Preparation method and application of NiCo2O4 nanozyme with multi-level through-pore characteristics

Abstract

The application discloses a preparation method and application of a NiCo2O4 nanometer enzyme with a multistage through-pore channel characteristic, and belongs to the technical field of biological materials. The NiCo2O4 nanometer enzyme with the multistage through-pore channel characteristic is prepared by constructing a gelatin composite hydrogel precursor loaded with metal salt, and combining a freeze-drying, rapid combustion and template etching process to cooperatively construct a "micropore-mesopore-macropore" multistage through-pore channel structure. The nanometer enzyme has rich pore channel structures and a great specific surface area, so that active sites are fully exposed, and the nanometer enzyme exhibits excellent POD catalytic activity, thereby exhibiting high sensitivity and stability in the application of glucose colorimetric sensing detection.

Description

Technical Field

[0001] This invention relates to the field of bionanomaterials technology, specifically to a NiCo2O4 nanozyme with multi-level through-pore characteristics, its preparation method, and its application in glucose detection. Background Technology

[0002] As one of the most important energy metabolism substrates in the human body, changes in glucose concentration are generally considered a crucial physiological indicator for assessing metabolic status and monitoring disease progression. Therefore, real-time and accurate monitoring of glucose concentrations in urine and blood is of great significance for disease diagnosis, disease assessment, and clinical intervention. Blood glucose monitoring is a core component of the daily management of diabetic patients. With the continuous rise in the prevalence of diabetes, it has become one of the leading chronic diseases causing death and disability worldwide. To achieve effective blood glucose control, reduce the risk of complications, and improve patients' quality of life, establishing accurate, rapid, convenient, and low-cost glucose detection methods is particularly critical. Therefore, the development of high-performance glucose sensors remains a research hotspot in the fields of biosensing and disease diagnosis.

[0003] Currently, the most commonly used clinical method for blood glucose detection is the enzymatic method based on glucose oxidase (GOx), which has high selectivity and sensitivity. However, natural enzymes are easily inactivated under conditions such as high temperature and low pH, and their preparation cost is relatively high. Furthermore, this method often relies on reagent kits or instruments, making the operation relatively cumbersome and unsuitable for immediate and portable detection. In recent years, nanozymes with enzyme-like catalytic functions have been considered a potential alternative, offering better stability, lower cost, and easier structural control. However, they still lag behind natural enzymes in terms of catalytic efficiency and selectivity.

[0004] Polymetallic oxides, especially bimetallic oxides, have proven to perform well in mimicking the activity of natural enzymes. Their advantage lies in their abundant redox active sites, which promote electron transfer and enhance catalytic reaction efficiency. Furthermore, in nanozyme design, abundant pore structures are key to improving catalytic activity and stability. They can significantly increase specific surface area and expose more active sites, promoting substrate adsorption and mass transfer, and accelerating reaction kinetics. However, constructing different pore sizes depends on different templates and formation mechanisms, which are prone to mutual interference, and the interoperability and structural stability of the pores are difficult to guarantee.

[0005] Against this backdrop, there is an urgent need to develop metal oxide peroxidases (PODs) with multi-level permeable channels that possess both high stability and high catalytic activity. These nanozymes hold promise for improving the sensitivity and accuracy of blood glucose detection and providing a new technological pathway for constructing low-cost, high-efficiency detection systems, thus possessing significant research value and application prospects. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned technical problems and provide a method for preparing NiCo2O4 nanozymes with multi-level through-pore characteristics and their applications. This method constructs a gelatin composite hydrogel precursor loaded with metal salts, and combines freeze-drying, rapid combustion, and template etching processes to synergistically construct a multi-level through-pore structure of "micropore-mesopore-macropore," fully exposing the active sites, significantly enhancing reaction kinetics, exhibiting excellent POD activity, and further achieving highly sensitive detection of glucose.

[0007] The technical solution adopted in this invention is as follows:

[0008] A method for preparing NiCo2O4 nanozymes with multi-level through-pore characteristics includes the following steps:

[0009] S1. Dissolve nickel nitrate hexahydrate, cobalt nitrate hexahydrate, and magnesium nitrate hexahydrate in deionized water by ultrasonication; then add tartaric acid and dissolve by ultrasonication to obtain mixture A;

[0010] S2. Disperse gelatin and ammonium bicarbonate in deionized water, stir until completely dissolved, and obtain mixed solution B and cool; then slowly add mixture A to solution B and stir a second time, then let stand at low temperature to form a hydrogel, rinse with deionized water and freeze dry to obtain the first solid.

[0011] S3. Preheat the muffle furnace to a high temperature, then quickly transfer the first solid into the muffle furnace for calcination to obtain the second solid. The second solid is acid-washed, centrifuged and washed with deionized water, and then vacuum-dried to obtain NiCo2O4 nanozyme with multi-level through-pore characteristics.

[0012] Preferably, in S1, the ratio of nickel nitrate hexahydrate, cobalt nitrate hexahydrate, magnesium nitrate hexahydrate, tartaric acid, and deionized water is: 2 mmol: 2-7 mmol: 5-10 mmol: 3-8 mmol: 15-30 mL.

[0013] Preferably, in S1, the ultrasound time is 10 minutes.

[0014] Preferably, in step S2, the ratio of gelatin, ammonium bicarbonate, and deionized water is 1-3g: 7-14mmol: 100mL.

[0015] Preferably, in step S2, the first stirring temperature is 50-70℃ and the first stirring time is 1-3h; the mixed solution B is cooled to 45℃; the second stirring temperature is room temperature and the second stirring time is 20min; the settling temperature is 4℃ and the settling time is 2h; the deionized water rinsing is performed twice; and the freeze-drying temperature is -80℃ and the time is 24h.

[0016] Preferably, in step S3, the ratio of the second solid to the HCl solution is 100 mg: 20-45 mL.

[0017] Preferably, in step S3, the preheating temperature of the muffle furnace is 500-600℃, and the holding time is 0.5-1.5h; the pickling conditions are: HCl concentration 1M, ultrasonic time 2min, and stirring at room temperature for 30min; the number of centrifugal washing cycles is 3; and the vacuum drying conditions are: temperature 60℃ and time 8h.

[0018] The present invention also provides NiCo2O4 nanozymes with multi-level through-pore characteristics obtained by the above preparation method.

[0019] The present invention also provides the application of the above-mentioned NiCo2O4 nanozyme with multi-level through-pore characteristics in glucose detection.

[0020] Preferably, the glucose detection is performed using an enzymatic colorimetric method, and the NiCo2O4 nanozyme with multi-level permeable channels is used as a catalyst.

[0021] In the structural design of bimetallic oxide peroxidases (PODs), this invention constructs a multi-level interconnected pore system of micropores, mesopores, and macropores. This significantly reduces mass transfer resistance and improves active site utilization throughout the entire process of substrate adsorption, diffusion to active sites, reaction, and product desorption, thereby enhancing overall catalytic performance. Specifically, micropores have a large specific surface area, which can accommodate and expose more catalytic sites. Their spatial confinement facilitates the enrichment of small molecules such as H2O2 on the pore walls, increasing the effective substrate concentration around the active sites and accelerating the reaction. Mesopores have lower diffusion resistance, allowing for more efficient transport of substrates to the internal micropore active sites, increasing the participation of inner-layer sites. Macropores provide low-resistance entry and exit channels, enhancing solution penetration and mass exchange, enabling substrates to quickly reach deep into the particle and facilitating timely product desorption, reducing the coverage and inhibition of active sites by product retention. Furthermore, when the multi-level pore system interconnects to form a pore network, substrates can rapidly enter the particle interior through macropores, then diffuse through mesopores and enter the micropores to react with the active sites. The generated products can also diffuse and escape rapidly along the original pathway. As a result, not only are the active sites more fully utilized, but intermediates / products are also less likely to accumulate and cover the active sites in the pores, making the reaction process more continuous and stable. This ultimately results in higher utilization of active sites and a faster reaction rate, thereby significantly improving the sensitivity and accuracy of glucose detection.

[0022] The beneficial effects of this invention are as follows:

[0023] (1) This invention successfully constructed a NiCo2O4 nanozyme with multi-level through-pore characteristics by introducing key components such as tartaric acid and a hydrogel framework. First, tartaric acid forms complexes with metal ions (Ni, Co, Mg), which are then uniformly distributed in the hydrogel network. During the preheating and calcination process in a muffle furnace, tartaric acid and gelatin burn rapidly, and the decomposition of ammonium bicarbonate generates a large amount of gas (such as CO2 and NH3). These gases escape rapidly from the material, causing local expansion and forming micropores and mesopores. In addition, through freeze-drying, the water in the material sublimates to form larger pore structures, which in turn provides framework support for the formation of macropores. The multi-level pores mentioned above are all constructed step by step based on the hydrogel framework. Finally, acid washing removes the MgO template, further enhancing the connectivity between pores, and ultimately constructing a multi-level pore network that penetrates micropores, mesopores, and macropores.

[0024] (2) The hierarchical through-pore structure constructed in this invention significantly enhances the catalytic activity of NiCo2O4 nanozymes. In the micropores, substrate molecules accumulate, increasing the effective concentration around the active sites and thus accelerating the reaction. Mesopores, by reducing diffusion resistance, efficiently transfer the substrate to the internal micropore active sites, improving the reaction efficiency of the inner sites. Macropores provide low-resistance channels, allowing the substrate to rapidly enter the material and quickly desorb the product, preventing product retention within the pores and inhibition of the active sites. The synergistic effect of the hierarchical channels ensures that the substrate can rapidly diffuse to the active sites, while the product can be removed in a timely manner, thereby improving the overall catalytic efficiency.

[0025] (3) The NiCo2O4 nanozyme prepared by this invention has a multi-level through-pore structure and a very large specific surface area (132.21 m²). 2 This nanozyme exhibits excellent POD catalytic activity. In the colorimetric detection of glucose, it achieves an extremely low limit of detection (LOD) of only 3.48 μM. These results demonstrate that it not only possesses excellent intrinsic POD enzyme activity but also exhibits extremely high sensitivity and promising market potential in the application of colorimetric glucose detection. Attached Figure Description

[0026] Figure 1 The image shows the X-ray powder diffraction (XRD) pattern of the sample.

[0027] Figure 2 This is a transmission electron microscope (TEM-EDS) mapping image of the sample;

[0028] Figure 3 The graph shows the sample's specific surface area and porosity as measured by a fully automated BET analyzer.

[0029] Figure 4The colorimetric test pattern of 3,3',5,5'-tetramethylbenzidine (TMB) in the sample is shown.

[0030] Figure 5 and Figure 6 This is a graph showing the relationship between the UV absorbance of the sample and the glucose concentration. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0032] Example 1: Preparation of NiCo2O4 nanozymes with multi-level through-pore characteristics

[0033] (1) 2 mmol Ni(NO3)2·6H2O (nickel nitrate hexahydrate), 4 mmol Co(NO3)2·6H2O (cobalt nitrate hexahydrate), and 8 mmol Mg(NO3)2·6H2O (magnesium nitrate hexahydrate) were ultrasonically dispersed in 20 mL of deionized water for 10 min. Then 6 mmol tartaric acid was added and the mixture was ultrasonically dissolved for 10 min to obtain mixture A.

[0034] (2) Add 2.0 g of gelatin and 10 mmol of NH4HCO3 (ammonium bicarbonate) to 100 mL of deionized water and stir rapidly at 60 °C for 2 h until completely dissolved to obtain solution B. After solution B cools to 45 °C, slowly add mixture A to solution B and stir continuously for 20 min. Then let it stand at 4 °C for 2 h to form a salt-loaded composite hydrogel. Take out the hydrogel and rinse it quickly twice with deionized water. Finally, freeze-dry the obtained hydrogel at -80 °C for 24 h to obtain the first solid.

[0035] (3) Preheat the muffle furnace to 550°C, then place the first solid in a quartz crucible and quickly transfer it into the muffle furnace. Keep it at this temperature for 1 hour to obtain the second solid. Add 100 mg of the second solid to 30 mL of 1 M HCl, sonicate for 2 minutes, and stir at room temperature for 30 minutes. Then centrifuge with deionized water, wash the precipitate three times until it is nearly neutral, and vacuum dry at 60°C for 8 hours to obtain NiCo2O4 nanozyme with multi-level through-pore characteristics, which is recorded as the sample of Example 1.

[0036] Example 2: Preparation of NiCo2O4 nanozymes with multi-level through-pore characteristics

[0037] (1) Disperse 2 mmol Ni(NO3)2·6H2O, 2 mmol Co(NO3)2·6H2O and 5 mmol Mg(NO3)2·6H2O in 15 mL of deionized water by ultrasonication for 10 min. Then add 3 mmol tartaric acid and dissolve by ultrasonication for 10 min to obtain mixture A.

[0038] (2) 1.0 g of gelatin and 7 mmol of NH4HCO3 were added to 100 mL of deionized water and stirred rapidly at 50 °C for 3 h until completely dissolved to obtain solution B. After solution B was cooled to 45 °C, mixture A was slowly added to solution B and stirred continuously for 20 min. Then, the mixture was allowed to stand at 4 °C for 2 h to form a salt-loaded composite hydrogel. The hydrogel was removed and rinsed twice with deionized water. Finally, the obtained hydrogel was freeze-dried at -80 °C for 24 h to obtain the first solid.

[0039] (3) Preheat the muffle furnace to 500°C, then place the first solid in a quartz crucible and quickly transfer it into the muffle furnace. Keep it at this temperature for 1.5 h to obtain the second solid. Add 100 mg of the second solid to 20 mL of 1 M HCl, sonicate for 2 min, and stir at room temperature for 30 min. Then centrifuge with deionized water, wash the precipitate three times until it is nearly neutral, and vacuum dry at 60°C for 8 h to obtain NiCo2O4 nanozyme with multi-level through-pore characteristics, which is recorded as the sample of Example 2.

[0040] Example 3: Preparation of NiCo2O4 nanozymes with multi-level through-pore characteristics

[0041] (1) Disperse 2 mmol Ni(NO3)2·6H2O, 7 mmol Co(NO3)2·6H2O and 10 mmol Mg(NO3)2·6H2O in 30 mL of deionized water by ultrasonication for 10 min. Then add 8 mmol tartaric acid and dissolve by ultrasonication for 10 min to obtain mixture A.

[0042] (2) 1.0 g of gelatin and 14 mmol of NH4HCO3 were added to 100 mL of deionized water and stirred rapidly at 70 °C for 1 h until completely dissolved to obtain solution B. After solution B was cooled to 45 °C, mixture A was slowly added to solution B and stirred continuously for 20 min. Then, the mixture was allowed to stand at 4 °C for 2 h to form a salt-loaded composite hydrogel. The hydrogel was removed and rinsed twice with deionized water. Finally, the obtained hydrogel was freeze-dried at -80 °C for 24 h to obtain the first solid.

[0043] (3) The muffle furnace was preheated to 600°C. The first solid was then placed in a quartz crucible and quickly transferred into the muffle furnace. The mixture was kept at this temperature for 0.5 h to obtain the second solid. 100 mg of the second solid was added to 45 mL of 1 M HCl and ultrasonically dispersed for 2 min. The mixture was then stirred at room temperature for 30 min. Subsequently, the precipitate was centrifuged with deionized water and washed three times until it was nearly neutral. The precipitate was then vacuum dried at 60°C for 8 h to obtain NiCo2O4 nanozyme with multi-level through-pore characteristics, which was recorded as the sample of Example 3.

[0044] Comparative Example 1: Preparation of NiCo Bimetallic Oxide Nanozymes

[0045] The preparation method is the same as in Example 1, except that tartaric acid is not added in Comparative Example 1.

[0046] Comparative Example 2: Preparation of NiCo Bimetallic Oxide Nanozymes

[0047] The preparation method is the same as in Example 1, except that ammonium bicarbonate is not added in Comparative Example 1.

[0048] Comparative Example 3: Preparation of NiCo Bimetallic Oxide Nanozymes

[0049] The preparation method is the same as in Example 1, except that the muffle furnace calcination method in Comparative Example 1 is changed to: first, the first solid is transferred to a quartz crucible and placed in a muffle furnace, then the heating rate is set to 5℃ / min, the calcination temperature is 550℃, and the holding time is 1h to obtain the second solid.

[0050] Characterization Analysis of Experimental Example 1

[0051] The samples prepared in Example 1 were analyzed by XRD (X-ray powder diffraction), TEM-EDS mapping (transmission electron microscopy and elemental distribution), and BET (fully automated specific surface area and porosity analysis). The analysis results are as follows: Figures 1-3 As shown in Table 2.

[0052] XRD and BET analyses were performed on the samples prepared in Examples 2-3 and Comparative Examples 1-3, respectively. The XRD and BET analysis results were expressed as phase composition and specific surface area (m²), respectively. 2 The formal characterization of pore volume (ml / g) and pore size (nm) is described in Tables 1-2.

[0053] The individual analysis results of Example 1 show that the XRD test results ( Figure 1 The results show that the characteristic peaks of the sample are highly matched with the standard card PDF#97-002-4211, confirming that the sample is composed of the NiCo2O4 phase.

[0054] Figure 2 The TEM-EDS mapping image of the sample from Example 1 is shown, in which Ni, Co and O elements are uniformly distributed, indicating that the NiCo bimetallic oxide was successfully synthesized. Figure 3 Table 2 shows the BET test results of the sample from Example 1, which demonstrates its extremely large specific surface area (132.21 m²). 2The NLDFT pore size distribution plots show that the differential pore volume curves exhibit clear distribution signals across a wide range of <2nm, 2~50nm, and >50nm, corresponding to the micropore, mesopore, and macropore structures of the material, respectively. The cumulative pore volume curves further confirm the pore volume contributions of micropores, mesopores, and macropores, clearly indicating that the synthesized material possesses a hierarchical pore system spanning micro-meso-macropores. Furthermore, the T-Plot micropore specific surface area reaches 61.95 m². 2 The / g indicates that approximately 47% of its total specific surface area comes from the contribution of micropores, demonstrating a highly developed microporous structure. The HK method-measured micropore volume of 0.06 ml / g further confirms the presence of micropores. The average pore diameter of 13.99 nm clearly indicates that the material is predominantly mesoporous. The presence of an H2-type hysteresis loop in the N2 adsorption-desorption curve is a hallmark of complex mesopores and micropores. Furthermore, the total pore volume is the cumulative volume of pores with diameters less than 195.6 nm, indicating the presence of macropores as well. In summary, the sample in Example 1 is composed of a NiCo2O4 phase, exhibiting a rich pore structure and a very large specific surface area. This is because tartaric acid can first complex with metal ions in the solution stage, resulting in a more uniform distribution, and then be uniformly immobilized in the network voids by gelatin hydrogel, forming a primary template. When the composite hydrogel is freeze-dried, the water in the system first crystallizes into ice crystals, which then sublimate, retaining the spaces they originally occupied, forming a larger pore structure. This ultimately transforms into a macroporous structure, constructing the macroscopic pore framework of the material and providing a foundation for the further formation of subsequent pore structures. Subsequently, the system undergoes vigorous combustion in a preheated muffle furnace. Tartaric acid, some gelatin, and ammonium bicarbonate rapidly decompose, producing large amounts of gases (CO2, N2, and NH3, etc.). These gases are rapidly released within the framework, causing localized expansion, typically at a scale of several to tens of nanometers, which is conducive to the formation of mesoporous structures. Gelatin and tartaric acid, after rapid combustion, locally generate nanoscale pores. Simultaneously, the NiCo2O4 grains formed during calcination are typically in the nanoscale range. These nanoparticles inevitably create interparticle gaps during accumulation, resulting in even smaller microporous structures. Finally, MgO is removed as a sacrificial template during acid washing, its original positions transformed into pores, and enhancing the direct connectivity of the pore structure. Because macropores, mesopores, and micropores are all formed stepwise within the hydrogel framework and exhibit a hierarchical spatial connection, they ultimately work together to form nanoparticles with multi-level through-pore characteristics.

[0055] In the comparative analysis results of Examples 2-3 and Comparative Examples 1-3, as shown in Table 1, the XRD test results of the samples obtained in Examples 2-3 and Comparative Examples 1-3 indicate that the samples are composed of the NiCo2O4 phase, which is consistent with the phase composition of the sample in Example 1. The BET test results show that the specific surface area of ​​Comparative Example 1 is 53.77 m².2 The tartaric acid concentration ( / g) decreased significantly compared to the sample in Example 1, with a marked reduction in both total pore volume and the proportion of micropores. This is because tartaric acid acts as both a complexing dispersant and a combustion pore-forming agent. The carboxyl / hydroxyl groups in its molecule coordinate with metal ions, resulting in more uniform nucleation. After high-temperature thermal shock, it burns rapidly, instantly generating a large amount of gas and producing a localized expansion effect, which helps form a rich mesoporous structure. Simultaneously, its rapid decomposition leaves behind tiny pores, further contributing to the formation of micropores.

[0056] The specific surface area of ​​Comparative Example 2 is 96.32 m². 2 The specific surface area was also reduced compared to the sample in Example 1, with both the total pore volume and the proportion of micropores decreasing. This is because NH4HCO3 readily decomposes thermally, releasing gases such as NH3 and CO2. These gases are rapidly generated within the material framework and escape outwards, which is conducive to the formation of mesopores. Furthermore, during the escape process, they can form through-channels in the pore walls, ultimately facilitating the construction of hierarchical porous nanoparticles with through-channels. The pores left by the fuel also contribute to the formation of micropores, ultimately increasing the specific surface area.

[0057] The specific surface area of ​​Comparative Example 3 is 84.16 m². 2 The specific surface area was reduced by g compared to the sample in Example 1, with a significant decrease in total pore volume and micropore ratio. This is because the rapid combustion reaction induced by preheating in a muffle furnace allows for the rapid combustion of tartaric acid and gelatin, accompanied by rapid gas release and a local expansion effect, which is conducive to mesopore formation. Similarly, the pores left by the fuel also contribute to the formation of micropores, ultimately increasing the specific surface area.

[0058] Table 1. XRD and specific surface area test results of Examples 1-3 and Comparative Examples 1-3

[0059]

[0060] Table 2. Test results of the pore structure in Example 1 and Comparative Examples 1-3

[0061]

[0062] Experimental Example 2: POD (Peroxidase) Activity Test

[0063] The POD activity of the samples obtained in Examples 1-3 and Comparative Examples 1-3 was tested. The test method was as follows: suspensions (1 mg / mL) of NiCo2O4 nanozymes, TMB (3,3',5,5'-tetramethylbenzidine) (1 mM), and H2O2 (60 mM) solutions were prepared using pure water, respectively. Subsequently, 30 µL of the suspensions from Examples 1-3 and Comparative Examples 1-3, 120 µL of H2O2, and 150 µL of TMB were mixed in 2700 µL of sodium acetate-acetic acid (NaAc-HAc, pH=4, 0.2 M) buffer solution. After incubation at room temperature for 30 min, the absorbance of the reaction solution was measured at 652 nm using a UV-Vis spectrophotometer to evaluate the POD activity of the NiCo2O4 nanozymes. The test results are as follows. Figure 4 As shown.

[0064] Figure 5 The POD enzyme activities of the samples from Examples 1-3 and Comparative Examples 1-3 were compared. The relative intensity of the absorption peak at 652 nm in the figure represents the order of POD enzyme activity, with the results showing that Example 1 > Example 2 > Example 3 > Comparative Example 2 > Comparative Example 3 > Comparative Example 1. This indicates that the NiCo2O4 nanoparticles prepared under the synthesis conditions of Example 1 (Sample 1) exhibit the most superior POD enzyme activity. This is because the abundant pore structure and large specific surface area facilitate repeated exposure of active sites, thereby enhancing POD enzyme activity.

[0065] Experimental Example 3: Detection of Glucose by Colorimetric Method

[0066] The glucose colorimetric method was used to detect the glucose content of the sample described in Example 1, and the application effect of the sample in detecting glucose was obtained. The test method is as follows, and the test results are shown in [the table below]. Figure 5 and Figure 6 .

[0067] Test method: Suspensions of NiCo2O4 nanozymes (1 mg / mL), GOD (glucose oxidase) suspension (1 mg / mL), TMB (0.8 mM), and glucose solutions (1 / 2 / 5 / 10 / 20 / 50 / 100 / 200 / 500 μM) obtained in Example 1 were prepared using pure water. Subsequently, 50 μL of GOD suspension and 150 μL of glucose solution were mixed in 2620 μL of NaAc-HAc (pH=4, 0.2 M) buffer solution and incubated at 37 °C for 20 min. Then, 30 μL of the suspension from Example 1 and 50 μL of TMB solution were added, and the mixture was incubated at room temperature for 30 min. The absorbance of the reaction solution was measured at 652 nm using a UV-Vis spectrophotometer to obtain absorbance curves corresponding to different glucose concentrations. Furthermore, a linear standard curve for colorimetric detection of glucose concentration was obtained.

[0068] Depend on Figure 5 As shown, the absorbance value at 652 nm gradually increases with the increase of glucose concentration. Figure 6 It can be seen that the detection of glucose concentrations exhibits a good linear relationship in the range of 1-500 μM, with the linear relationship being: y = 0.852x + 0.137. The detection limit is 3.48 μM. This indicates that the sample in Example 1 exhibits extremely high sensitivity in the colorimetric detection of glucose.

[0069] As can be seen from Comparative Example 1 and Examples 1-3, tartaric acid, as a complexing agent and fuel, can form a uniformly dispersed complex system with metal ions, inhibiting local enrichment of metal salts. Furthermore, it undergoes vigorous combustion when rapidly introduced into a muffle furnace at high temperatures, simultaneously providing instantaneous gaseous products and local expansion, inducing the formation of micropores and mesopores. Comparative Example 2 and Examples 1-3 show that NH4HCO3 easily decomposes during heating, releasing gases such as NH3, which can rapidly generate a large number of microbubbles and pore channels within the gel framework and metal salt distribution network, laying the foundation for the subsequent formation of interconnected hierarchical pores. Comparative Example 3 and Examples 1-3 demonstrate that the flame thermal shock strategy, employing preheating to 550°C in a muffle furnace followed by rapid sample delivery, can achieve rapid fuel combustion and gas release in a short time. In addition, freeze-drying facilitates the formation of macropores, while the hierarchical pore-forming based on the gel framework and the acid leaching to remove MgO to enhance pore connectivity ultimately result in NiCo2O4 nanoparticles with multi-level interconnected pore characteristics and sufficient exposure of active sites, providing a high specific surface area for its use as a POD nanozyme.

[0070] The specification and drawings of this invention are intended to be illustrative rather than restrictive. Based on this invention, those skilled in the art can make substitutions and modifications to some of the technical features without creative effort, and all such modifications are within the scope of protection of this invention.

Claims

1. A method for preparing NiCo2O4 nanozymes with multi-level through-pore characteristics, comprising the following steps: S1. Dissolve nickel nitrate hexahydrate, cobalt nitrate hexahydrate, and magnesium nitrate hexahydrate in deionized water by ultrasonication; then add tartaric acid and dissolve by ultrasonication to obtain mixture A; The ratio of nickel nitrate hexahydrate, cobalt nitrate hexahydrate, magnesium nitrate hexahydrate, tartaric acid, and deionized water is: 2 mmol: 2-7 mmol: 5-10 mmol: 3-8 mmol: 15-30 mL; S2. Disperse gelatin and ammonium bicarbonate in deionized water, stir until completely dissolved, and then cool the mixture to obtain mixed solution B. Then, mixture A was slowly added to solution B and stirred a second time. The mixture was then allowed to stand at low temperature to form a hydrogel. After rinsing with deionized water, the mixture was freeze-dried to obtain the first solid. The ratio of gelatin, ammonium bicarbonate and deionized water is 1-3g: 7-14mmol: 100mL; S3. Preheat the muffle furnace to 500-600℃, then quickly transfer the first solid into the muffle furnace for calcination, and hold for 0.5-1.5h to obtain the second solid. The second solid is acid-washed, centrifuged and washed with deionized water, and then vacuum-dried to obtain NiCo2O4 nanozyme with multi-level through-pore characteristics.

2. The preparation method according to claim 1, characterized in that, In S1, the ultrasound time is 10 minutes.

3. The preparation method according to claim 1, characterized in that, In step S2, the first stirring temperature is 50-70℃ and the first stirring time is 1-3h; the mixed solution B is cooled to 45℃; the second stirring temperature is room temperature and the second stirring time is 20min; the settling temperature is 4℃ and the settling time is 2h; the deionized water rinsing is performed twice; and the freeze-drying temperature is -80℃ and the time is 24h.

4. The preparation method according to claim 1, characterized in that, In S3, the ratio of the second solid to the HCl solution is 100mg:20-45mL.

5. The preparation method according to claim 1, characterized in that, In S3, the acid washing conditions are: HCl concentration 1M, ultrasonic time 2min, stirring at room temperature for 30min; centrifugal washing is performed 3 times; vacuum drying conditions are: temperature 60℃, time 8h.

6. The NiCo2O4 nanozyme with multi-level through-pore characteristics obtained by the preparation method according to any one of claims 1-5.

7. The application of the NiCo2O4 nanozyme with multi-level through-pore characteristics as described in claim 6 in glucose detection.

8. The application according to claim 7, characterized in that, The glucose detection method employs an enzymatic colorimetric method, with the NiCo2O4 nanozyme, which has multi-level permeable channels, used as a catalyst.

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