g Au-AMP NCs uniformly into 2 mL of Fe-containing solution. 2+ In guanosine-boric acid sol, the amount of Au-AMP NCs added makes it suitable for use in Fe-containing... 2+ The concentration of guanosine-boric acid sol was 100 μg·mL -1 After mixing evenly, the mixture was allowed to cool naturally to room temperature to obtain Fe-GB@Au hydrogel.

[0033] The Au-AMP NCs, Fe-GB@Au hydrogel, and Fe-GB hydrogel (containing Fe in step 2 of Example 1) obtained in this embodiment were compared. 2+ The guanosine-boric acid sol (obtained after natural cooling) underwent various performance tests. The specific test methods and results are as follows:

[0034] 1. Enzyme activity test

[0035] (1) Peroxidase-like activity test

[0036] 30 μL of Fe-GB hydrogel, 10 μL of 10 mM TMB aqueous solution, or 10 μL of 10 mM TMB aqueous solution and 10 μL of 50 mM H2O2 aqueous solution were added to NaAc-HAc buffer (20 mM, pH 4) to make a total solution volume of 200 μL. After reacting for 10 min, the UV-Vis absorption spectrum of the mixed solution was detected using a microplate reader. The results are shown in [Figure 1]. Figure 1 A.

[0037] 30 μL of Fe-GB hydrogel, 10 μL of 10 mM TMB aqueous solution, and 10 μL of 50 mM H2O2 aqueous solution were added to a 20 mM NaAc-HAc buffer (pH 1–8) to make a total volume of 200 μL. After reacting for 10 min, the absorbance of the mixed solution at 652 nm was measured using a microplate reader. The results are shown below. Figure 1 B.

[0038] (2) Glucose oxidase activity test

[0039] 10 μL 100 μg·mL -1 Au-AMP NCs aqueous solution, 10 μL of 10 mM ABTS aqueous solution, or 10 μL of 10 mM ABTS aqueous solution and 10 μL of 0.2 M glucose aqueous solution were added to NaAc-HAc buffer (20 mM, pH 4) to make a total volume of 200 μL. After reacting for 30 min, the UV-Vis absorption spectrum of the mixed solution was detected using a microplate reader. The results are shown in [Figure 1]. Figure 1 C.

[0040] 10 μL 100 μg·mL -1 Au-AMP NCs aqueous solution, 10 μL of 10 mM ABTS aqueous solution, and 10 μL of 0.2 M glucose aqueous solution were added to NaAc-HAc buffer (pH 1–8) to make a total volume of 200 μL. After reacting for 30 min, the absorbance of the mixed solution at 420 nm was measured using a microplate reader. The results are shown in the figure. Figure 1 D.

[0041] (3) Cascade catalytic glucose activity test

[0042] 30 μL of Fe-GB@Au hydrogel containing different concentrations of Au-AMP NCs (prepared in the same way as in Example 1, except that the amount of Au-AMP NCs added in step 3 is different), 10 μL of 10 mM TMB aqueous solution, and 10 μL of 0.2 M glucose aqueous solution were added to NaAc-HAc buffer (20 mM, pH 4) to make a total volume of 200 μL. After reacting for 30 min, the absorbance of the mixed solution at 652 nm was measured using a microplate reader. The results are shown in [Figure 1]. Figure 1 E.

[0043] 30 μL of Fe-GB@Au hydrogel (Au-AMP NCs concentration of 100 μg·mL) was added. -110 μL of 10 mM TMB aqueous solution and 10 μL of 0.2 M glucose aqueous solution were added to 20 mM NaAc-HAc buffer (pH 1–8) to make a total volume of 200 μL. After reacting for 30 min, the absorbance of the mixed solution at 652 nm was measured using an ELISA reader. The results are shown in the figure. Figure 1 F.

[0044] Depend on Figure 1 The results from A show that when H2O2 or Fe-GB hydrogel is added to TMB alone, there is no obvious absorption peak at 652 nm. Only when H2O2 and Fe-GB hydrogel are present simultaneously can TMB be oxidized to generate a blue product with a clear characteristic absorption peak at 652 nm. This indicates that the blue product is the result of Fe-GB hydrogel catalyzing the oxidation of TMB by H2O2, proving that Fe-GB hydrogel has POD-like activity. Figure 1 B investigated the variation of POD-like activity of Fe-GB hydrogel with pH value. Fe-GB hydrogel exhibited POD-like activity at pH ≤ 5, and its activity increased as pH value decreased, reaching its highest level at pH = 1. Figure 1 In C, when Au-AMP NCs, glucose, and ABTS are present simultaneously, ABTS can be oxidized to generate a green product, producing a characteristic absorption peak at 420 nm, indicating that Au-AMP NCs have POD-like and GOD-like activities. Figure 1 In D, Au-AMP NCs exhibited strong GOD-like activity at pH ≤ 5. Figure 1 E concluded that the optimal concentration of Au-AMP NCs in the Fe-GB@Au hydrogel was 100 μg·mL. -1 . Figure 1 F indicates that the Fe-GB@Au hydrogel cascade exhibits a strong ability to catalyze the production of ROS from glucose at pH ≤ 5, and this ability increases as pH decreases.

[0045] 2. Antibacterial performance test

[0046] (1) Antibacterial properties of different materials

[0047] The antibacterial activity of Fe-GB@Au hydrogel against E. coli and MRSA was evaluated using the plate coating method. First, bacteria stored at -80℃ were activated in tryptic soy broth for 12 h. The activated bacteria were then washed three times with PBS (pH = 7.4) before use. Different materials (PBS, GB hydrogel, Fe-GB hydrogel, Au-AMP NCs, and Fe-GB@Au hydrogel, respectively) and bacterial suspensions (1 × 10⁻⁶) were then prepared. 4 CFU·mL -1The mixture was incubated at 37°C for 3 h. The concentrations of the hydrogel and Au-AMP NCs were 2.0 mM (in Fe). 2+ Concentration meter, Fe-free 2+ The hydrogel retains the Fe 2+ (with consistent hydrogel volume) and 100 μg·mL -1 After incubation, 50 μL of the co-incubation solution was inoculated onto a solid culture medium. The bacterial colony count on the agar plate was observed after 12 h. The results are shown below. Figure 2 A and 2B. Simultaneously, after incubation, 100 μL of the co-incubation solution was stained with green fluorescent dye SYT09 and red fluorescent dye PI at 37℃ for 15 min, respectively. The bacteria were washed three times by centrifugation with sterile PBS. 5 µL of the bacterial suspension was added to a glass slide, and the survival of the bacteria after different treatments was observed under a fluorescence inverted microscope using a filter. The results are shown in [Figure 1]. Figure 2 F.

[0048] (2) Antibacterial properties of Fe-GB@Au hydrogels at different concentrations

[0049] Different volumes of Fe-GB@Au hydrogel and 1 mL of bacterial suspension (1 × 10⁻⁶) were mixed. 4 CFU·mL -1 The Fe-GB@Au hydrogels were mixed to achieve final concentrations of 0, 1.0, 1.5, 2.5, 5.0, 7.5, and 10.0 mM (based on Fe content). 2+ (using a concentration meter), incubate at 37℃ for 3 h, then inoculate 50 μL of the co-incubation solution onto a solid culture medium. Observe the bacterial colony count on the agar plate after 12 h. The results are shown in the figure. Figure 2 C, 2D, and 2E.

[0050] Depend on Figure 2 In results A and 2B, the bacterial colony count and bacterial activity in the GB hydrogel group were comparable to those in the PBS group, indicating that the GB hydrogel had no antibacterial ability. Compared to the PBS group, both the Fe-GB hydrogel group and the Au-AMP NCs group showed a decrease in bacterial colony count and bacterial activity, indicating that the Fe-GB hydrogel and Au-AMP NCs alone possessed some antibacterial activity. However, the Fe-GB@Au hydrogel group showed a significant reduction in bacterial colony count and a 100% decrease in bacterial activity, indicating that the Fe-GB@Au hydrogel exhibited the best antibacterial performance. Figure 2 C, 2D, and 2E show that E. coli was completely inactivated when Fe-GB@Au hydrogel was at 7.5 mM, and MRSA was completely inactivated after treatment with 10.0 mM Fe-GB@Au hydrogel. Figure 2In F, E. coli and MRSA treated with Fe-GB@Au hydrogel showed strong red fluorescence and almost no green fluorescence, while bacteria treated with Fe-GB hydrogel and Au-AMP NCs still showed relatively obvious green fluorescence and some red fluorescence. This indicates that Fe-GB hydrogel and Au-AMP NCs alone have certain antibacterial abilities, but Fe-GB@Au hydrogel has the strongest antibacterial ability.

[0051] 3. Biocompatibility testing

[0052] The cytotoxicity of Fe-GB@Au hydrogel was studied using mouse fibroblasts (L929) as an example. L929 cells were cultured at 1 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 96-well plates and cultured for 12 h. [The cells were then treated with different concentrations of Fe [container name]...] 2+ The Fe-GB@Au hydrogel (concentration 0–15 μM) was cultured in fresh 1640 medium for 24 h. After culture, the medium was discarded, and 5 mg / mL of the solution was added. -1 Incubate with 50 μL of MTT aqueous solution for 4 h. Finally, discard the MTT, add 150 μL of DMSO, shake and incubate for 15 min, and measure the absorbance of the solutions under different treatments at 490 nm. The results are shown in [Figure 1]. Figure 3 .

[0053] Figure 3 In the study, the viability of L929 cells was maintained above 94% under different concentrations of Fe-GB@Au hydrogel (0–15 μM), indicating that Fe-GB@Au hydrogel has low cytotoxicity and good biocompatibility with normal cells.

[0054] 4. Performance test for extending the shelf life of fruits

[0055] Preparation of Fe-GB@Au hydrogel film: 0.35 g PVA was added to every 6 mL of Fe-GB@Au sol, coated into a circular mold, and dried at 60℃ for 1.5 h to obtain Fe-GB@Au hydrogel film.

[0056] Preparation of Fe-GB@Au coating: 0.35 g PVA was added to every 6 mL of Fe-GB@Au sol and coated onto the inner wall of the food storage container. The container was then dried at 60°C for 1.5 h to form a Fe-GB@Au coating on the inner wall of the food storage container.

[0057] Application of Fe-GB@Au hydrogel in fruit preservation: A Fe-GB@Au hydrogel film was wrapped around the surface of strawberries and left to rest naturally; this was designated as the Fe-GB@Au film group. Strawberries were also placed in a preservation box with an inner wall coated with Fe-GB@Au and left to rest naturally; this was designated as the Fe-GB@Au coating group. Additionally, a blank group (strawberries left to rest naturally), a plastic film group (strawberries wrapped in plastic film and left to rest naturally), and a preservation box group (strawberries placed in a regular preservation box and left to rest naturally) were also established. The condition and weight of the strawberries were recorded daily by taking photos.

[0058] Figure 4 In the study, the strawberries in the blank group showed a dull color and dehydration on the second day; the strawberries in the plastic wrap group showed surface damage on the third day and mold on the fourth day; the strawberries in the food storage box group showed surface damage on the fourth day; while the strawberries in the Fe-GB@Au film group and the Fe-GB@Au coating group did not show obvious spoilage from the first to the eighth day, and only spoiled on the ninth day. Figure 5 Among the samples, strawberries with the Fe-GB@Au coating showed the least weight loss, with a weight loss of only 2% after 9 days. This indicates that the Fe-GB@Au coating can significantly mitigate microbial degradation and reduce water loss in strawberries.

[0059] 5. Biodegradability test

[0060] A 2 cm × 2 cm Fe-GB@Au hydrogel film and commercially available plastic wrap were buried in the soil of a pothos plant, and the weight was recorded every 5 days after digging it out.

[0061] Figure 6 In the study, after 30 days, the degradation rate of the Fe-GB@Au hydrogel film reached 97%, while the weight of commercially available plastic wrap remained at 91.2% of its original value.

[0062] To determine the technical solution of this invention, the inventors synthesized guanosine-boric acid hydrogels (M-GB) using different metal ions as active centers and investigated their peroxidase-like activity. The specific method was as follows: 500 mL of 0.2 M guanosine aqueous solution was mixed with 100 μL of 0.5 M boric acid aqueous solution, and after ultrasonic dispersion, 100 μL of 0.5 M KOH aqueous solution and 20 μL of 0.1 M metal-soluble salts (cobalt nitrate, nickel nitrate, ferric chloride, ferrous chloride, manganese chloride, and cadmium nitrate, respectively) were added sequentially. The mixture was heated in a 95°C water bath until the solution became clear and transparent, and then naturally cooled to room temperature to obtain guanosine-boric acid hydrogels containing metal ions, denoted as Co-GB, Ni-GB, Fe(III)-GB, Fe(II)-GB, Mn-GB, and Cd-GB, respectively. A hydrogel prepared without the addition of metal-soluble salts (denoted as GB) was used as a comparison. 30 μL of hydrogel, 10 μL of 10 mM TMB aqueous solution, and 10 μL of 50 mM H2O2 aqueous solution were mixed in NaAc-Hac buffer (20 mM, pH 4.0) to make a total solution volume of 200 μL. After reacting for 10 min, the absorbance of the mixed solution at 652 nm was measured using a microplate reader. The results are as follows: Figure 7 As shown.

[0063] Figure 7 Among the samples, only the Fe(III)-GB and Fe(II)-GB hydrogel groups showed a significant characteristic absorption peak of oxidized TMB at 652 nm, and the absorption peak intensity of the Fe(II)-GB hydrogel group was greater, indicating that only the Fe... 3+ and Fe 2+ When coordinated with guanosine-boric acid hydrogel, it exhibits peroxidase-like activity under acidic conditions, and the Fe(II)-GB hydrogel shows even stronger activity.

[0064] Furthermore, the inventors synthesized gold nanoclusters (Au@BSA) as ligands and formed a composite material with Fe-GB hydrogel, and investigated its cascade catalytic activity against glucose. Specifically, 1 mL of 24.3 mM HAuCl4 aqueous solution was added to 4 mL of 10 mM BSA aqueous solution and stirred at room temperature for 5 min, followed by rapid addition of 100 μL of 1 mg·mL⁻¹ HAuCl₄. -1 NaBH4 aqueous solution was reacted at room temperature for 30 min to obtain Au@BSA aqueous solution, which was then dried to obtain Au@BSA. 10 μL of 100 μg·mL⁻¹ -1 Natural glucose oxidase (GOD) aqueous solution or 10 μL 100 μg·mL -1Au@BSA aqueous solution, 10 μL of 10 mM ABTS aqueous solution, and 10 μL of 0.2 M glucose aqueous solution were mixed in NaAc-HAc buffer (20 mM, pH 4.0) to make a total solution volume of 200 μL. After reacting for 30 min, the absorbance of the mixed solution at 420 nm was measured using a microplate reader. The results are shown in the figure. Figure 8 A. Meanwhile, different amounts of Au@BSA were added to Fe-GB hydrogel to form composite materials with different concentrations of Au@BSA. The enzyme-like activity of the composite materials was investigated, and the results are shown in 8B.

[0065] Figure 8 In experiment A, during the catalytic oxidation of ABTS using glucose in a cascade reaction, the absorbance of the Au@BSA group at 420 nm was about three times that of the GOD group, indicating that Au@BSA has glucose oxidase-like activity at pH 4 and its activity is higher than that of natural glucose oxidase.

[0066] Figure 8 In B, the absorbance of the mixed solutions after adding different concentrations of Au@BSA to the Fe-GB hydrogel at 420 nm was comparable to that of the blank group, indicating that the addition of Au@BSA did not cause the composite material to exhibit glucose oxidase-like activity.

Claims

1. A metal-nucleoside nanoenzyme hydrogel with dual-enzyme activity, characterized in that, It comprises: a three-dimensional network framework formed by cross-linking of guanosine and boronic acid through hydrogen bonds and boronic acid ester bonds; and Fe dispersed and coordinated within the three-dimensional network framework. 2+ ; and gold-adenosine coordination nanoclusters embedded and dispersed in the pores of the three-dimensional network framework; the metal-nucleoside nanoenzyme hydrogel simultaneously possesses glucose oxidase-like activity and peroxidase-like activity.

2. The metal-nucleoside nanoenzyme hydrogel with dual enzyme activity according to claim 1, characterized in that, The gold-adenosine coordination nanoclusters are coordination nanoclusters formed by the hydrothermal reaction of chloroauric acid and adenosine monophosphate in the presence of sodium citrate.

3. A method for synthesizing a metal-nucleoside nanoenzyme hydrogel with dual enzyme activity as described in claim 1 or 2, characterized in that, Includes the following steps: Step 1: Chloroauric acid and adenosine monophosphate are mixed, sodium citrate is added, and the mixture is hydrothermally reacted in a sealed container at 80-120℃ for 10-60 min. After separation and drying, gold-adenosine monophosphate coordination nanoclusters are obtained. Step 2: Combine guanosine, boric acid, alkaline substances, and Fe 2+ Salts are mixed and heated until dissolved, forming a clear, transparent substance containing Fe. 2+ Guanosine-boric acid sol; Step 3: Add the gold-adenosine coordination nanoclusters from Step 1 to the Fe-containing nanoclusters from Step 2. 2+ The metal-nucleoside-borate sol was mixed evenly and then naturally cooled to room temperature to obtain the metal-nucleoside-based nanoenzyme hydrogel.

4. The method for synthesizing the metal-nucleoside nanoenzyme hydrogel with dual enzyme activity according to claim 3, characterized in that, In step 1, the molar ratio of chloroauric acid to adenosine monophosphate and sodium citrate is 3-1:1:20-30; the hydrothermal reaction temperature is 100℃ and the reaction time is 30 min.

5. The method for synthesizing the metal-nucleoside nanoenzyme hydrogel with dual enzyme activity according to claim 3, characterized in that, In step 2, the molar ratio of guanosine to boric acid and the alkaline substance is 1:0.0005-0.002:0.0005-0.002, where the alkaline substance is KOH and the Fe... 2+ The salt is ferrous sulfate or ferrous chloride; the Fe-containing salt... 2+ Fe in guanosine-boric acid sol 2 + The concentration is 1–10 mM; the heating temperature is 90–100 °C.

6. The method for synthesizing the metal-nucleoside nanoenzyme hydrogel with dual enzyme activity according to claim 3, characterized in that, The amount of gold-adenosine coordination nanoclusters added in step 3 is such that they are present in Fe... 2+ The concentration of guanosine-boric acid sol is 50–200 μg·mL. -1 .

7. The application of the metal-nucleoside nanoenzyme hydrogel with dual enzyme activity as described in claim 1 or 2 in the preparation of antibacterial materials.

8. The application of the metal-nucleoside nanoenzyme hydrogel with dual enzyme activity according to claim 7 in the preparation of antibacterial materials, characterized in that, The antimicrobial material is used to inhibit Escherichia coli and / or methicillin-resistant Staphylococcus aureus.

9. The application of the metal-nucleoside nanoenzyme hydrogel with dual enzyme activity as described in claim 1 or 2 in the preparation of fruit preservatives or fruit preservative coatings.

10. A biodegradable composite film, characterized in that, It is composed of a metal-nucleoside nanoenzyme hydrogel with dual enzyme activity as described in claim 1 or 2 and polyvinyl alcohol.

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