A method for preparing nanoenzyme solid hydrogels with molecular sieve structures and their application in real-time monitoring of H2S in food.

Abstract

This invention provides a method for preparing a nanoenzyme solid hydrogel with a molecular sieve structure and its application in real-time monitoring of H2S in food. It relates to the field of food safety detection technology, involving the assembly of a fluorescent donor, a metal-organic framework, a metal-semiconductor system, and a hydrogel to obtain a heterogeneous nanoenzyme solid hydrogel with a molecular sieve effect. The fluorescent donor is UCNPs, and the metal-organic framework includes the organic ligand 2-methylimidazole and the inorganic metal center Zn. 2+ The metal-semiconductor is Cu2O-Au; the hydrogel includes agar powder. First, a Cu2O-Au@UCNPs@ZIF molecular sieve nanozyme probe was constructed by organically integrating the metal-semiconductor heterojunction, UCNPs, and metal-organic framework ZIF-8 through an interface assembly strategy. This material was then combined with an agar hydrogel matrix to construct a visualized intelligent sensing gel. Low-cost on-site quantitative readout was achieved, and the platform was successfully applied to the monitoring of egg and pork freshness, demonstrating its potential in food safety monitoring.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, and in particular to a method for preparing a nanoenzyme solid hydrogel with a molecular sieve structure and its application in real-time monitoring of H2S in food. Background Technology

[0002] In the field of food safety, hydrogen sulfide (H2S) is a core metabolite produced by the breakdown of proteins under the action of microorganisms, and is recognized as one of the key endogenous biomarkers for assessing the freshness of meat, eggs, and dairy products. During food storage and logistics, the release level of H2S is significantly correlated with the degree of food spoilage. Therefore, developing technologies capable of in-situ, real-time monitoring of H2S has extremely high commercial value and social significance for ensuring food safety and preventing foodborne diseases.

[0003] To address these challenges, nanomaterial-driven H2S sensing technology has developed into a multifunctional field, with various detection platforms based on nanozyme catalysis for colorimetric or fluorescence analysis already available. To address the low catalytic efficiency of single components, constructing metal-semiconductor heterojunctions is an effective way to enhance nanozyme activity, and introducing the noble metal Au to construct Cu2O-Au complexes has become an ideal solution. However, in practical applications, these materials face severe challenges in resisting interference. Food matrices are complex in composition, and the widely present biological sulfur-containing molecules (such as cysteine, glutathione, and sulfur-containing proteins) have similar chemical activities to the target analyte H2S, easily generating non-specific signal interference, leading to serious deviations in detection results.

[0004] Existing sensing materials often lack effective steric hindrance protection or size filtering capabilities, allowing large-molecule bio-sulfur components to easily enter the active site and cause interference. Furthermore, liquid probes have limitations in real-time monitoring, such as volatility, difficulty in recovery, and inability to integrate with packaging. Therefore, constructing a solid hydrogel sensing platform with a molecular sieve structure, capable of accurately identifying small-molecule H2S while blocking large-molecule interfering substances, and possessing high stability, is a key focus in the field of intelligent food packaging and real-time monitoring. Summary of the Invention

[0005] This invention proposes a method for preparing nanoenzyme solid hydrogels with molecular sieve structures and their application in real-time monitoring of H2S in food, in order to solve the problems of poor anti-interference ability and difficulty in achieving real-time visualization detection of solid-state H2S in existing nanoenzyme sensing platforms.

[0006] In a first aspect, the present invention provides a method for preparing a nanoenzyme solid hydrogel with a molecular sieve structure, the method comprising the following steps: Step S1: The fluorescent donor, metal-semiconductor and metal-organic framework are assembled using an interface assembly strategy to obtain a heterostructured nanozyme Cu2O-Au@UCNPs@ZIF with molecular sieve effect; Step S2: Purify the product obtained in step S1 to obtain a molecular sieve nanozyme probe; Step S3: The molecular sieve nanozyme probe is immobilized in the hydrogel matrix, and after solidification and cutting, a nanozyme solid hydrogel with a molecular sieve structure is obtained.

[0007] The fluorescent donor is a UCNP with surface ligands removed, the metal-semiconductor is Cu2O-Au, the metal-organic framework includes the organic ligand 2-methylimidazole and the inorganic metal center zinc acetate dihydrate, and the hydrogel matrix includes agar powder.

[0008] Furthermore, the method for removing ligands from the surface of UCNPs includes the following steps: (1) Add 5 mL of OA-UCNPs cyclohexane solution with a concentration of 10 mg / mL to 5 mL of solution containing 0.01 MNOBF4 and shake at room temperature for 15 min; (2) After standing for 5 min, discard the upper cyclohexane and transfer the lower DMF phase into a centrifuge tube; (3) Add a mixed solvent of toluene and cyclohexane with a volume ratio of 1:1 to a centrifuge tube, centrifuge at 10000 rpm for 10 min, and collect the nanoparticles; (4) Redisperse with DMF and cyclohexane and wash twice more; (5) The obtained product was redispersed in water to obtain a UCNPs dispersion with the surface ligands removed.

[0009] Furthermore, the synthesis method of Cu2O-Au includes: (1) Place 100 mL of 0.01% HAuCl4 aqueous solution in a round-bottom flask, heat to boiling and stir continuously; (2) Quickly add 4 mL of 1% sodium citrate solution; (3) Maintain boiling and stirring until the solution turns wine red, then stop heating and allow it to cool naturally to room temperature; (4) Make up to 100 mL to obtain Au NPs. Transfer to a brown bottle and store at 4 °C protected from light; (5) Prepare Benedict's reagent. Dissolve 173 g of sodium citrate and 100 g of anhydrous sodium carbonate in 800 mL of water. Dissolve 17.3 g of copper sulfate in 100 mL of water and slowly add this solution to the above solution. Finally, dilute with water to 1 L. If there is a precipitate, filter it. (6) 2.5 mL of Au NPs solution was mixed sequentially with 0.5 mL of 5 wt% polyvinylpyrrolidone (PVP) solution, 250 μL of Benedict's reagent, and 18 μL of 0.1 M glucose solution; (7) Heat the mixture in a 100 °C water bath for 10 min; (8) When the solution color gradually changes from wine red to purple red, Cu2O-Au is synthesized.

[0010] Furthermore, the specific operation of the interface assembly strategy described in step S1 is as follows: (1) Mix the Cu2O-Au solution with the UCNPs dispersion with surface ligands removed until homogeneous; (2) Add zinc acetate dihydrate aqueous solution to the mixture and stir magnetically at room temperature for 30 min; (3) Inject 2-methylimidazole aqueous solution and let it stand for 30 min; (4) Centrifuge to collect the precipitate, and obtain Cu2O-Au@UCNPs@ZIF; (5) Finally, redisperse Cu2O-Au@UCNPs@ZIF in 1 mL of water for later use.

[0011] Further, in step (1), the amount of Cu2O-Au solution added is 650-850 μL, and the amount of UCNPs solution for removing surface ligands added is 90-110 μL with a concentration of 2.5 mg / mL.

[0012] Furthermore, the mass ratio of the organic ligand 2-methylimidazole to the inorganic metal center zinc acetate dihydrate is 1:1.5-1:3.5, and the 2-methylimidazole serves as the organic ligand, while the zinc acetate dihydrate serves as the inorganic metal center.

[0013] Further, the purification in step S2 involves centrifuging and washing the product obtained in step S1 and then redispersing it in water to obtain a molecular sieve nanozyme probe dispersion.

[0014] Furthermore, the specific method for immobilizing the molecular sieve nanozyme probe in the hydrogel matrix in step S3, followed by solidification and cutting to obtain a nanozyme solid hydrogel with a molecular sieve structure includes: (1) Dissolve agar powder in 5 mL of boiling water and stir until the solution becomes transparent to prepare agar stock solution; (2) Transfer the agar stock solution to a flat-bottomed petri dish with an inner diameter of 3.5 cm, and add the preheated Cu2O-Au@UCNPs@ZIF dispersion; (3) Place the mixture from step (2) in a 4 ℃ environment for 30 min; (4) Cut the composite hydrogel obtained in step (3) into square slices with a side length of 1×1 cm and store them in a refrigerator at 4℃ for later use.

[0015] Furthermore, the amount of agar powder added is 0.35-0.55 g; and the volume ratio of agar powder to Cu2O-Au@UCNPs@ZIF is (0.5-2.5):(2.5-0.5).

[0016] Secondly, the present invention also provides an application of the nanoenzyme solid hydrogel with molecular sieve structure prepared by the above preparation method in the preparation of a portable probe for real-time monitoring of H2S in food.

[0017] Compared with the prior art, the present invention has the following advantages: 1. Through surface ligand regulation and electrostatic driving, a hybrid core with Cu2O-Au as the catalytic center and UCNPs as the luminescent center was constructed and encapsulated in situ with a ZIF molecular sieve shell. This hierarchical design from the inside out ensures close interfacial contact between the catalytic center and the luminescent center. Taking advantage of the UCNPs' lack of background fluorescence and narrow emission bandwidth, the changes in nanozyme activity are converted into a highly sensitive fluorescence response, achieving effective amplification of optical signals and high signal-to-noise ratio output.

[0018] 2. This invention effectively shields large-volume bio-thiol interfering substances through the molecular sieve effect of ZIF-8, while simultaneously achieving directional penetration of small-molecule H2S. This design significantly improves the selectivity of the sensing platform in complex sulfur-containing matrices, ensuring the accuracy of the detection results.

[0019] 3. Compared with traditional liquid detection systems, this solid hydrogel platform effectively solves practical application problems such as the easy volatility of probes, difficulty in recycling, and inability to integrate with food packaging; it enables direct adhesion to the inner surface of food packaging such as meat and eggs, and achieves in-situ, real-time color warning by capturing H2S gas in the headspace, significantly improving the application value of the platform in smart packaging and cold chain logistics.

[0020] Based on the implementation methods provided in the above aspects, this application can be further combined to provide more implementation methods. Attached Figure Description

[0021] The above and other objects, features, and advantages of exemplary embodiments of the present invention will become readily apparent upon reading the following detailed description with reference to the accompanying drawings. In the drawings, several embodiments of the invention are illustrated by way of example and not limitation, with the same or corresponding reference numerals denoteing the same or corresponding parts, wherein: Figure 1The images shown are transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of the molecular sieve structured nanoenzyme solid hydrogel grown in situ according to the present invention. Figure 2 The emission spectra of core-shell UCNPs and Cu2O-Au@UCNPs@ZIF, and the normalized UV-vis absorption spectrum of Cu2O-Au, are shown in the preparation method of in-situ grown molecular sieve structure nanozymes provided in the embodiments of this application. Figure 3 This is to verify the peroxidase activity of the in-situ grown molecular sieve structure nanozyme provided in the embodiments of this application; Figure 4 This study verifies the feasibility of using the in-situ grown molecular sieve structured nanozyme Cu2O-Au@UCNPs@ZIF nanoprobe for H2S detection provided in the embodiments of this application. Figure 5 A schematic diagram of the response mechanism of the in-situ grown molecular sieve structured nanoenzyme Cu2O-Au@UCNPs@ZIF nanoprobe to H2S. Figure 6 A schematic diagram for assessing food freshness using smartphones, and the changes in color signals at 30 ℃, 4 ℃ and -20 ℃ during the preservation monitoring of pork and eggs; Figure 7 This is a structural model diagram of Cu2O-Au@UCNPs@ZIF. Detailed Implementation

[0022] The exemplary embodiments disclosed in this application will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of this application are shown in the drawings, it should be understood that this application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of this application and to fully convey the scope of this application to those skilled in the art. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0023] Nanomaterial-driven H2S sensing technology has developed into a multifunctional field, with various detection platforms based on nanozyme catalysis for colorimetric or fluorescence analysis. To address the low catalytic efficiency of single components, constructing metal-semiconductor heterojunctions is an effective way to enhance nanozyme activity, and introducing the noble metal Au to construct Cu2O-Au complexes has become an ideal solution. However, in practical applications, these materials face severe challenges in resisting interference. Food matrices are complex, and widely present biological sulfur-containing molecules (such as cysteine, glutathione, and sulfur-containing proteins) have similar chemical activities to the target analyte H2S, easily generating non-specific signal interference and leading to serious deviations in detection results.

[0024] To overcome this bottleneck, this application creatively proposes a method for preparing a portable probe using a molecular sieve-structured nanozyme solid hydrogel for real-time monitoring of H2S in eggs and pork. A Cu2O-Au@UCNPs@ZIF "all-in-one" nanozyme probe is constructed by organically integrating a metal-semiconductor heterojunction (Cu2O-Au), UCNPs, and a metal-organic framework (ZIF-8). This composite system not only leverages the enhanced enzyme activity generated by Cu2O-Au to achieve sensitive UV and colorimetric signal responses to H2S, but also combines the anti-interference capability of ZIF-8 with the anti-background fluorescence function of UCNPs, successfully constructing a sensing platform integrating "high activity, strong anti-interference, and tri-mode self-calibration." Expanding the application of this probe in portable hydrogel devices aims to provide a precise and reliable new analytical tool for real-time on-site monitoring of food freshness.

[0025] In a first aspect, the present invention provides a method for preparing a nanoenzyme solid hydrogel with a molecular sieve structure, the method comprising the following steps: Step S1: The fluorescent donor, metal-semiconductor, and metal-organic framework are assembled using an interfacial assembly strategy to obtain a heterostructured nanozyme Cu2O-Au@UCNPs@ZIF with a molecular sieve effect. The structural model of Cu2O-Au@UCNPs@ZIF is shown below. Figure 7 As shown; Step S2: Purify the product obtained in step S1 to obtain a molecular sieve nanozyme probe; Step S3: The molecular sieve nanozyme probe is immobilized in the hydrogel matrix, and after solidification and cutting, a nanozyme solid hydrogel with a molecular sieve structure is obtained.

[0026] The fluorescent donor is a UCNP with surface ligands removed, the metal-semiconductor is Cu2O-Au, the metal-organic framework includes the organic ligand 2-methylimidazole and the inorganic metal center zinc acetate dihydrate, and the hydrogel matrix includes agar powder.

[0027] To achieve interfacial self-assembly on the surface of oleic acid-UCNPs and improve their dispersibility in the aqueous phase to facilitate subsequent assembly processes, hydrophobic ligands on their surface were removed by a ligand removal method.

[0028] Optionally, the method for removing ligands from the surface of UCNPs includes the following steps: (1) Add 5 mL of OA-UCNPs solution with a concentration of 10 mg / mL cyclohexane to 5 mL of solution containing 0.01 MNOBF4 and shake at room temperature for 15 min; (2) After standing for 5 min, discard the upper cyclohexane and transfer the lower DMF phase into a centrifuge tube; (3) Add a mixed solvent of toluene and cyclohexane with a volume ratio of 1:1 to a centrifuge tube, centrifuge at 10000 rpm for 10 min, and collect the nanoparticles; (4) Redisperse with DMF and cyclohexane and wash twice more; (5) The obtained product is redispersed in water to obtain a UCNPs dispersion with surface ligands removed, namely NOBF4-UCNPs solution.

[0029] The method utilizes the reduction of Benedict's reagent with glucose under alkaline conditions, eliminating the need for high temperatures, high pressures, or toxic organic solvents (such as strong reducing agents or ligands), thus simplifying the process and lowering operational barriers and safety risks. The Cu₂O generated by the reduction of Benedict's reagent tightly binds with gold nanoparticles, forming a metal-semiconductor heterojunction Cu₂O-Au, providing highly catalytically active nanocrystalline nuclei. While other methods (such as high-temperature pyrolysis, hydrothermal methods, or conventional chemical reduction methods) can theoretically prepare Cu₂O-Au, the method described in this invention has irreplaceable advantages. Optionally, the synthesis method of Cu₂O-Au includes: (1) Place 100 mL of 0.01% HAuCl4 aqueous solution in a round-bottom flask, heat to boiling and stir continuously; (2) Quickly add 4 mL of 1% sodium citrate solution; (3) Maintain boiling and stirring until the solution turns wine red, then stop heating and allow it to cool naturally to room temperature; (4) Make up to 100 mL to obtain Au NPs. Transfer to a brown bottle and store at 4 °C protected from light.

[0030] (5) Prepare Benedict's reagent. Dissolve 173 g of sodium citrate and 100 g of anhydrous sodium carbonate in 800 mL of water. Dissolve 17.3 g of copper sulfate in 100 mL of water and slowly add this solution to the above solution. Finally, dilute with water to 1 L. If there is a precipitate, filter it. (6) 2.5 mL of Au NPs solution was mixed sequentially with 0.5 mL of 5 wt% polyvinylpyrrolidone (PVP) solution, 250 μL of Benedict's reagent, and 18 μL of 0.1 M glucose solution; (7) Heat the mixture in a 100 °C water bath for 10 min; (8) When the solution color gradually changes from wine red to purple red, Cu2O-Au is synthesized.

[0031] Optionally, the specific operation of the interface assembly strategy in step S1 is as follows: (1) Mix the Cu2O-Au solution with the UCNPs dispersion with surface ligands removed until homogeneous; (2) Add 7.5 μL of zinc acetate dihydrate aqueous solution to the mixture and stir magnetically at room temperature for 30 min; (3) Inject 25 μL of 2-methylimidazole aqueous solution and let it stand for 30 min; (4) Centrifuge to collect the precipitate and obtain Cu2O-Au@UCNPs@ZIF.

[0032] Optionally, the amount of Cu2O-Au solution added in step (1) is 650-850 μL. The amount of metal-semiconductor added can be 650 μL, 700 μL, 750 μL, 800 μL, 850 μL, or any value within the above volume range. This addition range ensures sufficient active sites and high sensitivity. In addition, if too much Cu2O-Au is added, it will cause the nanoparticles to be overloaded on the ZIF framework surface, causing severe agglomeration and even clogging the micropores of ZIF.

[0033] The amount of UCNPs solution added to remove surface ligands is 90-110 μL, and the amount of fluorescent donor added can be 90 μL, 95 μL, 100 μL, 105 μL, 110 μL, or any value within the above volume range; the concentration is 2.5 mg / mL. This range of UCNPs solution addition ensures optimal synergy between luminescence intensity, detection sensitivity, and structural stability in the multimodal sensing system.

[0034] Optionally, the mass ratio of the organic ligand 2-methylimidazole to the inorganic metal center zinc acetate dihydrate is 1:1.5 to 1:3.5, with 2-methylimidazole serving as the organic ligand and zinc acetate dihydrate as the inorganic metal center. The mass ratio of the organic ligand 2-methylimidazole to the inorganic metal center zinc acetate dihydrate can be 1:1.5; 1:2, 1:2.5, 1:3, 1:3.5, or any ratio within the above range. Limiting the mass ratio of 2-methylimidazole to zinc acetate dihydrate to the range of 1:1.5 to 1:3.5 is to achieve an optimal balance between framework integrity, crystal morphology, and mass transfer performance to the target analyte (H2S).

[0035] Optionally, attention should be paid to the order of sample addition after the reaction of the fluorescent donor, organic ligand, and inorganic metal center.

[0036] Optionally, the purification in step S2 involves centrifuging and washing the product obtained in step S1 and then redispersing it in water to obtain a molecular sieve nanozyme probe dispersion.

[0037] Optionally, the specific method for immobilizing the molecular sieve nanozyme probe in the hydrogel matrix in step S3, followed by solidification and cutting to obtain a nanozyme solid hydrogel with a molecular sieve structure includes: (1) Dissolve agar powder in 5 mL of boiling water and stir until the solution becomes transparent to prepare agar stock solution; (2) Transfer the agar stock solution to a flat-bottomed petri dish with an inner diameter of 3.5 cm, and add the preheated Cu2O-Au@UCNPs@ZIF dispersion; (3) Place the mixture from step (2) in a 4 ℃ environment for 30 min; (4) Cut the composite hydrogel obtained in step (3) into square slices with a side length of 1×1 cm and store them in a refrigerator at 4℃ for later use.

[0038] Optionally, the amount of agar powder added is 0.35-0.55 g; the amount of agar powder added can be 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, or any value within the above range; and the volume ratio of agar powder to Cu2O-Au@UCNPs@ZIF is (0.5-2.5):(2.5-0.5); the volume ratio of agar powder to Cu2O-Au@UCNPs@ZIF can be 0.5:2.5, 1:2, 1.5:1.5, 2:1, 2.5:0.5, or any ratio within the above range. Limiting the amount of agar powder added and its volume ratio to the composite material within the above range enables the prepared hydrogel film or sensing substrate to possess excellent mechanical strength, transparency, and air permeability. This ratio range not only effectively prevents the aggregation or loss of nanomaterials in the matrix, but also provides an efficient mass transfer channel for the target gas (H2S).

[0039] Secondly, the present invention also provides an application of the nanoenzyme solid hydrogel with molecular sieve structure prepared by the above preparation method in the preparation of a portable probe for real-time monitoring of H2S in food.

[0040] The method for preparing molecular sieve-structured nanozyme solid hydrogels provided by this invention constructs a hybrid core with Cu2O-Au as the catalytic center and UCNPs as the luminescent center through surface ligand regulation and electrostatic driving, and in-situ encapsulates a ZIF molecular sieve shell. This hierarchical design from the inside out ensures close interfacial contact between the catalytic center and the luminescent center. Utilizing the advantages of UCNPs—no background fluorescence and narrow emission bandwidth—the changes in nanozyme activity are converted into a highly sensitive fluorescence response, achieving effective amplification of the optical signal and high signal-to-noise ratio output. Through the molecular sieve effect of ZIF-8, large-volume bio-thiol interfering substances are effectively shielded, while directional penetration of small molecule H2S is achieved. This design significantly improves the selectivity of the sensing platform in complex sulfur-containing matrices, ensuring the accuracy of the detection results. This solid hydrogel platform effectively solves practical application problems such as probe volatility, difficulty in recovery, and inability to integrate with food packaging. This allows it to be directly attached to the inner surface of food packaging such as meat and eggs, and to achieve in-situ, real-time color-coded warnings by capturing H2S gas in the headspace, significantly enhancing the platform's application value in smart packaging and cold chain logistics.

[0041] Example 1 750 μL of Cu₂O-Au solution was mixed thoroughly with 100 μL of NOBF₄-UCNPs solution at a concentration of 2.50 mg / mL. Then, 7.50 μL of an aqueous solution containing 0.32 mg zinc acetate dihydrate was added, and the mixture was magnetically stirred at room temperature for 30 min. Next, 25 μL of an aqueous solution containing 0.88 mg 2-methylimidazole was slowly added, and the mixture was allowed to stand for 30 min. After the reaction was complete, the precipitate was collected by centrifugation at 8000 rpm for 5 min. Unreacted precursors and free particles were washed with deionized water, and the product was finally redispersed in 1 mL of water for later use, yielding a Cu₂O-Au@UCNPs@ZIF solution.

[0042] like Figure 1 As shown, the left image is a scanning electron microscope (SEM) image, and the right image is a transmission electron microscope (TEM) image. The left image reveals large polygonal or near-hexagonal prism crystals, consistent with typical crystal growth characteristics of ZIF-8. Numerous tiny nanoparticles are tightly wrapped or attached to the surface and interstices of these large particles, demonstrating that Cu2O-Au nanozymes and UCNPs have been successfully loaded and integrated into ZIF-8. The right TEM image clearly shows the interior and surface of ZIF-8, revealing a large number of near-spherical nanoparticles. The nanoparticles exhibit good dispersion on ZIF-8, without large-area physical aggregation. This uniform distribution contributes to the full exposure of active sites.

[0043] Figure 2The normalized spectra of the emission spectra of core-shell UCNPs, Cu2O-Au@UCNPs@ZIF, and the UV-vis absorption spectra of Cu2O-Au are shown in the molecular sieve structure nanozyme preparation method provided in this embodiment.

[0044] Experimental Procedure: Take 1 mL of Cu₂O-Au@UCNPs@ZIF solution and 1 mL of 0.25 mg / mL NOBF₄-UCNPs solution. Under 808 nm excitation, use a fluorescence spectrophotometer to detect the upconversion fluorescence emission spectrum in the range of 500–750 nm. Pipette 2 mL of Cu₂O-Au solution into a quartz cuvette. Use a UV-Vis spectrophotometer to detect the UV spectrum in the range of 500–750 nm. Perform normalization using data analysis software to obtain the following results: Figure 2 The normalized absorption / emission spectra of each component are shown.

[0045] The emission peak positions of Cu2O-Au@UCNPs@ZIF remained largely unchanged, but the overall emission intensity decreased slightly. This is attributed to the localized refractive index change and non-radiative energy dissipation channels introduced by the Cu2O-Au nanozyme and the ZIF shell, indicating that there is a certain electronic coupling between the components without disrupting the crystal phase and luminescent center of UCNPs. Combined with UV-vis spectroscopy analysis, this demonstrates that Cu2O-Au, UCNPs, and ZIF-8 have been successfully constructed into a stable hybrid structure.

[0046] Example 2 This embodiment provides a verification of the peroxidase activity of a molecular sieve-structured nanozyme, including: The peroxidase-like activity of Cu2O-Au@UCNPs@ZIF was verified by the TMB-H2O2 colorimetric reaction. During the reaction, the colorless substrate TMB was oxidized to generate the blue oxidized product oxTMB (maximum absorption wavelength λmax=650 nm).

[0047] Experimental Procedure: Experimental Group: 200 μL of nanoprobe solution, 200 μL of TMB solution (1 mM), and 200 μL of H2O2 solution (75 mM) were added sequentially to 400 μL of acetate-sodium acetate buffer (0.2 M, pH 4.0). Control Group 1: An equal volume of deionized water was added to replace the nanoprobe solution and H2O2; all other reagents were the same as in the experimental group. Control Group 2: An equal volume of deionized water was added to replace the nanoprobe solution; all other reagents were the same as in the experimental group. Control Group 3: An equal volume of deionized water was added to replace the H2O2 solution; all other reagents were the same as in the experimental group. Control Group 4: An equal volume of deionized water was added to replace the TMB solution; all other reagents were the same as in the experimental group. All reaction systems were incubated at 25 °C for 10 min, and the absorption spectra in the 400-800 nm range were detected by UV-Vis absorption spectroscopy.

[0048] like Figure 3 As shown, under the same conditions, no obvious color development was observed in the mixtures of TMB, TMB+H2O2, or Cu2O-Au@UCNPs@ZIF with TMB or H2O2. However, when Cu2O-Au@UCNPs@ZIF was added to TMB+H2O2, the solution rapidly changed from colorless to deep blue, exhibiting the characteristic absorption peak of oxTMB at 650 nm. This difference demonstrates that Cu2O-Au@UCNPs@ZIF possesses peroxidase activity.

[0049] Example 3 The fluorescence and absorption spectra of the provided molecular sieve structured nanozyme Cu2O-Au@UCNPs@ZIF were obtained under conditions of presence and absence of H2S.

[0050] Experimental Procedure: 200 μL of Cu₂O-Au@UCNPs@ZIF probe solution was mixed with 300 μL of Na₂S standard solution (0 and 125 μM), and incubated at room temperature for 10 min. Unbound Na₂S was removed by washing with water and centrifugation (10000 rpm, 1 min). 600 μL of acetate-sodium acetate buffer (0.2 M, pH 4.0), 200 μL of TMB solution (1 mM), and 200 μL of H₂O₂ solution (75 mM) were added to the washed composite material (Cu₂O-Au@UCNPs@ZIF-H₂S). After reacting at 25 °C in the dark for 20 min, the upconversion fluorescence emission spectrum in the range of 500–750 nm was recorded under 808 nm laser excitation. Simultaneously, the absorption spectrum (range 600–750 nm) and changes in the UV absorption peak of the mixture were detected using a UV-vis spectrophotometer.

[0051] By comparing the changes in fluorescence and UV absorption signals before and after the addition of H2S, the feasibility of using nanoprobes for H2S detection was verified. Figure 4 As shown, the left image is the UV-Vis absorption spectrum, and the right image is the upconversion fluorescence emission spectrum. The left image shows that after incubation with H₂S, the Cu₂O-Au@UCNPs@ZIF catalytic system exhibits absorption attenuation at 650 nm, which is manifested as a change in solution color from dark blue to light blue. Simultaneously, the 665 nm fluorescence signal of Cu₂O-Au@UCNPs@ZIF under 808 nm excitation is significantly recovered.

[0052] like Figure 5 The diagram shown illustrates the three-mode response mechanism of the in-situ grown molecular sieve structured nanoenzyme Cu2O-Au@UCNPs@ZIF to H2S.

[0053] Application of actual samples Experimental Procedure: Fresh pork was washed and diced, and eggs were shelled and homogenized. Samples were placed in sealed boxes. A 1 cm diameter pink hydrogel was affixed to a 2×3 cm square Tyvek paper. The Tyvek paper prevented direct contact of the hydrogel with food or leakage, while still allowing contact with H2S produced during vacuum packaging. The samples were then placed in sealed boxes containing either pork or egg liquid and stored at 30 ℃, 4 ℃, and -20 ℃ respectively. Color images were captured using an iPhone 15 Pro smartphone (48MP, Apple Inc., USA).

[0054] Appendix Figure 6 This diagram illustrates how a smartphone can be used to assess food freshness. Based on the changes in color signals at 30°C, 4°C, and -20°C during the preservation monitoring of pork and eggs using a smartphone, the results confirm that the probe responds well to different concentrations of H2S generated during the degradation of pork and eggs.

[0055] This invention discloses a method for preparing an in-situ grown molecular sieve structure nanoenzyme solid hydrogel. The detection process of this solid hydrogel is entirely based on the spontaneous sulfurization reaction between H2S gas and the nanomaterial Cu2O. The reaction products alter the inherent optical properties of the material, thereby achieving signal output without the need for any external detection instruments. This visual change from "pink to yellow" overcomes the complexity of traditional colorimetric methods that require comparison with standard color charts, improving the convenience and widespread potential of food safety monitoring.

[0056] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a nanoscale enzyme solid hydrogel having a molecular sieve structure, characterized in that, The method includes the following steps: Step S1: The fluorescent donor, metal-semiconductor and metal-organic framework are assembled using an interface assembly strategy to obtain a heterostructured nanozyme Cu2O-Au@UCNPs@ZIF with molecular sieve effect; Step S2: Purify the product obtained in step S1 to obtain a molecular sieve nanozyme probe; Step S3: The molecular sieve nanozyme probe is immobilized in the hydrogel matrix, and after solidification and cutting, a nanozyme solid hydrogel with a molecular sieve structure is obtained. The fluorescent donor is a UCNP with surface ligands removed, the metal-semiconductor is Cu2O-Au, the metal-organic framework includes the organic ligand 2-methylimidazole and the inorganic metal center zinc acetate dihydrate, and the hydrogel matrix includes agar powder.

2. The method for preparing a nanoenzyme solid hydrogel with a molecular sieve structure according to claim 1, characterized in that, The method for removing ligands from the surface of UCNPs includes the following steps: (1) Take 50 mg of OA-UCNPs solid particles, dissolve them in 5 mL of cyclohexane to obtain a solution with a concentration of 10 mg / mL, add it to 5 mL of NOBF4 solution containing 0.01 M, and shake at room temperature for 15 min; (2) After standing for 5 min, discard the upper cyclohexane and transfer the lower DMF phase into a centrifuge tube; (3) Add a mixture of toluene and cyclohexane in a volume ratio of 1:1 to a centrifuge tube, centrifuge at 10000 rpm for 10 min, and collect the nanoparticles; (4) Redisperse with DMF and cyclohexane and wash twice more; (5) The obtained product was redispersed in water to obtain a UCNPs dispersion with the surface ligands removed.

3. The method for preparing a nanoenzyme solid hydrogel with a molecular sieve structure according to claim 1, characterized in that, The synthesis method of Cu2O-Au includes: (1) Place 100 mL of 0.01% HAuCl4 aqueous solution in a round-bottom flask, heat to boiling and stir continuously; (2) Quickly add 4 mL of 1% sodium citrate solution; (3) Maintain boiling and stirring until the solution turns wine red, then stop heating and allow it to cool naturally to room temperature; (4) Make up to 100 mL to obtain Au NPs, transfer to a brown bottle and store at 4°C protected from light; (5) Prepare Benedict's reagent: Dissolve 173 g sodium citrate and 100 g anhydrous sodium carbonate in 800 mL of water; then dissolve 17.3 g copper sulfate in 100 mL of water, slowly add this solution to the above solution, and finally dilute with water to 1 L. If there is a precipitate, filter it. (6) 2.5 mL of Au NPs solution was mixed sequentially with 0.5 mL of 5 wt% polyvinylpyrrolidone (PVP) solution, 250 μL of Benedict's reagent, and 18 μL of 0.1 M glucose solution; (7) Heat the mixture in a 100 °C water bath for 10 min; (8) When the solution color gradually changes from wine red to purple red, Cu2O-Au is synthesized.

4. The method for preparing a nanoenzyme solid hydrogel with a molecular sieve structure according to claim 1, characterized in that, The specific operation of the interface assembly strategy described in step S1 is as follows: (1) Mix the Cu2O-Au solution with the UCNPs dispersion with surface ligands removed until homogeneous; (2) Add zinc acetate dihydrate aqueous solution to the mixture and stir magnetically at room temperature for 30 min; (3) Inject 2-methylimidazole aqueous solution and let it stand for 30 min; (4) Centrifuge to collect the precipitate, and obtain Cu2O-Au@UCNPs@ZIF; (5) Finally, redisperse Cu2O-Au@UCNPs@ZIF in 1 mL of water for later use.

5. The method for preparing a nanoenzyme solid hydrogel with a molecular sieve structure according to claim 4, characterized in that, The amount of Cu2O-Au solution added in step (1) is 650-850 μL, and the amount of UCNPs solution for removing surface ligands added is 90-110 μL with a concentration of 2.5 mg / mL.

6. The method for preparing a nanoenzyme solid hydrogel with a molecular sieve structure according to claim 4, characterized in that, The mass ratio of the organic ligand 2-methylimidazole to the inorganic metal center zinc acetate dihydrate is 1:1.5 to 1:3.5, and the 2-methylimidazole is the organic ligand, while the zinc acetate dihydrate is the inorganic metal center.

7. The method for preparing a nanoenzyme solid hydrogel with a molecular sieve structure according to claim 1, characterized in that, The purification in step S2 involves centrifuging and washing the product obtained in step S1, and then redispersing it in water to obtain a molecular sieve nanozyme probe dispersion.

8. The method for preparing a nanoenzyme solid hydrogel with a molecular sieve structure according to claim 1, characterized in that, The specific method for immobilizing the molecular sieve nanozyme probe in the hydrogel matrix in step S3, followed by solidification and cutting to obtain a nanozyme solid hydrogel with a molecular sieve structure includes: (1) Dissolve agar powder in 5 mL of boiling water and stir until the solution becomes transparent to prepare agar stock solution; (2) Transfer the agar stock solution to a flat-bottomed petri dish with an inner diameter of 3.5 cm, and add the preheated Cu2O-Au@UCNPs@ZIF dispersion; (3) Place the mixture from step (2) in a 4 ℃ environment for 30 min; (4) Cut the composite hydrogel obtained in step (3) into square slices with a side length of 1×1 cm and store them in a refrigerator at 4 ℃ for later use.

9. The method for preparing a nanoenzyme solid hydrogel with a molecular sieve structure according to claim 8, characterized in that, The amount of agar powder added is 0.35-0.55 g; and the volume ratio of the agar powder to Cu2O-Au@UCNPs@ZIF is (0.5-2.5):(2.5-0.5).

10. The application of a nanoenzyme solid hydrogel with a molecular sieve structure prepared by the preparation method according to any one of claims 1-9 in the preparation of a portable probe for real-time monitoring of H2S in food.

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