Cerium nano-enzyme hydrogel as well as preparation method and application thereof

Abstract

The invention discloses a cerium nano-enzyme hydrogel and a preparation method and application thereof, the preparation method comprises the following steps: adding a methanol solution of Ce (NO3) 3.6 H2O into a methanol solution of ZIF-8, stirring for 18-30 hours, then centrifuging, collecting a precipitate, washing and drying to obtain ZIF-8-Ce; the preparation method comprises the following steps: dissolving ZIF-8-Ce in water to obtain a ZIF-8-Ce solution, then adding sodium alginate powder to obtain a ZIF-8-Ce / SA solution, then dropwise adding the ZIF-8-Ce / SA solution into a CaCl2 solution, cross-linking for 5-10 minutes, and washing to obtain the ZIF-8-Ce / SA hydrogel. The prepared ZIF-8-Ce / SA hydrogel and cerium nano enzyme ZIF-8-Ce have excellent multi-enzyme activity, and the hydrogel is used as a three-dimensional network structure carrier and an ideal carrier to maintain the stability of nano enzyme activity, so that the detection process is easier and faster.

Description

Technical Field

[0001] The present invention relates to the field of nanomaterial technology, in particular to a cerium nanoenzyme hydrogel and a preparation method thereof, as well as its application in detecting heavy metal ions Hg 2+ Application in. Background Art

[0002] Hg in water 2+ Pollution can damage aquatic ecosystems, such as Hg 2+ It may accumulate in some aquatic plants and then accumulate through the food chain, affecting human health. 2+ The detection of Hg 2+ Conventional detection methods for Hg include atomic absorption spectroscopy, inductively coupled plasma mass spectrometry, anodic stripping voltammetry, and high performance liquid chromatography. However, the operation process usually requires tedious sample pretreatment and the use of expensive large instruments, which is not suitable for on-site real-time monitoring. Therefore, research and development of methods that facilitate on-site real-time monitoring of Hg 2+ A simple, economical and fast-response detection method for ions is crucial.

[0003] Nanozymes refer to a class of functional nanomaterials that have enzymatic properties such as catalytic efficiency and enzymatic reaction kinetics similar to those of natural enzymes. Nanozymes have gradually become a hot topic of research in the field of heavy metal detection due to their advantages such as low preparation cost, high atomic utilization, and controllable active sites.

[0004] In related technologies, such as in the literature [1], Li R et al. used discarded peanut shells as carbon, nitrogen and sulfur sources to synthesize three-dimensional hierarchical porous graphite carbon material Fe-N / SC single-atom nanozyme. Based on the good OXD mimetic properties of Fe-N / SC, a new type of "on-off-on" colorimetric sensor was proposed for rapid detection of Hg 2+ GSH has the ability to reduce Fe-N / SC, which can inhibit the oxidation of TMB, resulting in the reduction of blue oxTMB, thus closing the TMB+Fe-N / SC system. 2+ Afterwards, due to the sulfhydryl group of GSH and Hg 2+ Due to the high affinity between GSH and Hg, the TMB in the above system is released, thereby restoring the blue color and restoring the TMB+Fe-N / SC-GSH color system (turning on). The proposed colorimetric sensor has been successfully applied to GSH and Hg in real samples. 2+ detection and achieved good analytical results.

[0005] However, this method has the following defects: complex preparation process: it requires multiple steps to synthesize carbon-based materials, involving high-temperature carbonization and doping, which is time-consuming and energy-consuming; insufficient stability: nanozymes are prone to aggregation and inactivation, and their long-term storage performance is reduced; limited detection sensitivity: it relies on traditional spectrophotometers, making it difficult to achieve rapid on-site detection.

[0006] Literature [1]: Li R, He 2+ [J].Science of the Total Environment,2022(834-):834.DOI:10.1016 / j.scitotenv.2022.155428. Summary of the Invention

[0007] The present invention aims to solve one of the technical problems existing in the related art to at least a certain extent. To this end, the present invention provides a cerium nanozyme hydrogel and a preparation method and application thereof.

[0008] The present invention adopts the following technical solutions:

[0009] Firstly,

[0010] The present invention provides a method for preparing a cerium nanozyme hydrogel, comprising the following steps:

[0011] (1) Under stirring at 600-800 rpm, a methanol solution of Ce(NO3)3·6H2O with a concentration of 18 mM / mL was added to a methanol solution of ZIF-8 with a concentration of 0.1 mM / mL in a volume ratio of 1:2. The mixture was stirred at room temperature for 18-30 h, then centrifuged for 10-20 min, and the precipitate was collected, washed with methanol several times, and dried in air at 50-70°C for 8-15 h to prepare ZIF-8-Ce.

[0012] (2) The ZIF-8-Ce prepared in step (1) was dissolved in ultrapure water and ultrasonically dispersed to obtain a ZIF-8-Ce solution with a concentration of 1 mg / mL. Sodium alginate powder was then added to make the concentration of sodium alginate 10 mg / mL. The mixture was magnetically stirred for 2-4 h to obtain a ZIF-8-Ce / SA solution. CaCl2 was dissolved in ultrapure water to prepare a CaCl2 solution with a concentration of 10 mg / mL. The ZIF-8-Ce / SA solution was added dropwise to the CaCl2 solution, cross-linked for 5-10 min, and the mixture was taken out and rinsed with deionized water to obtain a ZIF-8-Ce / SA hydrogel.

[0013] In some embodiments, the methanol solution of Ce(NO3)3·6H2O with a concentration of 18 mM / mL is prepared by dissolving 90 mM Ce(NO3)3·6H2O in 5 mL of methanol solution and ultrasonically dispersing it for 15 minutes; the methanol solution of ZIF-8 with a concentration of 0.1 mM / mL is prepared by dissolving 1 mM ZIF-8 in 10 mL of methanol solution and ultrasonically dispersing it for 15 minutes.

[0014] In some embodiments, the method for preparing ZIF-8 comprises the following steps:

[0015] Under stirring at 600 rpm, a methanol solution of Zn(NO3)2·6H2O with a concentration of 5.04 mM / mL was added to a methanol solution of 2-MIM with a concentration of 5.16 mM / mL in a volume ratio of 1:1, stirred at room temperature for 1 h, and then centrifuged at 9500 rpm for 10 minutes. The fresh precipitate was washed with methanol, dried at 60°C, and set aside.

[0016] In some embodiments, the methanol solution of Zn(NO3)2·6H2O with a concentration of 5.04mM / mL is prepared by dissolving 25.2mM Zn(NO3)2·6H2O in 5mL of methanol solution and stirring at room temperature for 20min until completely dissolved; the methanol solution of 2-MIM (2-methylimidazole) with a concentration of 5.16mM / mL is prepared by dissolving 25.8mM 2-MIM in 5mL of methanol solution and stirring at room temperature for 20min until completely dissolved.

[0017] In some embodiments, the ZIF-8-Ce is spherical in shape and has a particle size of 250-350 nm.

[0018] Secondly,

[0019] An embodiment of the present invention provides a cerium nanozyme hydrogel prepared by the above method.

[0020] Thirdly,

[0021] The embodiment of the present invention provides a detection system based on cerium nanozyme hydrogel, comprising:

[0022] (1) the cerium nanozyme hydrogel described above;

[0023] (2) Cys solution dissolved in Tris-HCl buffer (pH = 8.5);

[0024] (3) TMB solution;

[0025] (4) HAc-NaAc buffer solution.

[0026] The detection principle of the detection system of the present invention is as follows: using the oxidase-like activity of cerium nanozymes, oxygen in the air is used as a green oxidant to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) into a blue product oxTMB. Under acidic conditions, cysteine ​​(Cys) has different reducing effects on oxidized TMB, thereby restoring the blue oxTMB to colorless. However, the addition of Hg 2+ Afterwards, due to the sulfhydryl group of Cys and Hg 2+ Due to the high affinity between the two, the TMB in the above system is released, the solution turns blue again, and the oxidase-like activity of the cerium nanozyme is also reactivated.

[0027] In some embodiments, the detection system further includes a smartphone installed with an application that performs color channel separation on the captured image, collects and obtains RGB values, and analyzes the concentration of the target substance based on changes in the RGB values.

[0028] Fourthly,

[0029] The present invention also provides the above-mentioned cerium nanozyme hydrogel or a detection system based on cerium nanozyme hydrogel for detecting Hg in water. 2+ application.

[0030] In some application embodiments, the application method includes:

[0031] First, 30 μL of Cys solution (400 μM) dissolved in Tris-HCl buffer (pH = 8.5) was poured into a centrifuge tube, and 10 μL of Hg 2+The standard working solution and the test solution were introduced into a centrifuge tube and incubated at 37°C for 10 minutes. Then, 50 μL of ZIF-8-Ce / SA hydrogel, 350 μL of HAc-NaAc buffer solution (pH = 4.0), and 10 μL of TMB (20 mM) were added to the system in sequence and reacted at 25-30°C (room temperature) for 20 minutes. The total volume was 450 μL. The RGB values ​​of the solution were read using the smartphone application Color Pickers (downloaded from the Apple App Store) to obtain the color values ​​of different standard Hg 2+ The R, G, B data of the concentration are prepared (R+G+B) / 3 and Hg 2+ Make a standard curve based on the concentration, derive a linear equation, measure the R, G, and B data of the test solution, and calculate the Hg of the test solution based on the linear equation. 2+ concentration.

[0032] In some embodiments, the Hg-containing medium contains Hg at different concentrations ranging from 60 to 1200 nmol / L. 2+ The concentration gradient of the standard working solution is 60 nmol / L, 300 nmol / L, 600 nmol / L, 900 nmol / L, and 1200 nmol / L.

[0033] The present invention has the following advantages and beneficial effects:

[0034] The cerium nanozyme ZIF-8-Ce prepared by the present invention has easily available raw materials and low cost; the preparation method is simple and only requires simple mechanical stirring to obtain it; experimental verification shows that the activity of the cerium nanozyme is still above 95% on the 90th day, and has high stability and good long-term activity.

[0035] The ZIF-8-Ce / SA hydrogel prepared by the present invention has excellent multi-enzyme activity. Cerium is evenly dispersed in the form of single atoms on the ZIF-8 carrier material, becoming the catalytic active center and endowing it with catalytic performance. As a three-dimensional network structure carrier, the hydrogel can be used as an ideal carrier to maintain the stability of nanoenzyme activity due to its porous structure, large surface area and good biocompatibility, making the detection process easier and faster. The multifunctional sensing and analysis platform constructed on this basis can be used for heavy metal Hg in water. 2+ The present invention can detect food safety issues quickly and with high sensitivity. Furthermore, the mobile phone application can separate the color channels of captured images, collect and obtain color feature values ​​such as RGB values, and analyze the concentration of the target substance based on changes in RGB values, enabling portable quantitative analysis. The detection system of the present application has good sensitivity and specificity, meets the needs of on-site testing, and has visual readability and good practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The SEM images of ZIF-8 and ZIF-8-Ce prepared in Example 1, wherein the left side is ZIF-8 and the right side is ZIF-8-Ce;

[0037] Figure 2 The effect of different Ce doping amounts on the oxidase-like activity of ZIF-8-Ce;

[0038] Figure 3 This is the SEM image of the ZIF-8-Ce / SA hydrogel prepared in Example 2;

[0039] Figure 4 This is the SEM image of the ZIF-8-Ce / SA hydrogel prepared in Comparative Example 5;

[0040] Figure 5 This is the SEM image of the ZIF-8-Ce / SA hydrogel prepared in Comparative Example 6;

[0041] Figure 6 Effects of different hydrogel systems prepared in Example 2 and Comparative Example 7 on the oxidase-like activity of cerium nanozyme;

[0042] Figure 7 is the UV-visible absorption spectrum of different systems;

[0043] In the figure, a is TMB, b is ZIF-8 / TMB, c is ZIF-8-Ce / TMB, and d is Hg 2+ -ZIF-8-Ce / TMB, e is Cys-ZIF-8-Ce / TMB, f is Hg 2+ -Cys-ZIF-8-Ce / TMB;

[0044] Figure 8 The effects of pH, temperature, time, and nanozyme concentration on the activity of ZIF-8-Ce-like oxidases;

[0045] Figure 9 is the absorbance difference (ΔA) and the Hg concentration 2+ The error bars represent the standard deviation of three independent measurements.

[0046] Figure 10 For different concentrations of Hg 2+ The corresponding photos of the sensor solution under daylight;

[0047] Figure 11 To evaluate the stability of ZIF-8-Ce within 90 days;

[0048] Figure 12 The ZIF-8-Ce-TMB-Cys reaction system was 2+(100 nM) and relative activities in the presence of other metal ions (10 μM);

[0049] Figure 13 The corresponding RGB values ​​and Hg measured for smartphones 2+ Concentration relationship;

[0050] Figure 14 For different Hg 2+ (R+G+B) / 3 value curve corresponding to concentration. DETAILED DESCRIPTION

[0051] Most of the methods for preparing cerium nanozymes in related technologies use solvent (hydro) thermal synthesis, for example:

[0052] Method 1. Preparation of CeO2-MIL(Fe): [Reference [2]]

[0053] PVP-CeO2, FeCl3·6H2O, and 2-aminoterephthalic acid were dissolved in 20 mL of ethanol. The resulting mixture was heated at 50°C for 180 minutes, and the product was centrifuged for 40 minutes. The obtained product was redissolved in 20 mL of ethanol containing FeCl3·6H2O and 2-aminoterephthalic acid. The mixture was heated again at 50°C for 2 hours, and the product was centrifuged in ethanol for 30 minutes. CeO2-MIL(Fe) was collected after dehydration at 60°C for 8 hours.

[0054] Method 2. Preparation of MIL-101(Fe)-NH2: [Reference [3]]

[0055] FeCl3·6H2O, CeCl3·7H2O, and NH2-BDC were dissolved in DMF (30 mL). The precipitate was transferred to a Teflon autoclave and reacted at 110°C for 20 h. The precipitate was then centrifuged at 8500 rpm for 6 min, rinsed three times with DMF and methanol (10 mL each), and dehydrated at 75°C for 30 min. The resulting sample was then dried in a vacuum oven at 150°C for 7 h.

[0056] Method 3. Preparation of Zn / Ce-ZIF: [Reference [4]]

[0057] Zn(NO₃)₂·6H₂O, Ce(NH₄)₂(NO₃)₆, hexadecyltrimethylammonium bromide (CTAB), and 2-methylimidazole were dissolved in a solution consisting of 40 mL of water and 10 mL of DMSO (99.9% purity). The mixture was magnetically stirred and heated at 50°C for 1.5 h. Then, 80 μL of a 1.25 M NaOH solution and 100 μL of a 0.98 M H₂O₂ solution were gradually added to the mixed solution. After stirring for 15 min, the collected solid was washed three times with deionized water and anhydrous ethanol. The resulting Zn / Ce-ZIF was then dried under vacuum.

[0058] Literature [2] Amalraj, Arunjegan, N.Mariyammal, and P.Perumal. "Highly EfficientPeroxidase-like Activity ofMetal Oxide Incorporated Metal Organic FrameworkCeO2-MIL(Fe) and its application for Colorimetric Detection ofMelamine andMercury ions via Induced Hydrogen and Covalent Bond." Analyst 147(2022).

[0059] Literature [3] Gao, Yang, and W. Liu. "Impact ofcerium doping on the peroxidase-like activity of metal–organic frameworks." Dalton T 54.6 (2025).

[0060] Literature [4] Liu, Peng, et al. "Highly specific colorimetric detection of sarcosine using surface molecular imprinted Zn / Ce-ZIF." Journal of ColloidAndInterface Science 681 (2025): 239-249.

[0061] Most of the methods for preparing cerium nanozymes in related technologies use solvent (water) thermal synthesis, which requires the application of certain high temperatures and high pressures. The method for preparing cerium nanozymes in the present invention is a room temperature stirring synthesis method, which is very simple.

[0062] In addition, the cerium nanozyme of the present invention is used as a bimetallic organic framework (MOFs) nanozyme to dope MOFs. There are two synthetic strategies for doping MOFs materials: water (solvent) thermal synthesis and PSM method. Most researchers use water (solvent) thermal synthesis in the preparation of bimetallic organic framework nanozymes. It requires mixing metal salts and organic connectors in a high boiling point solution in a Teflon-lined stainless steel autoclave, and then heating the mixture in an oven for 12-48 hours. This method has inherent synthetic defects. The reaction system is located in a closed container, and it is difficult to control the reaction process and reaction progress. The PSM method used in this application (a method of synthesizing an organic framework first and then performing chemical modification) is more convenient for observation and regulation during the reaction, and has controllability for the product morphology.

[0063] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0064] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0065] Where values ​​are described herein as ranges, it should be understood that such disclosure includes disclosure of all possible sub-ranges within that range, as well as specific values ​​falling within that range, regardless of whether a specific value or sub-range is explicitly stated.

[0066] The following are specific examples and comparative examples of the present invention.

[0067] Example 1

[0068] A method for preparing cerium nanozyme, comprising the following steps

[0069] 25.2 mM Zn(NO₃)₂·6H₂O and 25.8 mM 2-MIM were dissolved in 5 mL of methanol and stirred at room temperature for 20 minutes until completely dissolved. The Zn(NO₃)₂·6H₂O methanol solution was added dropwise to the 2-MIM methanol solution while stirring, and the mixture was stirred at 600 rpm for 1 hour at room temperature. The mixture was centrifuged at 9500 rpm for 10 minutes, and the fresh precipitate was washed with methanol and dried at 60°C for later use. Ce(NO₃)₃·6H₂O was used as a cerium source. The prepared 1 mM ZIF-8 and 90 mM Ce(NO₃)₃·6H₂O were dissolved in 10 mL and 5 mL of methanol, respectively, and ultrasonically dispersed at 40 kHz for 15 minutes. The Ce(NO₃)₃·6H₂O methanol solution was added dropwise to the ZIF-8 methanol solution while stirring, and the mixture was stirred at 800 rpm for 24 hours at room temperature. The mixture was centrifuged at 9500 rpm for 15 minutes, and the pale yellow precipitate was collected, washed with methanol several times, and dried in air at 60°C overnight to obtain ZIF-8-Ce.

[0070] Figure 1 The morphology of ZIF-8 and ZIF-8-Ce prepared in Example 1 was analyzed by SEM. Figure 1 ZIF-8 exhibits a complete rhombic dodecahedron structure, with uniform particle distribution and a smooth surface. ZIF-8-Ce nanoparticles are more spherical. This difference can be attributed to the partial replacement of zinc atoms by cerium atoms, which causes the original MOF structure to partially collapse, forming an irregular, dense grid structure. Ce-doped ZIF-8 increases in size. Using image J software to measure the diameter distribution of the two materials, it was found that the particle size of ZIF-8 is approximately 200 nm, while the particle size of ZIF-8-Ce nanomaterials is approximately 250-350 nm.

[0071] Example 2

[0072] A method for preparing cerium nanozyme hydrogel comprises the following steps

[0073] 0.01 g of the dried ZIF-8-Ce powder prepared in Example 1 was weighed and dissolved in 10 mL of ultrapure water. Ultrasonic dispersion was performed at 40 kHz for 5 min to obtain a ZIF-8-Ce solution. 0.1 g of SA powder was then weighed and dissolved in the ZIF-8-Ce solution. The solution was stirred magnetically at 200 rpm for 3 h to completely dissolve the sodium alginate in the ZIF-8-Ce solution to obtain Solution A. 1 g of CaCl2 was dissolved in 100 mL of ultrapure water to obtain Solution B. Solution A was added dropwise to Solution B using a syringe with an inner diameter of 1.8 mm and cross-linked for 5 min. The solution was then removed and rinsed with deionized water to obtain a ZIF-8-Ce / SA hydrogel.

[0074] Example 3

[0075] Cerium nanozyme-UV-visible spectrometer detection:

[0076] First, 50 μL of TMB (20 mM) and 100 μL of ZIF-8-Ce (1 mg / mL) were added to acetate buffer (pH = 4.0, 1810 μL) and incubated at 30°C for 20 min. Subsequently, 10 μL of Hg 2+ The standard series working solution and the mixed solution of Cys (30 μL, 2 mM) were added to the solution and incubated for 10 min. The absorbance at 650 nm was measured using a UV-visible spectrophotometer. The absorbance difference (ΔA) was compared with the Hg 2+ Make a standard curve with the concentration and derive a linear equation. Under the same conditions, measure the absorbance of the solution to be tested and calculate the Hg content of the solution to be tested according to the linear equation. 2+ Concentration. Among them, the absorbance difference (ΔA) refers to the different concentrations of Hg 2+ The absorbance obtained by detecting the sensor solution (or the mixed solution containing the solution to be tested) is subtracted from the absorbance measured when only the Cys solution is present.

[0077] Example 4

[0078] Portable sensitive hydrogel smartphone colorimetric sensor: First, a Cys solution (30 μL, 400 μM) dissolved in Tris-HCl buffer (pH = 8.5) was poured into a centrifuge tube, and 10 μL of HgCl2 solution containing different concentrations in the range of 60-1200 nmol / L was added to the centrifuge tube. 2+ The standard series working solutions and the test solutions were introduced into centrifuge tubes and incubated at 37°C for 10 min. Subsequently, the ZIF-8-Ce / SA hydrogel prepared in Example 2 (50 μL, a volume of a small ball injected with a 1.8 mm syringe needle), HAc-NaAc buffer solution (pH = 4.0, 350 μL) and TMB (10 μL, 20 mM) were added to the system in sequence and reacted at room temperature for 20 min. The total volume was 450 μL. The RGB value of the solution was read using the Color Pickers smartphone application (downloaded from the Apple App Store) to obtain the color values ​​of different standard Hg 2+ The R, G, B data of the concentration are prepared (R+G+B) / 3 and Hg 2+ The concentration of the standard curve is used to obtain a linear equation, and the R, G, and B data of the test solution are measured. The Hg content of the test solution is calculated according to the linear equation. 2+ concentration.

[0079] This application also studies the effect of different Ce doping amounts on the oxidase-like activity of ZIF-8-Ce

[0080] The ZIF-8-Ce prepared in Example 1 is ZIF-8-Ce-0.09M;

[0081] Comparative Example 1:

[0082] The difference from Example 1 is that Ce(NO3)3·6H2O is 60mM, and the prepared ZIF-8-Ce is ZIF-8-Ce-0.06M; Comparative Example 2:

[0083] The difference from Example 1 is that Ce(NO3)3·6H2O is 70mM, and the prepared ZIF-8-Ce is ZIF-8-Ce-0.07M; Comparative Example 3:

[0084] The difference from Example 1 is that Ce(NO3)3·6H2O is 80mM, and the prepared ZIF-8-Ce is ZIF-8-Ce-0.08M; Comparative Example 4:

[0085] The difference from Example 1 is that the concentration of Ce(NO3)3·6H2O is 100 mM, and the prepared ZIF-8-Ce is ZIF-8-Ce-0.1M;

[0086] from Figure 2 As can be seen in the figure, as the Ce doping content in ZIF-8 increases, the absorbance at 650nm increases rapidly and then decreases slowly. This may be due to the fact that the higher doping amount leads to an increase in the number of active Ce sites in the ZIF-8 framework. However, when the doping amount exceeds a certain critical value, the catalytic effect on the TMB oxidation reaction reaches a peak. Beyond this value, excessive Ce doping will cause changes in the ZIF-8-Ce crystal structure. These results indicate that the Ce doping level has a significant effect on the oxidase-like activity of MOF, which means that Ce doping is an effective strategy to regulate the oxidase-like activity of ZIF-8-Ce. ZIF-8-Ce doped with 0.09M Ce has the best catalytic activity.

[0087] Comparative Example 5

[0088] The difference from Example 2 is that in Comparative Example 5, the amount of CaCl2 added is 0.5 g; that is, the system is 1% SA hydrogel and 0.5% CaCl2.

[0089] Comparative Example 6

[0090] The difference from Example 2 is that in Comparative Example 6, the amount of CaCl2 added is 1.5 g; that is, the system is 1% SA hydrogel and 1.5% CaCl2.

[0091] pass Figure 3-Figure 5From the comparison, it can be seen that the hydrogel prepared in Example 2 (1% SA hydrogel, 1% CaCl2) is moderately cross-linked and has a uniform pore structure, while the hydrogel prepared in Comparative Example 5 has a low degree of cross-linking, large pores, and an unstable structure. The hydrogel prepared in Comparative Example 6 is excessively cross-linked, has a reduced porosity, and an unstable structure.

[0092] Comparative Example 7

[0093] The difference from Example 1 is that in Comparative Example 7, chitosan hydrogel is used.

[0094] A 10 mg / mL chitosan solution and a β-glycerophosphate solution were mixed in a 5:1 volume ratio to prepare a chitosan / β-glycerophosphate solution. 0.1 g of ZIF-8-Ce nanozyme was dispersed in 1 mL of ultrapure water and sonicated for 15 minutes to ensure uniform dispersion to prepare a nanozyme solution. 1 mL of the nanozyme solution was slowly added to 9 mL of the chitosan / β-glycerophosphate solution and stirred evenly. Then, 100 mL of a 1% glutaraldehyde solution was added dropwise as a crosslinker and stirred at room temperature for 45 minutes until a gel was formed.

[0095] In Example 2 of the present application, SA hydrogel was used to load ZIF-8-Ce nanozyme. In Comparative Example 7, chitosan hydrogel was used to load ZIF-8-Ce nanozyme. The effects of different hydrogels on the oxidase-like activity of the nanozyme were compared by observing the color development of the TMB colorimetric reaction and measuring the absorbance value at 650nm using an ultraviolet spectrophotometer.

[0096] The results are as follows Figure 6 As shown, through Figure 6 It can be seen that under the same conditions, the nanozyme loaded with chitosan hydrogel in comparative example 7 has a smaller absorbance value measured at 650 nm than the nanozyme loaded with SA hydrogel in example 2, proving that the ZIF-8-Ce nanozyme can better exhibit its oxidase-like activity in SA hydrogel than in chitosan hydrogel.

[0097] Figure 7 The oxidase-like activity of ZIF-8-Ce prepared in Example 1 was studied by catalyzing the classic reaction of TMB oxidation. Figure 7 As shown in the figure, when only TMB is present, no obvious absorption is observed at a wavelength of 650 nm (curve a). When ZIF-8 and TMB are added to the NaAc-HAc buffer (curve b), the color of the solution does not change, indicating that ZIF-8 has no mimic enzyme activity. However, after the addition of ZIF-8-Ce, the colorless TMB solution quickly turns blue, and at the same time, the characteristic absorption peak of oxTMB appears at a wavelength of 650 nm. This phenomenon indicates that ZIF-8-Ce has oxidase-like activity (curve c). In order to verify the feasibility of this strategy, ZIF-8-Ce, Cys and Hg were studied.2+ Catalytic activity in the presence of Hg 2+ Ions have no effect on the oxidase-like activity of ZIF-8-Ce nanozyme (curve d). After adding Cys, the redox reaction was greatly inhibited, and the color of the solution changed from blue to light blue and then to colorless (curve e). The results show that Cys inhibits the oxidase-like activity of ZIF-8-Ce. Since mercury ions have a strong affinity with the thiol group of Cys, adding Hg to the solution 2+ The absorbance increased and the color of the solution changed from colorless to light blue (curve f). These results indicate that the colorimetric sensor based on ZIF-8-Ce can be used to detect Hg 2+ .

[0098] Figure 8 The effects of different reaction conditions on the activity of the ZIF-8-Ce type oxidase prepared in Example 1 were studied. Figure 8 It can be seen that the catalytic activity of ZIF-8-Ce is highly dependent on pH. When the pH value is between 3.5 and 4.5, ZIF-8-Ce has a high oxidase-like activity. The catalytic activity of the ZIF-8-Ce nanozyme first increases and then decreases, with the highest activity at a pH of 4.0. Therefore, this application selects a pH value of 4.0. In the range of 10-60°C, the catalytic activity of the ZIF-8-Ce nanozyme first increases and then decreases, with the highest activity at 30°C. Therefore, this application selects a temperature of 30°C. In addition, this application also observes the effects of nanozyme concentration and reaction time on its catalytic ability. When the concentration of ZIF-8-Ce is 50μg / mL, the oxidase-like activity reaches the optimal value. As the reaction time increases, the measured absorbance value continues to increase (rapidly increasing within 0-10 minutes and slowly increasing within 10-20 minutes). When the reaction time reaches 20 minutes, the reaction system tends to be stable, so 20 minutes is selected as the reaction time.

[0099] Figure 9 is the absorbance difference (ΔA) and Hg 2+ Calibration curves between concentrations, e.g. Figure 9 As shown, the absorbance difference is related to Hg 2 + There is a good linear relationship between the concentrations of Hg 2+ In the concentration range of 0-2μM, the linear regression equation is A 650 =0.01171Hg(II)+0.00027, with a correlation coefficient of 0.9959. The limit of detection (LOD) was calculated as 0.91 nM based on 3σ / K, where σ is the standard deviation of the blank sample and K is the slope of the calibration curve.

[0100] Figure 10 For different concentrations of Hg2+ The corresponding photos of the sensor solution under sunlight; the solution composition: 50μLTMB (20mM), 100μL ZIF-8-Ce (1mg / mL), 30μLCys (2mM), 10μL different concentrations of Hg 2+ (0, 25, 50, 125, 250, 500, 750, 1000, 1500, 2000 nM), 1810 μL acetate buffer (0.1 M, pH = 4.0) total volume: 2 mL; Figure 10 It can be seen that the naked eye can directly distinguish Hg 2+ Concentration-dependent color change.

[0101] Figure 11 The researchers demonstrated that cerium nanozymes synthesized from the same batch were stored at room temperature and incubated with TMB (20 mM) for 20 minutes every 15 days. After 90 days of storage, the cerium nanozyme retained 95.1% of its initial catalytic activity. These results demonstrate the remarkable stability of cerium nanozymes.

[0102] Figure 12 The selectivity of the prepared ZIF-8-Ce-TMB-Cys system was studied under the optimized conditions. 2+ The concentration of nonspecific ions Co 2+ 、Fe 3+ 、Gd 2+ Mg 2+ 、Ni 2+ , Ca 2+ 、Al 3+ 、Mn 2+ 、Ba 2+ , Pb 2+ , K + 、Na + 、Zn 2+ and an equal amount of Hg as the specific analyte 2+ Colorimetric detection (Hg 2+ The concentration of the above-mentioned interfering substances was 100 nM and that of other ions was 10 μM. 2+ These results indicate that the sensing platform has good selectivity and anti-interference ability.

[0103] Figure 13 Shows that with the Hg 2+ As the concentration increases, the color of the ZIF-8-Ce-TMB-Cys hydrogel sensing platform undergoes a distinct gradient change. The image on the right shows the three primary color parameters of the solution read using the Color Pickers app on a smartphone. Figure 14Shows that with the Hg 2+ As the concentration increased, the value of (R+G+B) / 3 gradually increased, and had good linearity in the range of 0.015-1.2 μM, with an LOD of 21.53 nM.

[0104] This application also uses the standard addition method to measure Hg in water samples (tap water). 2+ (Standard amount: 1×10 -8 M, 1×10 -7 M, 5×10 -7 M, 1×10 -6 M) was used for detection. The established ZIF-8-Ce colorimetric sensing method and ICP-MS were used to detect aquaculture water (tap water), and the methods were validated. The collected water samples were filtered using a 0.22 μm filter membrane to remove suspended matter and impurities.

[0105] The test results are shown in Table 1. As can be seen from Table 1, the Hg 2+ The recoveries were between 97.76% and 105.89%, which were acceptable, with RSDs not exceeding 2.70%. 2+ The recovery rate was 88.47%-109.34%, and the RSD was less than 3.96%. Both analytical methods achieved ideal recovery rates and RSD values. At the same time, the results were consistent with the ICP-MS measurement results (93.86%-113.76%), confirming the effectiveness of the technology in detecting Hg in water samples. 2+ Excellent accuracy and reliability.

[0106] Table 1 Comparison of the results of UV-visible spectrometry and hydrogel smartphone mode with the results of the standard ICP-MS method

[0107]

[0108] Note: "_": not detected; " / ": not detected.

[0109] In the present invention, the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples without contradiction.

[0110] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for preparing a cerium nanozyme hydrogel, characterized in that: The steps include: (1) Under stirring at 600-800 rpm, a methanol solution of Ce(NO3)3·6H2O with a concentration of 18 mM / mL was added to a methanol solution of ZIF-8 with a concentration of 0.1 mM / mL in a volume ratio of 1:

2. The mixture was stirred at room temperature for 18-30 h, then centrifuged for 10-20 min, and the precipitate was collected, washed with methanol several times, and dried in air at 50-70°C for 8-15 h to prepare ZIF-8-Ce. (2) The ZIF-8-Ce prepared in step (1) was dissolved in ultrapure water and ultrasonically dispersed to obtain a ZIF-8-Ce solution with a concentration of 1 mg / mL. Sodium alginate powder was then added to make the concentration of sodium alginate 10 mg / mL. The mixture was magnetically stirred for 2-4 h to obtain a ZIF-8-Ce / SA solution. CaCl2 was dissolved in ultrapure water to prepare a CaCl2 solution with a concentration of 10 mg / mL. The ZIF-8-Ce / SA solution was added dropwise to the CaCl2 solution, cross-linked for 5-10 min, and the mixture was taken out and rinsed with deionized water to obtain a ZIF-8-Ce / SA hydrogel.

2. The method for preparing a cerium nanozyme hydrogel according to claim 1, characterized in that: The methanol solution of Ce(NO3)3·6H2O with a concentration of 18 mM / mL is prepared by dissolving 90 mM Ce(NO3)3·6H2O in 5 mL of methanol solution and ultrasonically dispersing it for 15 minutes; the methanol solution of ZIF-8 with a concentration of 0.1 mM / mL is prepared by dissolving 1 mM ZIF-8 in 10 mL of methanol solution and ultrasonically dispersing it for 15 minutes.

3. The method for preparing a cerium nanozyme hydrogel according to claim 1, characterized in that: The preparation method of ZIF-8 comprises the following steps: Under stirring at 600 rpm, a methanol solution of Zn(NO3)2·6H2O with a concentration of 5.04 mM / mL was added to a methanol solution of 2-MIM with a concentration of 5.16 mM / mL in a volume ratio of 1:1, stirred at room temperature for 1 h, and then centrifuged at 9500 rpm for 10 minutes. The fresh precipitate was washed with methanol, dried at 60°C, and set aside.

4. The method for preparing a cerium nanozyme hydrogel according to claim 3, characterized in that: The methanol solution of Zn(NO3)2·6H2O with a concentration of 5.04mM / mL is prepared by dissolving 25.2mM Zn(NO3)2·6H2O in 5mL of methanol solution and stirring at room temperature for 20 minutes until it is completely dissolved; the methanol solution of 2-MIM with a concentration of 5.16mM / mL is prepared by dissolving 25.8mM 2-MIM in 5mL of methanol solution and stirring at room temperature for 20 minutes until it is completely dissolved.

5. The method for preparing a cerium nanozyme hydrogel according to claim 1, wherein: The ZIF-8-Ce is spherical in shape and has a particle size of 250-350 nm.

6. A cerium nanozyme hydrogel, characterized in that The invention is prepared by the preparation method according to any one of claims 1 to 5.

7. A detection system based on cerium nanozyme hydrogel, characterized in that: include: (1) The cerium nanozyme hydrogel according to claim 6; (2) Cys solution dissolved in Tris-HCl buffer (pH = 8.5); (3) TMB solution; (4) HAc-NaAc buffer solution.

8. The cerium nanozyme hydrogel-based detection system according to claim 7, characterized in that: The detection system also includes a smart phone, which is installed with an application. The application performs color channel separation on the collected image, collects and obtains RGB values, and analyzes the concentration of the target substance based on changes in the RGB values.

9. The cerium nanozyme hydrogel according to claim 6 or the detection system based on the cerium nanozyme hydrogel according to claim 7 is used to detect Hg in water. 2+ application.

10. The use according to claim 9, characterized in that Application methods include: First, 30 μL of Cys solution (400 μM) dissolved in Tris-HCl buffer (pH = 8.5) was poured into a centrifuge tube, and 10 μL of Hg 2+ The standard working solution and the test solution were introduced into a centrifuge tube and incubated at 37°C for 10 minutes. Then, 50 μL of ZIF-8-Ce / SA hydrogel, 350 μL of HAc-NaAc buffer solution (pH = 4.0), and 10 μL of TMB (20 mM) were added to the system in sequence and reacted at 25-30°C for 20 minutes. The RGB value of the solution was read using the Color Pickers of the smartphone application to obtain the color of different standard Hg 2+ The R, G, B data of the concentration are prepared (R+G+B) / 3 and Hg 2+ The concentration of the standard curve is used to obtain a linear equation, and the R, G, and B data of the test solution are measured. The Hg content of the test solution is calculated according to the linear equation. 2+ concentration.

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