Carbon dot nanoszyme and hydrogel loaded with carbon dot nanoszyme and application thereof in detecting freshness of products

Abstract

This application belongs to the field of carbon dot nanozyme technology, and more specifically, relates to carbon dot nanozymes and hydrogels loaded with carbon dot nanozymes, as well as their application in detecting product freshness. This application uses disodium ethylenediaminetetraacetate, 2,4-dihydroxybenzoic acid, iron source, cobalt source, and phosphorus source as raw materials, and synthesizes them by heating to obtain green luminescent iron-cobalt-phosphorus co-doped carbon dot nanozymes. The preparation method is simple, and the obtained carbon dot nanozymes have high quantum yield. Their fluorescence color and intensity are sensitive to pH, and they exhibit excellent peroxidase mimicry activity, which can be used to detect pH and Hx. This application also provides hydrogels loaded with the above-mentioned carbon dot nanozymes that have dual pH and Hx responsiveness. These hydrogels can achieve visual monitoring of food freshness, especially aquatic product freshness, through morphological and color changes, providing a new device for detecting aquatic product freshness and possessing significant innovation and application value in the field of food safety testing.

Description

Technical Field

[0001] This application belongs to the field of carbon dot nanozyme technology, and more specifically, relates to carbon dot nanozymes and hydrogels loaded with carbon dot nanozymes and their application in detecting product freshness. Background Technology

[0002] During the spoilage stage of aquatic products, nitrogenous substances decompose into alkaline amines, causing the pH value to rise continuously. pH is a crucial indicator of aquatic product freshness. Currently, methods such as acidity meters, near-infrared spectroscopy, probe pH meters, and colorimetric array sensors are commonly used to measure the pH of aquatic products. These methods often require pretreatment of the actual samples, such as crushing and centrifugation, which are cumbersome, inefficient, and time-consuming. Furthermore, the volatilization of amines can affect the accuracy of the results, making it difficult to achieve large-scale and long-term monitoring of aquatic product freshness. While smart indicator tags are currently a highly efficient and non-destructive testing technology for visually monitoring aquatic product freshness, they typically rely on colorimetric or fluorescence channels to detect pH changes. This can be difficult for colorblind individuals and those with color insensitivity to color to interpret.

[0003] Hypoxanthine (Hx) is one of the substances widely found in the early stages of aquatic product spoilage. It accumulates gradually after the death of aquatic products and has become one of the most popular indicators for evaluating the freshness of aquatic products in recent years. Currently, mass spectrometry, near-infrared spectroscopy, electronic nose, electronic tongue, computer vision, and gas chromatography-mass spectrometry (GC-MS) are commonly used to detect Hx. The detection results are accurate and reliable, but the detection steps are cumbersome and the equipment requirements are high, making it difficult to conduct rapid large-scale detection. Establishing colorimetric or fluorescence analysis methods based on enzyme-linked reactions to achieve visual detection of Hx is a sensitive and feasible strategy. However, this strategy requires xanthine oxidase (XOD) to first oxidize and decompose Hx into H2O2, and then utilize the peroxidase (POD) properties of nanomaterials to oxidize H2O2 into reactive oxygen species that react with the chromogenic substrate. Among these methods, biological enzymes are expensive and unstable, and the catalytic reaction involving two enzymes is time-consuming, with relatively stringent requirements for detection conditions. The emergence of detection methods based on the direct inverse regulation of nanozyme activity by Hx provides a new strategy for the rapid and accurate detection of Hx. The study found that Hx can significantly reduce the POD activity of cubic platinum nanomaterials (PVP-PTNC) and strongly inhibit the formation of the blue product ox-TMB. This allows for the quantitative detection of Hx without the use of natural enzymes (XOD), overcoming the limitation of Hx relying on secondary conversion by oxidases.

[0004] Aquatic products have extremely high nutritional value and are an important source of high-quality protein. However, aquatic products contain a large amount of water and active endogenous proteases, making them highly susceptible to spoilage after death. This leads to significant changes in pH and Hx. pH and Hx are important indicators for evaluating the freshness of aquatic products, but current technologies generally detect pH or Hx separately, and cannot detect both simultaneously. Single-indicator detection is easily affected by environmental interference, resulting in inaccurate results. Multi-indicator detection can maximize the accuracy of the test. Therefore, developing a highly efficient and sensitive detection technology that can simultaneously and rapidly capture changes in both pH and Hx in aquatic products has great application value in the field of aquatic product safety testing. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this application is to provide carbon dot nanozymes and hydrogels loaded with carbon dot nanozymes and their application in detecting product freshness. It aims to solve the technical problems of existing technologies, such as cumbersome steps for detecting pH and Hx indicators, low accuracy of detection results, difficulty in rapid large-scale detection, and inability to detect pH and Hx indicators simultaneously.

[0006] To achieve the above objectives, this application provides a method for preparing carbon dot nanozymes, comprising the following steps:

[0007] Disodium ethylenediaminetetraacetate, 2,4-dihydroxybenzoic acid, iron source, cobalt source, phosphorus source and water were mixed evenly and heated to carry out the synthesis reaction. The reaction solution was centrifuged, dialyzed and freeze-dried to obtain carbon dot nanozymes.

[0008] Preferably, the iron source is one or more of ferric chloride hexahydrate, ferric sulfate, and ferric nitrate.

[0009] Preferably, the cobalt source is VB. 12 One or more of cobalt chloride, cobalt sulfate, and cobalt nitrate.

[0010] Preferably, the phosphorus source is one or more of phosphoric acid, sodium dihydrogen phosphate, and sodium pyrophosphate.

[0011] Preferably, the mass ratio of the above-mentioned disodium ethylenediaminetetraacetate, 2,4-dihydroxybenzoic acid, iron source, cobalt source and phosphorus source is (0.05-0.1):(0.05-0.1):(0.01-0.05):(0.01-0.05):(8-10).

[0012] Preferably, the concentration of the disodium ethylenediaminetetraacetate in the above reaction solution is 2 mg / mL to 4 mg / mL.

[0013] Preferably, the temperature of the above synthesis reaction is 180℃~220℃, and the synthesis reaction time is 1h~5h.

[0014] This application also provides a carbon dot nanozyme prepared according to the above preparation method.

[0015] This application also provides the application of the above-mentioned carbon dot nanozyme in the detection of pH and / or hypoxanthine.

[0016] On the other hand, this application provides a hydrogel loaded with the above-mentioned carbon dot nanozyme.

[0017] Preferably, the preparation method of the above-mentioned hydrogel includes the following steps:

[0018] The aqueous solution of the above carbon nanozyme, chromogenic substrate solution, H2O2 solution, PBS buffer solution, sodium alginate, gelatin, and chitosan were mixed evenly to obtain a blue polymer solution; then the blue polymer solution was mixed with CaCl2 solution and allowed to stand for crosslinking to obtain a hydrogel.

[0019] Preferably, the hydrogel includes at least one of hydrogel film and hydrogel sphere.

[0020] Preferably, the concentration of carbon dot nanozymes in the aqueous solution of the above-mentioned carbon dot nanozymes is 10 mg / mL to 20 mg / mL; and / or,

[0021] The concentration of the chromogenic substrate in the above-mentioned chromogenic substrate solution is 5 mM to 20 mM; and / or,

[0022] The chromogenic substrate described above is 3,3',5,5'-tetramethylbenzidine; and / or,

[0023] The mass percentage of H2O2 in the above H2O2 solution is 0.3wt% to 3wt%.

[0024] Preferably, the volume ratio of the above-mentioned aqueous solution of carbon nanozyme, the above-mentioned chromogenic substrate solution and the above-mentioned H2O2 solution is (0.5-1.5):(4-6):(0.5-1.5).

[0025] Preferably, the pH of the PBS buffer solution is 3 to 5.

[0026] Preferably, in the above-mentioned blue polymer solution, the mass percentage of the sodium alginate solution is 0.8 wt% to 1.5 wt%; and / or, the mass percentage of the gelatin is 0.45 wt% to 1 wt%; and / or, the mass percentage of the chitosan solution is 0.2 wt% to 0.42 wt%.

[0027] Furthermore, this application also provides the application of the above-mentioned hydrogel in the rapid visual detection of pH and / or hypoxanthine to determine product freshness.

[0028] In summary, compared with the prior art, the technical solutions conceived in this application have the following main technical advantages:

[0029] (1) The method for preparing carbon dot nanozymes provided in this application uses disodium ethylenediaminetetraacetate, 2,4-dihydroxybenzoic acid, iron source, cobalt source, and phosphorus source as raw materials, and carries out a synthesis reaction by heating to prepare green luminescent iron, cobalt, and phosphorus co-doped carbon dot nanozymes (Fe,Co,P-CDs nanozymes). The process is simple and easy to implement. The prepared carbon dot nanozymes have high quantum yield, and their fluorescence color and fluorescence intensity are sensitive to pH. They also have excellent peroxidase mimicry activity and can be used to detect pH and Hx. Furthermore, a pH and hypoxanthine detection sensing platform can be constructed based on Fe,Co,P-CDs nanozymes, which can be used to monitor the freshness of aquatic products.

[0030] (2) Compared with P-CDs nanozymes, Fe,P-CDs nanozymes and Co,P-CDs nanozymes, the Fe,Co,P-CDs nanozymes provided in this application have excellent peroxidase mimicry activity, which is even better than the simple sum of the technical effects of Fe,P-CDs nanozymes and Co,P-CDs nanozymes.

[0031] (3) The hydrogel loaded with the above-mentioned Fe,Co,P-CDs nanozymes provided in this application combines the safe, reliable Fe,Co,P-CDs nanozymes with great detection potential with a smart responsive gel to prepare an integrated portable smart hydrogel with dual pH and Hx responsiveness. It can realize the visual monitoring of food freshness, especially the freshness of aquatic products, and provide a new device for the detection of aquatic product freshness. It has significant innovation and application value in the field of food safety detection.

[0032] (4) In the preferred embodiment, the hydrogel film provided in this application has excellent pH response sensitivity. It maintains morphological stability at pH less than 8 and transforms from a solid to a sol state at pH 8. Without loading other active materials, it can quickly capture pH signals solely through visually observable morphological changes, overcoming the limitation of smart labels indicating food freshness relying solely on color and fluorescence changes. This provides a new perspective for the public to quickly judge food freshness. The aforementioned hydrogel film also exhibits excellent Hx responsiveness, showing two obvious visual changes when aquatic products spoil: breakage and dissolution, and complete fading of the blue color. It can accurately evaluate the freshness of aquatic products through morphological and color changes, quickly achieving naked-eye visual online monitoring of aquatic product freshness. Furthermore, the aforementioned hydrogel also possesses excellent stability, biocompatibility, and portability, is safe and reliable, and is applicable to various scenarios. Attached Figure Description

[0033] Figure 1These are the ultraviolet absorption and emission spectra of the Fe,Co,P-CDs nanozymes prepared in this application;

[0034] Figure 2 The infrared spectrum of the Fe,Co,P-CDs nanozyme prepared in this application is shown.

[0035] Figure 3 This is a transmission electron microscope image of the Fe,Co,P-CDs nanozyme prepared in this application;

[0036] Figure 4 The peroxidase and oxidase activities of the Fe,Co,P-CDs nanozyme prepared in this application;

[0037] Figure 5 The peroxidase activity of the Fe,Co,P-CDs nanozyme prepared in this application was simulated at different pH values.

[0038] Figure 6 The steady-state kinetics of the Fe,Co,P-CDs nanozyme prepared in this application under different concentrations of TMB are measured, wherein (a) is the steady-state kinetics measurement and (b) is the Lineweaver-Burk plot.

[0039] Figure 7 The steady-state kinetics of the Fe,Co,P-CDs nanozyme prepared in this application under different concentrations of H2O2 are measured, wherein (a) is the steady-state kinetics measurement and (b) is the Lineweaver-Burk plot.

[0040] Figure 8 This is the effect of detecting Hx based on the sensing platform of Fe,Co,P-CDs nanozymes. Content (a) is the ultraviolet absorption spectrum of Fe,Co,P-CDs nanozymes mixed with different concentrations of Hx at a wavelength of 652nm. Content (b) is the linear relationship between the difference in absorbance of Fe,Co,P-CDs nanozymes and the concentration of Hx before and after the addition of Hx.

[0041] Figure 9 The effect of different non-target analytes on the absorbance of the Fe, Co, P-CDs sensing platform at 652 nm wavelength;

[0042] Figure 10 The fluorescence spectra of Fe,Co,P-CDs nanozymes under 370 nm excitation at different pH values;

[0043] Figure 11 The effect of Fe,Co,P-CDs nanozymes on pH detection is shown in section (a) which represents the fluorescence intensity ratio of Fe,Co,P-CDs nanozymes (I 425 / I 505The linear relationship between pH and the fluorescence intensity of shrimp samples stored at 37℃ and 25℃ for 0-12 hours is shown in section (b). 425 / I 505 );

[0044] Figure 12 The UV-Vis spectra of the oxidation of TMB by P-CDs nanozymes, Co,P-CDs nanozymes, Fe,P-CDs nanozymes and Fe,Co,P-CDs nanozymes prepared in the embodiments of this application are shown.

[0045] Figure 13 The changes of the CDs-SCG hydrogel film prepared in the embodiments of this application in buffer solutions of different pH values ​​are shown, wherein (a) is the weight change and (b) is the morphological change.

[0046] Figure 14 This application describes the effect of CDs-SCG hydrogel spheres prepared in the embodiments of this application on Hx detection, wherein content (a) shows the linear relationship between the response signal ΔR / B of CDs-SCG hydrogel spheres and the concentration of Hx, and content (b) shows the quantitative detection of Hx content of shrimp placed at 25℃ for different times by CDs-SCG hydrogel spheres.

[0047] Figure 15 The CDs-SCG hydrogel film prepared in this application embodiment is used to monitor the freshness of shrimp placed at 25°C for different times. Contents (a), (b), (c), (d), and (e) are placed for 0h, 3h, 6h, 9h, and 12h, respectively. Content (f) is a comparison of the morphology and color of the CDs-SCG hydrogel film attached to the shrimp surface at the beginning and after 9h. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0049] This application provides a method for preparing carbon dot nanozymes, comprising the following steps:

[0050] Disodium ethylenediaminetetraacetate, 2,4-dihydroxybenzoic acid, iron source, cobalt source, phosphorus source and deionizer were mixed evenly and heated to carry out the synthesis reaction. The reaction solution was centrifuged, dialyzed and freeze-dried to obtain carbon dot nanozyme (i.e. Fe,Co,P-CDs nanozyme).

[0051] In some embodiments, the iron source is one or more of ferric chloride hexahydrate, ferric sulfate, and ferric nitrate. In some embodiments, the cobalt source is VB. 12One or more of cobalt chloride, cobalt sulfate, and cobalt nitrate. In some embodiments, the phosphorus source is one or more of phosphoric acid, sodium dihydrogen phosphate, and sodium pyrophosphate.

[0052] In some embodiments, the mass ratio of the above-mentioned disodium ethylenediaminetetraacetate, 2,4-dihydroxybenzoic acid, iron source, cobalt source and phosphorus source is (0.05-0.1):(0.05-0.1):(0.01-0.05):(0.01-0.05):(8-11).

[0053] In some embodiments, the concentration of disodium ethylenediaminetetraacetate in the above reaction solution is 2 mg / mL to 4 mg / mL.

[0054] In a preferred embodiment, the mass ratio of the above-mentioned disodium ethylenediaminetetraacetate, 2,4-dihydroxybenzoic acid, iron source, cobalt source and phosphorus source is (0.06-0.08):(0.06-0.08):(0.01-0.05):(0.01-0.05):(9-10).

[0055] In a preferred embodiment, the concentration of the disodium ethylenediaminetetraacetate in the above reaction solution is 2.4 mg / mL to 3.2 mg / mL.

[0056] In some embodiments, the temperature of the above-mentioned synthesis reaction is 180℃~220℃, and the reaction time is 1h~5h. It should be understood that this application does not limit the above-mentioned heating method, and it can be, but is not limited to, hydrothermal method or microwave method.

[0057] In some embodiments, a dialysis bag with a molecular weight cutoff of 500 Da is used to remove minute impurities by dialysis to obtain a solution containing green fluorescent carbon dots, which is then freeze-dried to obtain carbon dot nanozymes.

[0058] This application also provides carbon dot nanozymes prepared using the above-described preparation method.

[0059] The carbon dot nanozyme prepared in this application can be used to detect the pH value of a sample. The fluorescence intensity of the carbon dot nanozyme prepared in this application is pH-sensitive. When pH < 6, the emission peak wavelength of the carbon dot nanozyme at an excitation wavelength of 370 nm is 500 nm; when pH < 6 < pH < 7, the emission peak wavelength at an excitation wavelength of 370 nm is 505 nm; when pH = 7, two emission peaks appear at an excitation wavelength of 370 nm, with wavelengths of 425 nm and 505 nm, respectively; when pH > 7, the intensity of the emission peak at the 425 nm emission wavelength at an excitation wavelength of 370 nm is enhanced. The fluorescence color of the carbon dot nanozyme prepared in this application is pH-sensitive. When pH is less than 7, the carbon dot nanozyme appears green under 365 nm ultraviolet light irradiation; when pH is greater than 7, it appears blue-green under 365 nm ultraviolet light irradiation. The specific steps for detecting the pH value of the sample are as follows:

[0060] S1. Mix the above-mentioned carbon nanozyme with PBS buffers of different pH values ​​until homogeneous, and measure the ratio of the fluorescence intensity at 425 nm emission wavelength to that at 505 nm emission wavelength under an excitation wavelength of 370 nm (I 425 / I 505 );

[0061] S2, with pH as the independent variable, the fluorescence intensity ratio is I. 425 / I 505 As the dependent variable, the analysis yielded the ratio of pH value to fluorescence intensity I. 425 / I 505 Relationship;

[0062] S3. Process the sample to be tested. After centrifugation to obtain the supernatant, mix it thoroughly with the carbon nanozyme and measure the fluorescence intensity ratio at an excitation wavelength of 370 nm. 425 / I 505 ;

[0063] S4. The fluorescence intensity of the supernatant of the sample to be tested is compared with I. 425 / I 505 Substituting into the above formula, the pH value of the supernatant of the sample to be tested is obtained.

[0064] The carbon dot nanozyme prepared in this application can be used to detect the hypoxanthine concentration in a sample. The carbon dot nanozyme prepared in this application possesses peroxide-mimicking enzyme activity, enabling the substrate 3,3',5,5'-tetramethylbenzidine (TMB) to be oxidized to oxTMB by H₂O₂, resulting in a vivid color change in the solution and thus facilitating the detection of hypoxanthine concentration in the sample. The specific steps for detecting the hypoxanthine concentration in the sample are as follows:

[0065] S1. The aqueous solution of the above carbon nanoparticles and hypoxanthine standard solutions of different concentrations were placed in a 37°C water bath for treatment, wherein the concentration of the hypoxanthine standard solutions was greater than or equal to 0. Then, TMB solution, H2O2 solution and PBS were added and mixed evenly. The pH of the mixture was adjusted to 4 and placed in a 37°C water bath for treatment. The absorbance (A0) of the mixture when the concentration of the hypoxanthine standard solution was 0 and the absorbance (A) of the mixture of hypoxanthine standard solutions of different concentrations were measured at 652 nm. The absorbance difference A0-A was calculated.

[0066] S2. Using hypoxanthine concentration as the independent variable and absorbance difference A0-A as the dependent variable, analyze and obtain the relationship between hypoxanthine concentration and absorbance difference A0-A.

[0067] S3. Process the sample to be tested. After centrifuging to obtain the supernatant, place it in a 37°C water bath with the above-mentioned aqueous solution of carbon nanozyme. Then add TMB solution, H2O2 solution and PBS, mix well, adjust the pH of the mixture to 4, place it in a 37°C water bath, and measure the absorbance A of the mixture at 652 nm. n Calculate the absorbance difference A0-A n ;

[0068] S4. Calculate the absorbance difference A0-A of the supernatant of the sample to be tested. n Substituting into the above formula, the concentration of hypoxanthine in the supernatant of the sample to be tested is obtained.

[0069] In some embodiments, the aforementioned sample or test sample may be, but is not limited to, biological samples or environmental samples. Further, the aforementioned biological samples or environmental samples may be food samples (fresh fruits or vegetables, meat, aquatic products), soil samples, freshwater samples, wastewater samples, or combinations thereof.

[0070] It is understood that this application does not limit the methods of analysis described above, and may include, but is not limited to, linear analysis and regression analysis.

[0071] This application also provides a hydrogel loaded with the above-mentioned carbon dot nanozyme.

[0072] In some embodiments, the preparation method of the above-mentioned hydrogel includes the following steps:

[0073] The aqueous solution of the above carbon nanozyme, chromogenic substrate solution, H2O2 solution, PBS buffer solution, sodium alginate, gelatin and chitosan were mixed evenly to obtain a blue polymer solution; then the above blue polymer solution was mixed with CaCl2 solution and allowed to stand for crosslinking to obtain a hydrogel.

[0074] In some embodiments, the mass percentage of sodium alginate in the aforementioned blue polymer solution is 0.8 wt% to 1.5 wt%; and / or, the mass percentage of gelatin is 0.45 wt% to 1 wt%; and / or, the mass percentage of chitosan is 0.2 wt% to 0.42 wt%. This application regulates the pH responsiveness of the hydrogel by synergistically adjusting the mass percentages of sodium alginate, gelatin, and chitosan in the blue polymer solution. This allows the hydrogel to maintain morphological stability at pH less than 8 and to transform from a solid state to a sol state at pH 8, enabling it to rapidly capture pH changes when food, especially aquatic products, spoils.

[0075] In some embodiments, the concentration of carbon dot nanozymes in the aqueous solution of the above-mentioned carbon dot nanozymes is 10 mg / mL to 20 mg / mL.

[0076] In some embodiments, the chromogenic substrate solution is an organic solution of 3,3',5,5'-tetramethylbenzidine. In some embodiments, the concentration of the chromogenic substrate in the chromogenic substrate solution is 5 mM to 20 mM.

[0077] In some embodiments, the mass percentage of H2O2 in the above H2O2 solution is 0.3wt% to 3wt%.

[0078] In some embodiments, the volume ratio of the above-mentioned aqueous solution of carbon nanozyme, the above-mentioned chromogenic substrate solution and the above-mentioned H2O2 solution is (0.5-1.5):(4-6):(0.5-1.5), preferably (0.8-1.2):(4.5-5.5):(0.8-1.2), and more preferably 1:5:1.

[0079] This application adjusts the sensitivity of the hydrogel for detecting Hx by modifying the concentration of carbon nanozyme in the aqueous solution, the concentration of the chromogenic substrate in the chromogenic substrate solution, the mass percentage of H2O2 in the H2O2 solution, and the volume ratio of these three components. It is understood that any adjustments to these parameters by those skilled in the art based on the freshness criteria of the product to be tested are within the scope of protection of this application.

[0080] In some embodiments, the pH of the PBS buffer solution is 3 to 5.

[0081] This application does not limit the application of the above-mentioned hydrogels, but includes, but is not limited to, at least one of hydrogel films and hydrogel spheres.

[0082] In some embodiments, the preparation method of the above-mentioned hydrogel film includes the following steps: after casting and smoothing the above-mentioned blue polymer solution in a container, CaCl2 solution is added to completely immerse the above-mentioned blue polymer solution, and the solution is allowed to stand for crosslinking and then cut to obtain a hydrogel film, namely CDs-CSG hydrogel film.

[0083] In some embodiments, the preparation method of the above-mentioned hydrogel spheres includes the following steps: dropping the above-mentioned blue polymer solution into a CaCl2 solution, allowing it to stand for crosslinking, and obtaining hydrogel spheres, namely CDs-CSG hydrogel spheres.

[0084] This application does not limit the size of the hydrogel spheres or the thickness of the hydrogel film, etc., and those skilled in the art can choose appropriate size and thickness according to the needs of actual application scenarios.

[0085] The hydrogel film provided in this application enables naked-eye visualization of sample pH. In a specific embodiment of this application, the inventors discovered through experiments that when the aforementioned hydrogel film with a thickness of 0.5 mm to 1 mm is used to detect pH values, it can rapidly break down and dissolve when pH = 8.0, transforming from a solid gel to a sol state, thus quickly capturing pH changes.

[0086] The hydrogel loaded with carbon dot nanozymes provided in this application can be used to detect the concentration of Hx in a sample. The specific steps for detecting the concentration of Hx in a sample using the above-mentioned hydrogel spheres are as follows:

[0087] S1. Mix the above CDs-CSG hydrogel balls, Hx standard solutions of different concentrations and PBS buffer, adjust the pH of the mixture to 4.0, incubate at 37℃, take out the CDs-CSG hydrogel balls, take pictures with a smartphone to read the color difference value ΔR / B value of the CDs-CSG hydrogel balls before and after incubation in Hx standard solutions of different concentrations, and convert it into a digital value in RGB color mode as the judgment standard;

[0088] S2. Process the sample to be tested. After centrifuging to obtain the supernatant, repeat step S1. Use a smartphone to take a picture to obtain the color difference value ΔR / B of CDs-CSG gel beads before and after incubation in the supernatant of the sample to be tested. Convert the value to a digital value in RGB color mode and compare it with the above judgment criteria to determine the concentration of Hx in the sample to be tested.

[0089] This application also provides the application of the above-mentioned hydrogel in the rapid visual detection of pH and / or hypoxanthine to determine product freshness. It should be understood that the above-mentioned products include, but are not limited to, aquatic products. In a specific embodiment of this application, the prepared hydrogel film can rapidly capture changes in pH and Hx, rapidly break down and dissolve at pH = 8.0, and the blue color of the film quickly and completely fades when Hx ≥ 118 mg / kg (the standard for judging sub-freshness of shrimp meat).

[0090] In practical applications, the hydrogel film provided in this application can exhibit two obvious changes visible to the naked eye when aquatic products spoil: breakage and melting, and complete fading. By observing these changes in morphology and color, the freshness of aquatic products can be accurately evaluated, enabling rapid online monitoring of aquatic product freshness with naked-eye visualization.

[0091] It is understandable that when the freshness evaluation standard of the product to be tested is higher or lower than that of shrimp meat, those skilled in the art can adapt the content of carbon nanoparticles, chromogenic substrates, and H2O2 in the hydrogel to make the prepared hydrogel spheres and hydrogel films accurately respond to the freshness of the product to be tested, all of which are within the scope of protection of this application.

[0092] The hydrogel provided in this application can detect the freshness of aquatic products, especially shrimp. Through morphological changes in the hydrogel film, it rapidly responds to pH levels when aquatic products spoil, providing strong support for visual judgment of freshness. This overcomes the limitations of smart labels that rely solely on color and fluorescence changes to indicate food freshness, offering the public an unprecedented new perspective for quickly assessing food freshness. Furthermore, it can rapidly and accurately respond to changes in Hx based on the fading of the blue color of the hydrogel film or hydrogel spheres. Therefore, the hydrogel provided in this application achieves visual detection of aquatic product freshness based on a dual response of pH and Hx, providing a new device for aquatic product freshness detection and possessing enormous potential in the field of food safety testing.

[0093] The above technical solutions are described in detail below with reference to specific embodiments. It should be understood that these are merely exemplary and not intended to limit this application. Materials of the same or similar type, model, quality, properties, or function as the reagents and instruments described below can be used to implement this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0094] The following is an example:

[0095] Example 1

[0096] 1. Preparation of green luminescent iron-cobalt-phosphorus co-doped carbon nanoparticle nanozymes (Fe,Co,P-CDs):

[0097] Add 0.0731g of disodium ethylenediaminetetraacetate, 0.0771g of 2,4-dihydroxybenzoic acid, and 0.03g of vitamin B. 120.04 g of FeCl3·6H2O was dissolved in 20 mL of 5 M phosphoric acid solution. The mixed solution was transferred to a 25 mL polytetrafluoroethylene-lined reactor and purified water was added to fill the reactor. The reaction was carried out at 200 °C for 3 h. After the reaction was completed, the insoluble matter was removed by centrifugation, and the sample was dialyzed through a 500 Da dialysis bag for 4 h. The solid sample was then freeze-dried to obtain the solid sample.

[0098] 2. Detection of Fe, Co, P-CDs nanozymes

[0099] 2.1 Fluorescence and UV spectroscopy scans were performed on the above Fe,Co,P-CDs nanozymes:

[0100] The Fe,Co,P-CDs nanozymes were dispersed and dissolved in deionized water to obtain Fe,Co,P-CDs nanozyme aqueous solutions. Then, the emission wavelength and fluorescence intensity were measured in the detection cell of a fluorescence spectrophotometer (FL 8500, PerkinElmer Company), and the absorbance at different wavelengths was measured in the detection cell of a UV-Vis spectrophotometer (Lambda-35, PerkinElmer Company).

[0101] like Figure 1 As shown, the Fe,Co,P-CDs nanozymes exhibit two main absorption peaks at 273 nm and 435 nm, which are attributed to the π→π* transition of aromatic C=C and the n→π* transition of C=O, respectively. Furthermore, the fluorescence spectrum of the Fe,Co,P-CDs nanozymes shows an emission wavelength of approximately 505 nm at an excitation wavelength of 430 nm. Figure 1 The illustration in the upper left corner shows that the aqueous solution of Fe,Co,P-CDs nanozyme appears yellow in sunlight (left side of the illustration) and exhibits bright green fluorescence under 365nm ultraviolet light (right side of the illustration).

[0102] Using Rhodamine 6G as a reference (QYs = 95% in ethanol), the QYs of Fe,Co,P-CDs nanozymes were obtained by comparing the combined fluorescence intensity and absorbance values ​​of aqueous solutions of Fe,Co,P-CDs nanozymes (excited at 430 nm). The formula for calculating relative QYs is as follows.

[0103] φ x =φ ST (Grad x / Grad ST )(η x 2 / η ST 2 )×100%

[0104] In the formula, Φ represents QYs, Grad is the gradient of combined fluorescence intensity and absorbance, η is the refractive index (1.33 for water, 1.36 for ethanol), ST represents the standard, and x represents the sample. By minimizing the reabsorption effect and keeping the absorbance at the excitation wavelength below 0.1, the quantum yield of Fe,Co,P-CDs nanozymes reached as high as 48.76% after testing.

[0105] 2.2 Infrared spectroscopy was performed on the above Fe,Co,P-CDs nanozymes:

[0106] Fourier transform infrared spectroscopy (FT-IR) was used to detect the transmittance of the above Fe,Co,P-CDs nanozyme samples at different wavelengths to explore the surface groups of Fe,Co,P-CDs nanozymes.

[0107] like Figure 2 As shown, at 3447cm -1 and 3129cm -1 The CD absorption bands observed at 2936 cm⁻¹ are due to the stretching vibrations of the hydroxyl (OH) group and the amino (NH) group. -1 and 2454cm -1 The peak at 1644 cm⁻¹ is due to the stretching vibrations of the CH and C-C bonds present on Fe, Co, P-CDs. -1 and 1357cm -1 The stretching frequency originates from C=O / C=N and CN / COO - This confirmed that the carboxyl group of EDTA underwent a dehydration and condensation reaction with the hydroxyl group of 2,4-DHBA. Other major peaks were observed at 1715 cm⁻¹. -1 Vibration at C=O at 1140cm -1 Tensile vibration of COC at 617cm. -1 The characteristic peak of out-of-plane bending vibration at 1072 cm⁻¹ -1 955cm -1 and 862cm -1 Vibration at PO / P=O, 527cm -1 The strong absorption peak at that location indicates the presence of Fe-O.

[0108] 2.3 Observe the morphology and size distribution of the above Fe, Co, P-CDs nanozymes:

[0109] The purified sample was dispersed and dissolved in deionized water, and 10 μL was dropped onto a 300-mesh copper grid for observation using a transmission electron microscope (JEM-2100, JEOL Ltd., Japan).

[0110] like Figure 3 As shown, the Fe,Co,P-CDs nanozymes are uniform in size and well dispersed, with an average particle size of approximately 2.43 nm. As can be seen from the left inset (HRTEM image), the Fe,Co,P-CDs nanozymes have obvious and clear lattice stripes with a lattice spacing of 0.12 nm.

[0111] 2.4 Detection of peroxidase and oxidase mimicry activities of the above Fe,Co,P-CDs nanozymes

[0112] Using TMB as a chromogenic substrate, the peroxidase and oxidase activities of Fe,Co,P-CDs were preliminarily evaluated. A mixture of 50 μL of a 2 mg / mL Fe,Co,P-CDs nanozyme aqueous solution, 100 μL of a 5 mM TMB solution, and 50 μL of a 0.3% H2O2 solution was added. Then, 820 μL of a 20 mM pH 4 PBS buffer solution was added. The mixture was then subjected to hydrothermal treatment at 37 °C for 20 min, and the absorbance at 652 nm was recorded to determine the peroxidase-mimicking activity of the Fe,Co,P-CDs nanozyme.

[0113] The oxidase-simulating activity of Fe,Co,P-CDs nanozymes was tested according to the above steps. No H2O2 solution was added to the test system. The mixed solution was placed at 37℃ for hydrothermal treatment for 20 min, and its absorbance at 652 nm was recorded to determine the oxidase-simulating activity of Fe,Co,P-CDs nanozymes.

[0114] like Figure 4 As shown, in the presence of H2O2, Fe,Co,P-CDs can catalyze the oxidation of TMB to blue (centrifuge tube 1 in the right inset), exhibiting a typical characteristic absorption peak at 652 nm (red line 1), indicating that Fe,Co,P-CDs possess peroxidase-mimicking activity. In the absence of H2O2, the Fe,Co,P-CDs nanozyme requires a reaction at 37℃ for 20 min to catalyze the oxidation of TMB, turning the chromogenic substrate pale blue (centrifuge tube 4 in the right inset), exhibiting a lower absorption peak at 652 nm (purple line 4), indicating that Fe,Co,P-CDs possess weaker oxidase activity.

[0115] 2.5 Relationship between peroxidase-mimicking activity and pH value of the above Fe,Co,P-CDs nanozymes:

[0116] 50 μL of Fe,Co,P-CDs nanozyme aqueous solution with a concentration of 2 mg / mL, 100 μL of TMB solution with a concentration of 5 mM and 50 μL of H2O2 solution (H2O2 mass percentage of 0.3%) were mixed, and 820 μL of PBS buffer solution (concentration of 20 mM) with pH values ​​of 2, 3, 4, 5, 6 and 7 were added. The mixture was then placed at 37℃ for hydrothermal treatment for 20 min, and its UV absorption at 652 nm was recorded.

[0117] Figure 5 (The inset shows the colors of solutions with pH values ​​from 2 to 7). In PBS buffer at different pH values, the ultraviolet absorption intensity of Fe,Co,P-CDs nanozymes at 652 nm varies greatly. The optimal pH for the catalytic reaction of Fe,Co,P-CDs nanozymes is 4.0. pH values ​​greater than or less than 4 affect the catalytic activity of Fe,Co,P-CDs. The results indicate that the peroxidase-mimicking activity of Fe,Co,P-CDs nanozymes is affected by pH.

[0118] 2.6 The steady-state kinetics of the above Fe, Co, P-CDs nanozymes under different concentrations of TMB and H2O2 and the corresponding Lineweaver-Burk assays were determined:

[0119] The Vmax and Km of Fe,Co,P-CDs nanozymes were determined by steady-state kinetics, such as... Figure 6 Content (a), Figure 6 As shown in content (b), the Vmax and Km of TMB are 8.83 × 10⁻⁶. -5 mM s -1 and 1.28mM; such as Figure 7 Content (a), Figure 7 As shown in content (b), the Vmax and Km of H2O2 are 4.61 × 10⁻⁶. -5 mM s -1 And 0.13mM.

[0120] 2.7 The effect of the above Fe,Co,P-CDs nanozyme on the detection of hypoxanthine (Hx):

[0121] 20 μL of hypoxanthine (Hx) standard solutions of different concentrations and 50 μL of Fe,Co,P-CDs nanozyme aqueous solution with a concentration of 2 mg / mL were placed in a water bath at 37℃ for 30 min. Then, 100 μL of 5 mM TMB solution, 50 μL of H2O2 solution (H2O2 mass percentage 0.3%) and 800 μL of PBS buffer solution (pH=4) were added, and the mixture was placed in a water bath at 37℃ for 20 min. The UV-Vis absorbance of the mixture at a wavelength of 652 nm was recorded as A. 652 .

[0122] Figure 8 As shown in (a), with the increase of Hx concentration, the absorbance at 652 nm gradually decreases and the solution color gradually becomes lighter (the inset shows the corresponding solution color). The reason for this may be that the physical adsorption of Hx on Fe,Co,P-CDs causes the surface of Fe,Co,P-CDs with catalytic active sites to be coated with Hx. Therefore, the catalytic activity of Fe,Co,P-CDs nanozymes decreases as the number of active sites decreases. Figure 8 Content (b) shows the curve of the absorbance difference before and after the addition of Hx as a function of Hx concentration. It can be seen that when the Hx concentration is 0.3 μM to 400.0 μM, there is a linear relationship between the absorbance difference (A0-A) and the Hx concentration, with the equation y = 0.0014x + 0.0041 and the correlation coefficient (R) 2 The value is 0.9961, where x is the Hx concentration, y is the difference in absorbance at 652nm wavelength before and after the addition of Hx (A0-A), and the detection limit (LOD) of Hx is 0.3μM.

[0123] 2.8 Evaluation of the specificity of the Hx detection method based on the Fe,Co,P-CDs nanozyme sensing platform:

[0124] Non-target analytes (including lactose, glucose (Glc)) and common ions such as Mg2+ are selected in the detection reaction. 2+ Ca 2+ Fe 3+ Na + K + Co 2+ Mn 2+ Zn 2+ Cr 2+ Ag + The selectivity of Fe,Co,P-CDs nanozyme sensing platform for Hx was studied, and the detection method was the same as in 2.7.

[0125] Figure 9 Among the non-target analytes, the Fe,Co,P-CDs nanozyme colorimetric sensing platform showed the largest difference in absorbance values ​​for Hx, indicating that the detection platform exhibits excellent selective response to Hx.

[0126] 2.9 Detection of Hx in actual shrimp samples based on Fe,Co,P-CDs nanoenzyme sensing platform:

[0127] Live shrimp were randomly grouped and placed at room temperature for 3 hours and 6 hours, with three replicates for each treatment group. The shrimp were ground to purity using a mortar and pestle. 2g of shrimp sample was mixed with 10mL of distilled water, vortexed for 3 minutes, sonicated for 15 minutes, and centrifuged at 5000rpm for 5 minutes. The supernatant was collected, and TCA (10% by mass) was added and stirred thoroughly. The mixture was centrifuged to remove precipitated proteins and filtered through a 0.45μm polyethersulfone membrane to obtain the shrimp extract. 20μL of the shrimp extracts placed for 3 hours and 6 hours were taken, and Hx standard solution with final concentrations of 10μM, 20μM, and 40μM was added to each extract, respectively. Shrimp extract and shrimp extract with different final concentrations of Hx standard solution were placed in a 37℃ water bath for 30 min. Then, 50 μL of Fe,Co,P-CDs nanozyme aqueous solution (2 mg / mL), 100 μL of 5 mM TMB solution, 50 μL of H2O2 solution (H2O2 mass percentage 0.3%), and 780 μL of PBS buffer solution (pH=4) were added, and the mixture was placed in a 37℃ water bath for 20 min. The absorbance signal of the mixture at 652 nm was recorded. Substituting the results into the equation in section 2.7, the original amount of Hx and the measured amount of Hx were obtained. The recovery rate was calculated as ((measured amount of Hx - original amount of Hx) / amount of Hx added) × 100%. The results of evaluating the effectiveness of the Fe,Co,P-CDs nanozyme-based biosensing platform using the standard addition method are shown in Table 1.

[0128] Table 1. Analysis results of Hx and added Hx in shrimp extract.

[0129]

[0130] In Table 1, the recovery rate of Hx ranged from 95.30% to 107.15%, and the relative standard deviation (RSD) ranged from 2.88% to 5.94%, indicating that the biosensing platform has good recovery rate and reproducibility.

[0131] 2.10 The effect of the above Fe, Co, P-CDs nanozyme on pH detection:

[0132] Take 900 μL of PBS (20 mM) buffer solution at different pH values, add 100 μL of Fe,Co,P-CDs nanozyme aqueous solution at a concentration of 2 mg / mL, and mix thoroughly. Record the emission wavelength of the above mixed solution under 370 nm excitation using a fluorometer, and record the ratio of the fluorescence intensity at 425 nm to the fluorescence intensity at 505 nm (Ig). 425 / I 505 The sensitivity of the fluorescence properties of Fe,Co,P-CDs nanozymes to pH was investigated.

[0133] like Figure 10As shown, the fluorescence color and intensity of Fe,Co,P-CDs changed as the pH value increased from 2.0 to 13.0. When the pH value was less than 6, an emission peak appeared at 500 nm, and at this time, the fluorescence color of the Fe,Co,P-CDs nanozyme was green under 365 nm ultraviolet irradiation (top left inset). When the pH value was greater than 6, the emission peak red-shifted from 500 nm to 505 nm, and the intensity increased. When the pH value was 7, a new fluorescence emission peak appeared at a wavelength of 425 nm, and the fluorescence color changed from green to bluish-green. Further increasing the pH value increased the intensity of the fluorescence emission peak at 425 nm.

[0134] Depend on Figure 11 As shown in content (a), the fluorescence intensity ratio of Fe,Co,P-CDs nanozymes at 425 nm and 505 nm is (I 425 / I 505 The relationship between pH and pH can be expressed as two continuous linear relationships. When pH is 6–7, y = 0.4319x - 2.5039 (correlation coefficient R). 2 =0.9970); when pH is 7-9, y = 0.7154x - 4.499 (correlation coefficient R). 2 =0.9959), where x is the pH value and y is the fluorescence intensity ratio I. 425 / I 505 It can be seen that as the pH value increases, the intensity of the fluorescence emission peak at 425 nm increases, and the fluorescence intensity is higher than that of I. 425 / I 505 Increase.

[0135] Live shrimp were randomly grouped and placed at 25℃ and 37℃ for 0h, 2h, 4h, 6h, 8h, 10h, and 12h, respectively. The shrimp were then crushed, and 1g of shrimp meat was added to 10mL of pre-cooled distilled water. The mixture was vortexed for 1min, sonicated for 20min, and centrifuged at 4000rpm for 5min. 900μL of the supernatant was taken, and 100μL of a 2mg / mL Fe,Co,P-CDs nanozyme solution was added. The emission wavelength of the mixture under 370nm excitation was recorded using a fluorometer, and the ratio of the fluorescence intensity at 425nm to that at 505nm was recorded (If). 425 / I 505 Then, substitute the values ​​into the linear relationship described above to calculate the pH value of the supernatant of each sample.

[0136] Microbial spoilage of proteins in animal products can cause changes in pH levels, such as... Figure 11As shown in section (b), with prolonged storage time, the pH value of the sample increased, and the fluorescence color changed from blue-green to blue, indicating significant spoilage of the shrimp meat. Compared with the sample stored at 25°C, the pH value change of the sample stored at 37°C was more pronounced. These results further demonstrate that the fluorescence characteristics of Fe,Co,P-CDs nanozymes can sensitively and accurately indicate pH changes in shrimp meat.

[0137] Comparative Example 1

[0138] 0.0731 g of disodium ethylenediaminetetraacetate and 0.0771 g of 2,4-dihydroxybenzoic acid were dissolved in 20 mL of 5 M phosphoric acid solution. The mixed solution was transferred to a 25 mL polytetrafluoroethylene-lined reactor, and purified water was added to fill the reactor. The reaction was carried out at 200 °C for 3 h. After the reaction was completed, the insoluble matter was removed by centrifugation, and the sample was dialyzed through a 500 Da dialysis bag for 4 h. The sample was then freeze-dried to obtain a solid P-CDs sample.

[0139] Comparative Example 2

[0140] 0.0731 g of disodium ethylenediaminetetraacetate, 0.0771 g of 2,4-dihydroxybenzoic acid, and 0.04 g of FeCl3·6H2O were dissolved in 20 mL of 5M phosphoric acid solution. The mixed solution was transferred to a 25 mL polytetrafluoroethylene-lined reactor, and purified water was added to fill the reactor. The reaction was carried out at 200 °C for 3 h. After the reaction was completed, the insoluble matter was removed by centrifugation, and the sample was dialyzed through a 500 Da dialysis bag for 4 h. The sample was then freeze-dried to obtain a solid Fe,P-CDs sample.

[0141] Comparative Example 3

[0142] 0.0731g of disodium ethylenediaminetetraacetate, 0.0771g of 2,4-dihydroxybenzoic acid, and 0.03g of vitamin B12 were added. 12 Dissolve in 20 mL of 5 M phosphoric acid solution. Transfer the mixture to a 25 mL polytetrafluoroethylene-lined reactor and add purified water to fill the reactor. React at 200 °C for 3 h. After the reaction is complete, centrifuge to remove insoluble matter, dialyze through a 500 Da dialysis bag for 4 h, and freeze-dry to obtain a solid Co,P-CDs sample.

[0143] The peroxidase mimicry activities of P-CDs nanozymes, Fe,P-CDs nanozymes, and Co,P-CDs nanozymes were detected according to the method provided in Example 1, and the results are as follows: Figure 12 It can be seen that P-CDs nanozymes do not have peroxidase-mimicking activity, while Fe,Co,P-CDs nanozymes have significantly higher peroxidase-mimicking activity than Fe,P-CDs nanozymes and Co,P-CDs nanozymes. This may be due to synergistic effects among the components of Fe,Co,P-CDs nanozymes.

[0144] Example 2

[0145] Dissolve 0.05 g of disodium ethylenediaminetetraacetate, 0.05 g of 2,4-dihydroxybenzoic acid, 0.02 g of cobalt nitrate, and 0.01 g of FeCl3·6H2O in 13.5 mL of 5 M sodium dihydrogen phosphate solution. Transfer the mixed solution to a 25 mL polytetrafluoroethylene-lined reactor and add purified water to fill the reactor. React at 220 °C for 1 h. After the reaction is complete, centrifuge to remove insoluble matter, dialyze through a 500 Da dialysis bag for 2 h, and freeze-dry to obtain solid sample 1.

[0146] Dissolve 0.06 g of disodium ethylenediaminetetraacetate, 0.07 g of 2,4-dihydroxybenzoic acid, 0.01 g of cobalt chloride, and 0.03 g of ferric nitrate in 20 mL of 5 M phosphoric acid solution. Transfer the mixture to a 25 mL polytetrafluoroethylene-lined reactor and add purified water to fill the reactor. React at 180 °C for 5 h. After the reaction is complete, centrifuge to remove insoluble matter, dialyze through a 500 Da dialysis bag for 4 h, and freeze-dry to obtain solid sample 2.

[0147] Dissolve 0.1 g of disodium ethylenediaminetetraacetate, 0.1 g of 2,4-dihydroxybenzoic acid, 0.05 g of cobalt sulfate, and 0.05 g of ferric sulfate in 20 mL of 2M sodium pyrophosphate solution. Transfer the mixed solution to a 25 mL polytetrafluoroethylene-lined reactor and add purified water to fill the reactor. React at 200 °C for 4 h. After the reaction is complete, centrifuge to remove insoluble matter, dialyze through a 500 Da dialysis bag for 5 h, and freeze-dry to obtain solid sample 3.

[0148] The samples were tested according to the method in Example 1. The results showed that the aqueous solutions of the samples were all yellow under sunlight and exhibited bright green fluorescence under 365 nm ultraviolet light. In PBS buffer solutions with pH values ​​ranging from 2.0 to 13.0, the fluorescence color of the mixed solutions containing the samples changed. When the pH was greater than 7, the fluorescence color of the mixed solutions changed from green to blue-green, and a new fluorescence emission peak appeared on top of the original emission peak. The intensity of this emission peak increased with increasing pH, indicating that the fluorescence characteristics of the samples were all pH-sensitive. Furthermore, the peroxidase mimicry activity test results showed that the samples all possessed peroxidase mimicry activity, which was comparable to that of the carbon dot nanozyme prepared in Example 1.

[0149] Example 3

[0150] Preparation of hydrogel loaded with carbon dot nanozymes: A 20 mM TMB solution was prepared by dissolving TMB in methyl sulfoxide. Sodium alginate (1.5 wt% of the total blue polymer solution) was completely dissolved in PBS at pH 4.0. Gelatin (0.45 wt% of the total blue polymer solution) was added and stirred at 45°C until completely dissolved. Chitosan (0.42 wt% of the total blue polymer solution) was then added and stirred thoroughly. In a specific ratio, 15 μL of an aqueous solution of carbon dot nanozymes (20 mg / mL), 75 μL of TMB solution (20 mM), and 15 μL of H₂O₂ solution (3 wt% H₂O₂) were added to each gram of blue polymer solution. The mixture was stirred in a water bath at 37°C for 60 min to obtain a blue polymer solution. This blue polymer solution was then mixed with a 0.1 mol / L CaCl₂ solution and allowed to stand for crosslinking for 15 min to obtain the CDs-SCG hydrogel.

[0151] Preparation of hydrogel spheres: The above blue polymer solution was taken with a syringe and dripped into CaCl2 solution (concentration of 0.1 mol / L) by extrusion. After standing for crosslinking for 15 min, CDs-SCG hydrogel spheres with a diameter of 0.5 cm were obtained.

[0152] Preparation of hydrogel film: The above blue polymer solution was cast and smoothed in a petri dish using a casting method. Then, CaCl2 solution was added along the edge of the petri dish until the polymer solution was completely submerged. After standing for crosslinking for 15 minutes, the film was taken out and cut into circular CDs-SCG hydrogel films with a thickness of 0.5 mm and a diameter of 4.5 cm using a mold.

[0153] Experiments revealed that when the mass percentage of sodium alginate in the blue polymer solution was less than 0.8 wt%, the resulting hydrogel film exhibited extremely poor mechanical properties and unstable morphology. When the mass percentage of sodium alginate was greater than 1.5 wt%, the hydrogel film dissolved rapidly and completely in an alkaline buffer solution with a pH of 7.5, making it unsuitable for detecting the freshness of aquatic products. When the mass percentage of chitosan in the blue polymer solution was less than 0.2 wt%, the resulting hydrogel film required more than 24 hours to fully swell and break down in an alkaline buffer solution with a pH ≥ 8.0, failing to respond quickly to pH changes when aquatic products spoiled. When the mass percentage of gelatin in the blue polymer solution was less than 0.45 wt%, the resulting hydrogel film exhibited poor flexibility and plasticity, as well as poor mechanical properties. When the mass percentage of gelatin was greater than 1 wt%, the hydrogel film required a long time to completely dissolve and break down in an alkaline buffer solution with a pH of 8.0, also failing to respond quickly to pH changes when aquatic products spoiled.

[0154] The above-mentioned CDs-SCG hydrogel film is affected by pH value:

[0155] The pH of aquatic products such as shrimp is generally 7.1–7.4. In the initial post-mortem period, the pH of shrimp tends to decrease due to the degradation of glycogen and the generation of acids. Subsequently, the pH gradually increases as nitrogenous substances such as proteins decompose into amines. Studies generally consider pH = 7.8 to be the critical value for determining whether shrimp are edible. If CDs-CSG hydrogel films are used for online monitoring of aquatic product freshness, the CDs-CSG hydrogel films need to remain stable in the pH environment when the aquatic products are fresh, and respond rapidly to pH when the aquatic products spoil. Furthermore, considering that the loading of CDs may affect the pH responsiveness of the gel, it is necessary to examine the changes in the weight and morphology of the CDs-CSG hydrogel film over time at pH 6.5–8.0.

[0156] like Figure 13 As shown in content (a), the CDs-CSG hydrogel film initially absorbs water and swells in the solution, causing a slight increase in weight. However, as the pH increases, the electrostatic repulsion continuously increases, leading to continuous dissolution of the film and a decreasing weight trend. The CDs-CSG hydrogel film continuously dissolves, and the weight shows a decreasing region, as shown in... Figure 13 As shown in content (b), the CDs-CSG hydrogel film exhibits a morphological change from a solid gel to a sol, indicating that the CDs-SCG hydrogel film has pH responsiveness.

[0157] Combination Figure 13 Content (a) and Figure 13 As shown in section (b), the CDs-CSG hydrogel film exhibits damage and degradation after 20 minutes in an environment with pH 8.0, and its weight begins to decrease. After 50 minutes, it transforms from a solid gel to a sol, completely dispersed in the buffer solution, and its weight completely degrades to zero. When the CDs-CSG hydrogel film is placed in an environment with pH 7.5 for 3 hours, slight damage and minor degradation occur, but it still maintains a relatively intact and stable morphology. The morphology and weight of the CDs-CSG hydrogel film placed in environments with pH 6.5 and 7.0 show almost no change. This indicates that the CDs-CSG hydrogel film prepared in this application can rapidly respond to pH during the spoilage of aquatic products through its pH responsiveness, providing strong support for the visual sensory judgment of freshness.

[0158] The above CDs-SCG hydrogel beads showed the following effect on Hx detection:

[0159] Different concentrations of Hx, CDs-CSG hydrogel beads, and PBS at pH 4.0 were mixed in EP tubes and incubated at 37°C for 60 min. The CDs-CSG hydrogel beads were then removed. The R / B values ​​of the CDs-CSG hydrogel beads before and after incubation were recorded using a smartphone, and the quantitative relationship between the color difference value ΔR / B and the Hx concentration was calculated.

[0160] like Figure 14 As shown in (a), as the concentration of Hx increases from 1.5 μM to 1500 μM, the response signal ΔR / B also increases. ΔR / B shows a good linear relationship with the Hx concentration, with the regression equation being y = 0.10567x + 0.00374 and the correlation coefficient (R²) being [missing information]. 2 The threshold value is 0.9996, and the detection limit is 1.48 μM (LOD = 3σ / S), where x is the logarithm of the Hx concentration and y is the ΔR / B of the CDs-CSG hydrogel spheres. A smartphone can be used as a terminal reader to capture images and convert the color signals into digital values ​​of an RGB color pattern to accurately determine the Hx concentration.

[0161] The above CDs-SCG hydrogel's effect on detecting Hx in actual samples:

[0162] The quantitative detection of Hx in shrimp using CDs-CSG hydrogel beads was performed as follows: Fresh shrimp were euthanized and placed at 25℃ for 0, 3, 6, 9, and 12 hours, and samples were taken for Hx content detection. The sampling method was as follows: 2g of shelled shrimp meat from shrimp placed at different times was minced, 10mL of purified water was added, shaken for 1 min, sonicated for 15 min, centrifuged at 4000 rpm for 5 min, and the supernatant was collected. 10% trichloroacetic acid was added, shaken for 1 min, centrifuged at 4000 rpm for 10 min, and the supernatant was filtered through a 45μM polyethersulfone (PES) filter membrane to obtain the extracted Hx sample solution. The Hx content was detected by mixing the Hx sample solution, CDs-CSG hydrogel beads, and PBS (pH=4) in an EP tube and incubating at 37℃ for 60 min. The CDs-CSG hydrogel beads were then removed. The R / B values ​​of the CDs-CSG hydrogel beads before and after incubation were read using a smartphone.

[0163] Generally, the relationship between Hx content in shrimp meat and its freshness is considered to be: Freshness: ≤72 mg / kg; Sub-freshness: 72~118 mg / kg; Decay: ≥118 mg / kg. The results of Hx detection in shrimp meat using CDs-CSG hydrogel beads are shown below. Figure 14 Content (b) shows that Hx was detected to be greater than 118 mg / kg at 9h, and the shrimp meat had a noticeable off-odor and discoloration, indicating that the shrimp had already spoiled and could no longer be eaten.

[0164] The above-mentioned CDs-CSG gel film was used for online monitoring of the spoilage of actual samples:

[0165] After euthanizing fresh prawns, CDs-CSG hydrogel films were gently attached to the surface of the prawns. Using SCP gel films (without pH and Hx responsiveness) as a control, the prawns with hydrogel films were placed in petri dishes, sealed with plastic wrap, and placed at 25°C for 0, 3, 6, 9, and 12 hours, respectively. The color and morphological changes of the hydrogel films were observed and recorded.

[0166] like Figure 15 Content (a), Figure 15 Content (b) Figure 15 Content (c) Figure 15 Content (d) Figure 15 As shown in section (e), with prolonged storage time, the shrimp continuously decomposed, the blue color of the hydrogel film gradually disappeared, and the gel gradually dissolved. When the shrimp were placed at 25°C for 9 hours, as... Figure 15 As shown in section (f), the blue color of the hydrogel film completely disappeared, and compared with the control hydrogel film, the hydrogel transformed into a sol-like state. The morphology of the hydrogel showed a clear and visible change. At this point, the shrimp exhibited a distinct off-odor, and the shell color changed from the glossy bluish-green of fresh shrimp to a dull grayish-white, even showing noticeable black spots. This indicated that the shrimp had spoiled and was no longer edible, suggesting that the Hx content in the shrimp was >118 mg / kg and the pH was ≥8.0. This result is consistent with the results of quantitative Hx detection using hydrogel spheres to indicate shrimp freshness, demonstrating that the CDs-CSG hydrogel film can accurately evaluate shrimp freshness through morphological and color changes, and can serve as a reliable and powerful tool for long-term online monitoring of aquatic product freshness.

[0167] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing carbon dot nanozymes, characterized in that, Includes the following steps: Disodium ethylenediaminetetraacetate, 2,4-dihydroxybenzoic acid, iron source, cobalt source, phosphorus source and water were mixed evenly and heated to carry out the synthesis reaction. The reaction solution was centrifuged, dialyzed and freeze-dried to obtain carbon dot nanozymes. The iron source is one or more of ferric chloride hexahydrate, ferric sulfate, and ferric nitrate; the cobalt source is VB. 12 One or more of cobalt chloride, cobalt sulfate, and cobalt nitrate; the phosphorus source is one or more of phosphoric acid, sodium dihydrogen phosphate, and sodium pyrophosphate. The mass ratio of the disodium ethylenediaminetetraacetate, 2,4-dihydroxybenzoic acid, iron source, cobalt source, and phosphorus source is (0.05~0.1):(0.05~0.1):(0.01~0.05):(0.01~0.05):(8~11). The synthesis reaction is carried out at a temperature of 180℃ to 220℃ for 1 hour to 5 hours.

2. The preparation method according to claim 1, characterized in that, The concentration of disodium ethylenediaminetetraacetate in the reaction solution is 2 mg / mL to 4 mg / mL.

3. A carbon dot nanozyme prepared according to the preparation method of claim 1 or 2.

4. The application of the carbon dot nanozyme according to claim 3 in the detection of pH and / or hypoxanthine.

5. A hydrogel loaded with the carbon dot nanozyme of claim 3.

6. The hydrogel according to claim 5, characterized in that, The preparation method of the hydrogel includes the following steps: The aqueous solution of the carbon dot nanozyme, chromogenic substrate solution, H2O2 solution, PBS buffer solution, sodium alginate, gelatin, and chitosan were mixed evenly to obtain a blue polymer solution; then the blue polymer solution was mixed with CaCl2 solution and allowed to stand for crosslinking to obtain a hydrogel. The hydrogel includes at least one of hydrogel film and hydrogel sphere; In the blue polymer solution, the mass percentage of sodium alginate is 0.8wt%~1.5wt%; the mass percentage of gelatin is 0.45wt%~1wt%; and the mass percentage of chitosan is 0.2wt%~0.42wt%.

7. The hydrogel according to claim 6, characterized in that, The concentration of carbon dot nanozymes in the aqueous solution is 10 mg / mL to 20 mg / mL; and / or, The concentration of the chromogenic substrate in the chromogenic substrate solution is 5 mM to 20 mM; and / or, The chromogenic substrate is 3,3',5,5'-tetramethylbenzidine; and / or... The H2O2 solution contains 0.3wt% to 3wt% by mass; and / or, The volume ratio of the aqueous solution of the carbon nanoparticle nanozyme, the chromogenic substrate solution, and the H2O2 solution is (0.5~1.5):(4~6):(0.5~1.5); and / or, The pH of the PBS buffer solution is 3-5.

8. The application of the hydrogel according to claim 6 or 7 in the rapid visual detection of pH and / or hypoxanthine to determine product freshness.

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