Supported double-nano enzyme based on nanoflowers, preparation of supported double-nano enzyme and application of supported double-nano enzyme in colorimetric detection of uric acid

Abstract

The invention discloses a supported double nano-enzyme based on nanoflowers, preparation of the supported double nano-enzyme and application of the supported double nano-enzyme in uric acid colorimetric detection. The supported double nano-enzyme is prepared by immobilizing peroxidase-like nano-enzyme in a uricase nanoflower structure. According to the invention, the peroxidase-like nano-enzyme is immobilized in a natural uricase nano-flower structure to construct the supported double nano-enzyme, so that the stability and the loading capacity of the peroxidase-like nano-enzyme are remarkably improved, and the transmission distance between a substrate and an intermediate product H2O2 in a cascade reaction is greatly shortened, thereby greatly improving the catalytic efficiency and the reaction rate. The method effectively overcomes the core problems of high dependence on natural enzyme, high cost, poor stability and low cascade reaction efficiency in the prior art. When being used for colorimetric detection of uric acid, the kit shows high sensitivity, good selectivity and anti-interference capability, and lays a solid foundation for developing portable uric acid detection equipment which is low in cost, high in performance and suitable for primary medical treatment and family use.

Description

(I) Technical Field

[0001] This invention relates to a nanoflower-based supported dual nanozyme, its preparation, and its application in the colorimetric detection of uric acid. (II) Background Technology

[0002] Uric acid (UA) is the final product of purine nucleotide metabolism in the human body. At normal physiological concentrations, uric acid acts as an endogenous antioxidant, playing a positive role in maintaining vascular endothelial function and scavenging free radicals. However, when serum uric acid levels are abnormally elevated due to metabolic disorders (hyperuricemia), urate microcrystals precipitate and deposit in joints, soft tissues, kidneys, and other sites, leading to a series of serious diseases such as gouty arthritis, uric acid kidney stones, and chronic kidney disease. Furthermore, numerous epidemiological studies have confirmed that hyperuricemia is an independent risk factor for hypertension, metabolic syndrome, type II diabetes, and cardiovascular and cerebrovascular diseases, and is significantly associated with all-cause mortality. Therefore, achieving rapid and accurate detection of uric acid concentration in body fluids is of crucial medical value for early screening, clinical diagnosis, treatment efficacy monitoring, and epidemiological research of hyperuricemia and gout. Among numerous biological samples, urine is a highly attractive screening and monitoring method due to its non-invasive, convenient, and readily available collection, as well as the correlation between uric acid levels and serum uric acid concentrations.

[0003] Currently, the commonly used methods for uric acid detection in clinical practice mainly include the following three: First, the uricase-peroxidase method, which is the most mainstream method in current clinical biochemical analysis. Its principle is that uric acid, under the catalysis of uricase, generates allantoin, CO2, and H2O2. Subsequently, H2O2 reacts with the chromogenic substrate under the catalysis of peroxidase (such as horseradish peroxidase) to generate quinone imines, a colored substance. The uric acid concentration is quantified by measuring the change in absorbance. Although this method has high sensitivity and specificity, it heavily relies on expensive, difficult-to-extract, and unstable natural enzymes (sensitive to temperature and pH), resulting in high reagent costs and inconvenient storage and transportation, making it difficult to popularize in primary healthcare or home settings. Second, the electrochemical method, which is based on the direct electro-oxidation of uric acid on the electrode surface or the indirect catalysis of the enzyme-modified electrode. It has the advantage of high sensitivity, but the electrode is easily interfered with by electroactive substances such as ascorbic acid, dopamine, and urea in urine, leading to decreased selectivity. Furthermore, the electrode modification process is complex, and reproducibility is difficult to guarantee. Third, chromatographic methods, represented by high performance liquid chromatography, are recognized as the "gold standard" for uric acid detection, with extremely high accuracy and specificity. However, this method relies on large and expensive instruments, requires professional operators and complex sample pretreatment procedures, and takes a long time to analyze, which cannot meet the needs of rapid, point-of-care testing (POCT).

[0004] Therefore, developing a novel, low-cost, easy-to-operate, rapid, sensitive, and selective technology suitable for uric acid detection has become a pressing practical problem in the field of in vitro diagnostics. Colorimetric methods are favored due to their simple equipment (requiring only visual inspection or a portable spectrometer), intuitive readings, and ease of implementation in point-of-care testing (POCT). However, the core challenge lies in how to reduce dependence on natural enzymes and construct an efficient, stable, and specific catalytic system.

[0005] In recent years, the discovery of nanozymes has provided a revolutionary new approach to solving the aforementioned problems. As a class of functional nanomaterials with enzyme-like catalytic activity, nanozymes can mimic the catalytic functions of natural enzymes such as oxidases and catalases, exhibiting significant advantages such as high stability, low cost, large-scale preparation, and ease of modification. In uric acid detection applications, researchers have attempted to use various nanozymes, including those made of noble metals, metal oxides, and carbon-based nanozymes, to mimic peroxidase (POD) activity for catalyzing the colorimetric reaction between H2O2 and the chromogenic substrate. However, constructing a complete nanozyme detection system still faces severe challenges. Most studies merely replace POD with nanozymes, still relying on natural uricase to oxidize uric acid to generate H2O2, failing to fundamentally solve the bottlenecks of high cost and poor stability of natural enzymes. Simply mixing two nanozymes with uricase-like and POD-like activities respectively results in spatial separation, leading to low transport efficiency of substrate and intermediate product H2O2, causing slow cascade reaction kinetics and insufficient overall catalytic efficiency.

[0006] Inspired by natural multi-enzyme complexes, precisely co-locating or immobilizing multiple enzymes within a finite nanospace can significantly shorten substrate transport distances and dramatically improve the efficiency and rate of cascade reactions. In recent years, organic-inorganic hybrid nanoflowers have emerged as a novel biomimetic immobilization technology. Their typical preparation process involves the coordination and self-assembly of metal ions with functional groups in protein (enzyme) molecules, followed by co-crystallization growth with phosphate ions to form micro / nanomaterials with a hierarchical petal-like structure. This structure endows nanoflowers with three significant advantages: First, their ultra-high specific surface area greatly increases enzyme loading; second, the confinement and synergistic effects tightly immobilize different enzymes within the petal space, mimicking intracellular multi-enzyme complexes and significantly promoting the channelization and transport of intermediate products, resulting in an order-of-magnitude increase in cascade reaction rates; third, the inorganic crystals form a robust "nanocage" that encapsulates enzyme molecules, enabling them to withstand harsh environments such as high temperatures, extreme pH levels, and organic solvents, exhibiting storage and operational stability far exceeding that of free enzymes and traditionally immobilized enzymes. Based on these advantages, assembling two nanozymes with different enzyme activities (or nanozymes and natural enzymes) together in a single nanoflower to construct an efficient and stable cascade catalytic platform has become a cutting-edge research direction. (III) Summary of the Invention

[0007] The purpose of this invention is to provide a supported dual nanozyme based on nanoflowers, its preparation, and its application in uric acid colorimetric detection. This invention utilizes organic-inorganic hybrid self-assembly technology to immobilize a peroxidase-like nanozyme within a natural uricase nanoflower structure, constructing an integrated supported dual nanozyme catalytic platform. This structure leverages the high specific surface area and confinement effect of nanoflowers to significantly improve the stability and loading capacity of the peroxidase-like nanozyme, and greatly shortens the transport distance between the substrate and the intermediate product H₂O₂ in the cascade reaction, thereby significantly improving catalytic efficiency and reaction rate. This method effectively overcomes the core challenges of existing technologies, such as high dependence on natural enzymes, high cost, poor stability, and low cascade reaction efficiency. When applied to uric acid colorimetric detection, it exhibits high sensitivity, good selectivity, and anti-interference ability, making it suitable for complex urine environments. This lays a solid foundation for developing low-cost, high-performance portable uric acid detection devices suitable for primary healthcare and home use.

[0008] The technical solution adopted in this invention is:

[0009] This invention provides a supported dual nanozyme based on nanoflowers, wherein the supported dual nanozyme is prepared by immobilizing a peroxidase-like nanozyme in a uricase nanoflower structure; the peroxidase-like nanozyme is prepared by Ce(NO3)3·6H2O, citric acid monohydrate and ferrous sulfate.

[0010] Further, the supported dual nanozymes are prepared according to the following steps: uricase nanoflowers and peroxidase-like nanozymes are added to a buffer solution of pH 4-9 (preferably pH 4.0, 0.1M acetate-sodium acetate buffer) to form a reaction system. After thorough mixing and separation under an external magnetic field, the mixture is repeatedly washed with a buffer solution of pH 4-9 (preferably pH 4.0, 0.1M acetate-sodium acetate buffer), pre-frozen in an ultra-low temperature freezer at -80℃ for 8 hours, and then freeze-dried in a freeze dryer at -70℃ for 12 hours to obtain the supported dual nanozymes, denoted as LTDNs. In the reaction system, the mass ratio of uricase nanoflowers to peroxidase-like nanozymes is 1:1-4 (preferably 1:1), and preferably, the concentrations of both uricase nanoflowers and peroxidase-like nanozymes are 0.02 mg / mL; the mixing time is 3-30 min (preferably 5 min).

[0011] Furthermore, the preparation method of the uricase nanoflower is as follows: uricase is added to PBS buffer (preferably 3mM, pH 7.4), and then an aqueous solution of metal salt is added. After incubation at 20-30℃ for 30-40h (preferably 25℃, 36h), the suspension is centrifuged (preferably at 5000rpm for 5min). The resulting precipitate is washed with ultrapure water (preferably 3 times). The washed precipitate is pre-frozen in an ultra-low temperature freezer at -80℃ for 8h and then transferred to a freeze dryer at -70℃ for 12h to obtain uricase nanoflower. The metal salt is one or more of CaCl2, MnCl2, ZnCl2, FeCl2 or FeCl3, preferably MnCl2.

[0012] Furthermore, the concentration of the metal salt aqueous solution is 0.1-1 mol / L (preferably 0.12 mol / L), and the volume used is 0.1-1 mL / g (preferably 0.6 mL / g) based on the mass of uricase; the volume used of the PBS buffer is 1-10 mL / mg (preferably 5 mL / mg) based on the mass of uricase.

[0013] Further, the preparation method of the peroxidase-like nanozyme is as follows: (1) Dissolve Ce(NO3)3·6H2O and citric acid monohydrate in ultrapure water and adjust the pH to 9-10; transfer the above solution to a high-pressure hydrothermal reactor lined with polytetrafluoroethylene, and maintain the temperature at 150-200℃ for 4-8h (preferably 180℃, 6h); after the reaction, take out the product and add an appropriate amount of ethanol to induce precipitation, collect the precipitate by centrifugation, and then sequentially rinse with ultrapure water and... Ethanol was used to wash away impurities; the washed precipitate was pre-frozen in an ultra-low temperature freezer at -80℃ for 8 hours and then transferred to a freeze dryer at -70℃ for 12 hours; after complete drying, the product was ground into a fine powder, namely CeO2QDs nanozyme; the mass ratio of citric acid monohydrate to Ce(NO3)3·6H2O was 1:1-3 (preferably 1:2); the volume of ultrapure water used for dissolution was 30-50 mL / g (preferably 35 mL / g) based on the mass of citric acid monohydrate.

[0014] (2) The CeO2 QDs nanozyme was dispersed in a 0.1-0.5 mol / L (preferably 0.25 mol / L) NaOH aqueous solution and stirred vigorously at 60-100℃ for 20-50 min (preferably 80℃ for 30 min). A solution of ferrous sulfate and ultrapure water was slowly added and stirred vigorously in air for 20-50 min (preferably 30 min) to generate a black precipitate. The precipitate was collected by separating it with a magnet and washed with ultrapure water and ethanol in sequence until the precipitate was neutral after washing. The obtained precipitate was pre-frozen in an ultra-low temperature freezer at -80℃ for 8 h and then transferred to a freeze dryer at -70℃ for 12 h to obtain the Fe3O4-doped CeO2 QDs nanozyme, i.e., peroxidase-like nanozyme, denoted as Fe3O4@CeO2 QDs nanozyme. The volumetric volume of the NaOH aqueous solution, calculated based on the mass of the CeO2 QDs nanozyme, is 100-300 mL / g (preferably 200 mL / g); the volumetric volume of the ferrous sulfate, calculated based on the mass of the CeO2 QDs nanozyme, is 0.3-1 g / g (preferably 0.6 g / g); and the volumetric volume of the ultrapure water, calculated based on the mass of the CeO2 QDs nanozyme, is 50-150 mL / g (preferably 100 mL / g). The solution is added slowly at a rate of 1 drop / second.

[0015] The present invention also provides an application of the aforementioned supported dual nanozyme in the detection of uric acid.

[0016] Furthermore, the application method is as follows: a uric acid colorimetric detection system is constructed using the supported dual nanozyme, with the final concentration composition being: 5-50 μg / mL LTDNs, 5-200 μM uric acid (UA), 0.5-5 mM TMB (3,3',5,5'-tetramethylbenzidine), and acetate-sodium acetate buffer (0.1 M, pH 4.0-9.0); the reaction is carried out in a water bath at 20-45℃ for 5-30 min (preferably 37℃ for 10 min), and the absorbance value at 652 nm is detected. The uric acid concentration is calculated based on the standard curve of uric acid standard concentration versus absorbance value.

[0017] Furthermore, the final concentration composition of the uric acid colorimetric detection system is optimized as follows: 20 μg / mL LTDNs, 50 μM uric acid, 1 mM TMB, and acetate-sodium acetate buffer (0.1 M, pH 4.0).

[0018] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0019] To address the core pain points of existing uric acid detection technologies, such as strong dependence on natural enzymes, high cost, poor stability, and low efficiency of cascade reactions, this invention creatively proposes a method for preparing uric acid nanozymes by immobilizing natural uricase on nanoflower structures, and further assembling peroxidase-like nanozymes within a single nanoflower to form LTDNs, and applying LTDNs to the colorimetric detection of uric acid.

[0020] This invention significantly enhances the resistance to environmental stresses such as heat and pH by immobilizing natural uricase and peroxidase-like nanozymes within a nanoflower structure, thereby improving temperature and storage stability and effectively extending the lifespan of the detection reagent. Secondly, the ultra-high specific surface area and confined microenvironment provided by the three-dimensional nanoflower structure greatly promote substrate transport and the efficient utilization of the intermediate product H2O2, significantly increasing the cascade reaction rate and thus improving detection sensitivity. Thirdly, this invention partially uses peroxidase-like nanozymes to replace expensive natural HRP, effectively reducing costs while maintaining high catalytic activity. Finally, this integrated catalytic platform constructed using a "one-pot" method avoids the problems of poor compatibility and long reaction diffusion paths inherent in traditional physically mixed enzymes, exhibiting good anti-interference ability and repeatability in urine, laying a solid foundation for developing low-cost, high-performance uric acid POCT devices suitable for primary healthcare and home use. (iv) Description of the attached drawings

[0021] Figure 1 This is a schematic diagram of the preparation of LTDNs and the detection of uric acid in urine according to the present invention; A: the growth process of nanoflowers; B: the formation process of LTDNs; C: the catalytic principle diagram of LTDNs.

[0022] Figure 2 For Ca 2+ -UOx-NFs、Mn 2+ -UOx-NFs、Zn 2+ -UOx-NFs, Fe 2+ -UOx-NFs, Fe 3+ Scanning electron microscope (SEM) images of -UOx-NFs, CeO2 QDs, Fe3O4@CeO2 QDs, and LTDNs.

[0023] Figure 3 Fourier transform infrared spectroscopy (FT-IR) images of CeO2 QDs, Fe3O4@CeO2 QDs, and LTDNs.

[0024] Figure 4 A: Comparison of particle size and potential; A: Uricase nanoflowers induced by different metal ions (Ca 2+ -UOx-NFs、Mn 2+ -UOx-NFs、Zn 2+ -UOx-NFs, Fe 2+ -UOx-NFs, Fe 3+ Comparison of particle size of -UOx-NFs; B: LTDNs and free nanozymes (CeO2 QDs, Fe3O4@CeO2 QDs, Mn 2+Comparison of particle size of -UOx-NFs; C: Uricase nanoflowers induced by different metal ions (Ca 2+ -UOx-NFs、Mn 2+ -UOx-NFs、Zn 2+ -UOx-NFs, Fe 2+ -UOx-NFs, Fe 3+ Potential comparison of -UOx-NFs; D: LTDNs and free nanozymes (CeO2 QDs, Fe3O4@CeO2 QDs, Mn 2+ Potential comparison of -UOx-NFs).

[0025] Figure 5 The linearity (A) and sensitivity (B) of the uric acid colorimetric detection system and the detection system image (C, from left to right, uric acid concentrations are 1, 10, 40, 45, 50, 55 μM).

[0026] Figure 6 This study aimed to analyze the specificity of the uric acid colorimetric detection system. (V) Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0028] Example 1: Preparation of uricase nanoflowers

[0029] Take 1 mL of PBS (3 mM, pH 7.4) containing 0.2 mg of natural uricase (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., catalog number U837834), and add 20 μL of 0.12 mol / L aqueous solutions of CaCl2, MnCl2, ZnCl2, FeCl2, and FeCl3, respectively. Incubate at 25 °C for 36 h. After incubation, centrifuge the suspension at 5000 rpm for 5 min, and wash the resulting precipitate three times with ultrapure water. Pre-freeze the washed precipitate in an ultra-low temperature freezer at -80 °C for 8 h, then transfer it to a freeze dryer at -70 °C for 12 h to obtain 0.3 mg of uricase nanoflowers, denoted as Ca... 2+ -UOx-NFs、Mn 2+ -UOx-NFs、Zn 2+ -UOx-NFs, Fe 2+ -UOx-NFs, Fe 3+ -UOx-NFs, store at 4℃ for later use. Scanning electron microscope (SEM) images are shown below. Figure 2 As shown.

[0030] Example 2: Comparison of the activity of different types of uricase nanoflower enzymes

[0031] 0.25 mg of different types of UOx-NFs prepared in Example 1 were added to 150 μL of uric acid aqueous solution (50 μM) and 3 mL of sodium borate buffer (10 mM, pH 8) to form a 3.2 mL reaction system. The reaction system was incubated at 37°C for 20 minutes, and UOx-NFs were removed by centrifugation. The absorbance at 293 nm (A293) of the supernatant was measured. A reaction system without UOx-NFs served as a control group. Each sample was measured in triplicate. Enzyme activity was compared by the absorbance at 293 nm; higher absorbance indicated lower enzyme activity. Table 1 shows that Fe... 2+ -UOx-NFs exhibit the highest absorbance but the lowest relative enzyme activity. Mn 2+ -UOx-NFs and Ca 2 + The absorbance of -UOx-NFs was similar, and their enzyme activities were not significantly different. Based on the surface morphology results observed by SEM for both, Mn was selected. 2+ -UOx-NFs were selected as the optimal UOx-NFs. They were further combined with Fe3O4@CeO2 QDs nanozyme quantum dots to form a supported dual nanozyme.

[0032] Table 1 Comparison of different types of uricase nanoflower enzyme activity (A293)

[0033]

[0034] Example 3: Preparation of Fe3O4@CeO2 QDs nanozymes

[0035] Accurately weigh 2.0 g of Ce(NO3)3·6H2O and 1.0 g of citric acid monohydrate and dissolve them in 35 mL of ultrapure water. Then, adjust the pH of the solution to 9–10 using 4.3 mL of NaOH aqueous solution (5 mol / L). Transfer the solution to a high-pressure hydrothermal reactor lined with polytetrafluoroethylene and maintain the temperature at 180 °C for 6 h. After the reaction, remove the product and add an appropriate amount of ethanol to induce precipitation. Collect the precipitate by centrifugation and wash it sequentially with ultrapure water and ethanol to remove impurities. Pre-freeze the washed precipitate in an ultra-low temperature freezer at -80 °C for 8 h, then transfer it to a freeze dryer at -70 °C for 12 h. After complete drying, 0.871 g of product is obtained. Grind the product into a fine powder, which is CeO2QDs nanozyme. Scanning electron microscope (SEM) image is shown below. Figure 2 As shown.

[0036] 0.5 g of CeO2 QDs nanozyme was dispersed in 100 mL of 0.25 mol / L NaOH aqueous solution and stirred vigorously at 80 °C for 30 min to obtain a CeO2 nanozyme suspension. 0.3023 g of ferrous sulfate heptahydrate was dissolved in 50 mL of ultrapure water to prepare a solution, which was then slowly added dropwise at a rate of 1 drop / 1 second to the CeO2 nanozyme suspension. The solution was stirred vigorously in air for 30 min, resulting in a black precipitate. The precipitate was collected using a magnet and washed three times sequentially with ultrapure water and ethanol until the precipitate was neutral. The obtained precipitate was pre-frozen in an ultra-low temperature freezer at -80 °C for 8 h and then freeze-dried in a freeze dryer at -70 °C for 12 h to obtain 0.2811 g of Fe3O4-doped CeO2 QDs nanozyme, designated Fe3O4@CeO2 QDs nanozyme, i.e., peroxidase-like nanozyme, for later use. Scanning electron microscope (SEM) images are shown below. Figure 2 As shown.

[0037] Example 4: Preparation of supported dual nanozymes

[0038] The Mn prepared in Example 1 with a final concentration of 0.02 mg / mL was vortexed. 2+ -UOx-NFs were thoroughly mixed with 0.02 mg / mL Fe3O4@CeO2 QDs nanozymes and pH 4.0 acetate-sodium acetate buffer (0.1 M) in a 2 mL system for 5 min. After separation under an external magnetic field, the mixture was washed three times with pH 4.0 acetate-sodium acetate buffer (0.1 M), and the product was collected. It was then pre-frozen at -80°C for 8 h and then freeze-dried at -70°C for 12 h to obtain 0.07 mg of the supported dual nanozyme complex LTDNs. Scanning electron microscopy (SEM) images are shown below. Figure 2 As shown.

[0039] Example 5: Characterization of supported dual nanozymes

[0040] 1. Load capacity detection

[0041] SEM was used to observe CeO2 QDs nanozymes, Fe3O4@CeO2 QDs nanozymes, and Ca... 2+ -UOx-NFs、Mn 2+ -UOx-NFs、Zn 2+ -UOx-NFs, Fe 2+ -UOx-NFs, Fe 3+ The shape and surface morphology of UOx-NFs and LTDNs are used to determine whether a large specific surface area is formed, which indicates enhanced load-bearing capacity.

[0042] Using a toothpick, a suitable amount of powder sample was applied to the conductive adhesive, and any samples that did not adhere firmly were removed. To reduce the charging effect of the sample, gold nanoparticles were sprayed onto the sample before SEM analysis. SEM images are shown below. Figure 2 .Depend on Figure 2 As shown in Figure A, CeO2 QDs exhibit partially aggregated spherical nanocrystals. The main reason for this phenomenon is that QDs with higher surface energy tend to aggregate to reduce their surface energy. More independent QDs, such as... Figure 2 As shown in circle A, the size is approximately 5nm. From Figure 2 As can be seen in B, the Fe3O4@CeO2 QDs nanocomposite material formed by CeO2 QDs and Fe3O4 nanoparticles consists of spherical particles with a size of approximately 20 nm. Due to the combined effects of van der Waals forces, electrostatic forces, and magnetic attraction, the Fe3O4@CeO2 QDs nanocomposite material tends to form clustered aggregates. From... Figure 2 It is evident from C, D, and G that Ca is involved. 2+ Mn 2+ and Fe 3+ The induced synthesized UOx-NFs exhibit a distinct flower-like spike structure, differing only in their specific morphology, among which Mn 2+ -UOx-NFs are the most regular. Figure 2 The E and F results indicate that Zn 2+ -UOx-NFs, Fe 2+ -UOx-NFs have similar structures, both being dumbbell-shaped with nearly spherical ends and a rod-like middle section. Figure 2 As can be seen from H, LTDNs exhibit an irregular shape and a rough surface. In addition, it is clear that round particles are loaded on larger sheet-like structures to form aggregate units, and multiple small units aggregate to form an aggregate structure with a large specific surface area.

[0043] 2. Identification of the structure of dual-loaded nanozymes

[0044] The structures of CeO2 QDs nanozymes, Fe3O4@CeO2 QDs nanozymes, and LTDNs were analyzed using FT-IR to determine whether a supported dual nanozyme was formed. 1g of KBr solid powder was ground into a fine powder using a mortar and pestle. A suitable amount of the fine powder was placed in a hydraulic press and pressed into a thin, transparent sheet as a background signal for detection in the sample cell. Small amounts of the prepared CeO2 QDs nanozymes, Fe3O4@CeO2 QDs nanozymes, and LTDNs were added to approximately 15% KBr solid powder, ground into fine powder, and then pressed into thin sheets using a hydraulic press. FT-IR spectrophotometry was used to analyze the structures of CeO2 QDs nanozymes, Fe3O4@CeO2 QDs nanozymes, and LTDNs in four scans at a depth of 0.01 cm⁻¹. -1 Automatic signal collection resolution, in the range of 400-4000cm. -1The infrared spectra of the above nanoscale samples were recorded within the range of 3204 cm⁻¹. -1 The broad peak at 1557cm appeared -1 The absorption peak at 1376 cm⁻¹ is mainly attributed to the stretching vibration of hydroxyl groups adsorbed in water on the material surface. -1 The absorption peak at 570-635 cm⁻¹ is caused by the stretching vibration of the O-Ce-O bond. -1 The absorption peak at that location reflects the stretching vibration of Fe-O. Figure 3 ).

[0045] 3. Particle size and potential detection

[0046] LTDNs, CeO2 QDs nanozymes, Fe3O4@CeO2 QDs nanozymes, and Ca were analyzed using a Zata potentiometric analyzer and particle size analyzer. 2+ -UOx-NFs、Mn 2+ -UOx-NFs、Zn 2+ -UOx-NFs, Fe 2+ -UOx-NFs, Fe 3+ The particle size and potential of -UOx-NFs are shown in the figure. Figure 4 .

[0047] Place the powder samples into their respective sample containers. Add anhydrous ethanol to prepare an appropriate concentration, and sonicate for 5 minutes to ensure uniform particle distribution. Place the samples in the sample cell and perform three repeated measurements to improve data accuracy. The nanozyme particle size ( Figure 4 Analysis of A and B shows that Ca synthesized by induction from five different metal ions 2+ -UOx-NFs, Fe 3+ -UOx-NFs、Mn 2+ -UOx-NFs、Zn 2+ -UOx-NFs and Fe 2+ The particle sizes of -UOx-NFs nanozymes and CeO2 nanozymes before and after Fe3O4 modification are mainly concentrated in the range of 100 nm to 1000 nm, and exhibit different distribution characteristics. Among them, Ca 2+ -UOx-NFs, Fe 2+ -UOx-NFs、Mn 2+ -UOx-NFs、Zn 2+ -UOx-NFs and Fe 3+ The average particle sizes of -UOx-NFs were 568 nm, 1406 nm, 661 nm, 420 nm, and 310 nm, respectively. The particle size distribution of Fe3O4@CeO2 QDs ranged from 229 to 420 nm, with an average particle size of 310 nm. The average particle size of LTDNs was approximately 660 nm.

[0048] Zeta potential is an important parameter characterizing the surface charge properties of nanoparticles, and is commonly used to assess the stability of particles in solution. A comparison of the potentials of uric acid nanozymes induced by different ions is shown in the figure. Figure 4 As can be seen from C), the uric acid nanoflowers induced by different ions all carry a negative charge, and the values ​​are all relatively large, indicating that the prepared uric acid nanoflowers have high stability in solution. From the LTDNs potential comparison diagram ( Figure 4 From D), we can see that CeO2QDs have the lowest Zeta potential (approximately -32 mV), indicating that they easily aggregate in solution. Fe3O4@CeO2 has a Zeta potential of approximately 5 mV, indicating that its surface carries a positive charge, while Mn... 2+ The Zeta potential of -UOx-NFs is approximately -18 mV, indicating that their surfaces carry a negative charge. Since these two nanozymes have opposite surface charges, the electrostatic attraction between them may cause them to attract and bind to each other in solution.

[0049] Example 6: Enzyme activity assay method for LTDNs

[0050] 0.07 mg of LTDNs prepared according to the method in Example 4 was added to 2 mL of acetate-sodium acetate buffer (0.1 M, pH 4.0) containing 50 μM uric acid (UA) and 1 mM TMB (3,3',5,5'-tetramethylbenzidine). The reaction was carried out in a water bath at 37°C for 10 min. After separating the LTDNs using an external magnetic field, the UV-Vis absorption spectrum of the supernatant at 652 nm was measured. The enzyme activity was assessed by measuring the absorbance value at 652 nm; a higher absorbance value indicates stronger activity.

[0051] Example 7: Temperature stability of LTDNs

[0052] To compare the temperature stability of LTDNs, 0.07 mg of LTDNs prepared according to the method of Example 4 (experimental group) and a mixture of 0.035 mg of uricase (UOx) and 0.035 mg of horseradish peroxidase (HRP, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number P105526) (control group) were stored at constant temperatures of 20, 25, 30, 35 and 40 °C, respectively. After one day, the enzymes were isolated and the absorbance values ​​were measured according to the method of Example 6 to compare the relative enzyme activities.

[0053] Relative enzyme activity is defined as the ratio of absorbance after one day of storage to absorbance when it was first placed in the container (day 0).

[0054] As shown in Table 2, the initial absorbance (enzyme activity) of LTDNs was slightly lower than that of natural enzymes. After one day of storage, the relative enzyme activity of LTDNs remained above 85% at various temperatures. The relative enzyme activity was even higher at lower temperatures. At the same temperature, the stability of natural enzymes was relatively worse, retaining at least 88% of their activity at low temperatures (<35℃). With further increases in temperature, the relative enzyme activity decreased significantly, reaching only 53.18% at 45℃. Therefore, LTDNs exhibit better temperature stability than natural enzymes.

[0055] Table 2 Comparison of temperature stability of LTDNs and natural enzymes

[0056]

[0057]

[0058] Example 8: Storage stability of supported dual nanozymes

[0059] To compare the storage stability of the supported dual nanozymes, 0.07 mg of the supported dual nanozymes (LTDNs) prepared by the method in Example 4 and 0.07 mg of UOx / HRP (mixed at a mass ratio of 1:1 as in Example 7) were placed at a constant temperature of 25°C. The enzymes were isolated after 2, 4, 6, 8, and 10 days, and their absorbance values ​​were measured according to the method in Example 6 to compare their relative enzyme activities. Relative enzyme activity was defined as the ratio of enzyme activity after a certain number of days to that on day 0. As shown in Table 3, the relative enzyme activities of both LTDNs and UOx / HRP decreased with prolonged storage time, and the decrease in relative enzyme activity of the natural enzyme UOx / HRP was significantly greater than that of the supported dual nanozymes LTDNs. After 10 days of storage, the relative enzyme activity of UOx / HRP was only 46.49%, while that of LTDNs remained at 77.69%. The longer the storage period, the greater the difference in relative enzyme activity between LTDNs and UOx / HRP. In comparison, loaded dual nanozymes have a significant advantage over natural enzymes in terms of storage stability.

[0060] Table 3 Comparison of storage stability of LTDNs and natural enzymes

[0061]

[0062] Example 9: Uric acid colorimetric detection system and its linearity and sensitivity

[0063] 1. Linearity and sensitivity of the uric acid colorimetric detection system

[0064] The final concentration composition of the uric acid colorimetric assay system was: 20 μg / mL LTDNs, different concentrations (1, 10, 40, 45, 50, 55 μM) of uric acid, 1 mM TMB, and 2 mL of acetate-sodium acetate buffer (0.1 M, pH 4.0). The reaction was carried out at 37℃ for 10 min. After separating the supported dual nanozymes using an external magnetic field, the UV-Vis absorption spectrum of the supernatant was measured at wavelengths of 400–800 nm. Three parallel assays were performed. Results are shown below. Figure 5 B.

[0065] Using uric acid concentration as the x-axis, OD 652 Using the values ​​as the ordinate, linear fitting was performed using Origin 2019 software. The results are shown below. Figure 5 In step A, the detection limit is calculated according to the formula CL = 3Sb / M (K = 3, Sb is the blank standard deviation, and M is the slope of the standard curve).

[0066] from Figure 5 As can be clearly seen from Figure C, the color of the solution after color development gradually deepens visibly with increasing UA concentration. Figure 5 The results show that, within the 1-55 μM range, the UA concentration and the OD value (652 nm) of this colorimetric detection system exhibit a good linear relationship, with the linear equation being y = 0.0052x + 0.382, r 2 The value was 0.996, and the detection limit was 0.37 μM.

[0067] 2. Comparison of uric acid colorimetric detection system with literature

[0068] The detection system constructed in step 1 was compared with detection systems reported in the literature, and the results are shown in Table 4. Analysis of the data in the table shows that the detection range of this method is basically consistent with existing detection methods, and the detection limit is lower. The one-step method used in this strategy is simpler. Therefore, this method has significant advantages.

[0069] Table 4 Comparison with other nanozyme systems for uric acid detection

[0070]

[0071] Example 10: Reusability

[0072] The final concentration composition of the uric acid colorimetric detection system was as follows: LTDNs prepared according to the method in Example 4 with a final concentration of 20 μg / mL, containing 50 μM UA, 1 mM TMB, and 2 mL of acetate-sodium acetate buffer (0.1 M, pH 4.0). The reaction was carried out in a water bath at 37°C for 10 min. After separating the LTDNs using an external magnetic field, the OD of the supernatant was measured. 652The uric acid concentration was calculated based on the standard curve from Example 9. The LTDNs were washed three times with buffer solution before being added to the next batch of reaction. The OD value from the first wash was... 652 The value is defined as 100% relative enzyme activity. The relative enzyme activity of the nth time is defined as the OD of the supernatant of the nth time. 652 Value and OD of the first supernatant 652 The ratio of values ​​(Table 5).

[0073] Table 5. Effect of Number of Reuses on Relative Enzyme Activity

[0074]

[0075]

[0076] Reusability is a significant advantage of nanocatalytic materials. This example investigated the reusability of LTDNs in a colorimetric detection system. Table 5 shows that after three uses, the relative enzyme activity of the LTDNs remained at more than half, demonstrating strong biocatalytic activity. This strongly suggests that uricase immobilized with a nanoflower structure and Fe3O4@CeO2 QDs exhibit excellent reusability and can significantly reduce the difficulty of enzyme recovery and detection costs.

[0077] Example 11: Specificity Detection

[0078] To investigate whether other reducing substances in the human body interfere with uric acid detection, and thus determine the specificity of LTDNs for uric acid, we selected several interfering substances that may exist in human biological samples—glucose, D-fructose, sucrose, tyrosine, L-lysine, L-cysteine, dopamine, melatonin, urea, and sodium ascorbate—for control experiments.

[0079] According to the uric acid colorimetric detection system established in Example 10, the above-mentioned interfering substance was used instead of uric acid for detection. Three parallel experiments were performed to measure the absorbance of the above samples at 652 nm. Figure 6 As shown, only uric acid can clearly develop a color in the TMB colorimetric solution, while other interfering substances cannot. This experimental result indicates that the colorimetric detection system of LTDNs has excellent anti-interference capabilities. Therefore, this method can selectively detect the uric acid content in human urine.

[0080] Example 12: Analysis of actual samples

[0081] This experiment collected urine samples from three laboratory personnel (sample collection complied with voluntary and informed consent requirements) for recovery analysis. Because UA is prone to precipitation, the collected urine samples were tested within 10 minutes. The specific operating procedures were as follows:

[0082] First, dilute the three urine samples 1000-fold with acetate-sodium acetate buffer (0.1M, pH 4.0) and set aside. Take 1 mL of the diluted urine sample, add 20 μg / mL LTDNs and 1 mM TMB to a final concentration, and make up to 2 mL with acetate-sodium acetate buffer (0.1M, pH 4.0). Incubate at 37°C for 10 min. Perform three replicates for each experimental group. Measure the absorbance at 652 nm and calculate the initial UA concentration (i.e., the concentration before addition) in the diluted urine sample based on the standard curve plotted in Example 9.

[0083] Take another 1 mL of each of the three diluted urine samples, and add 20 μg / mL LTDNs and 1 mMMTMB to each sample. Then add 2 μM, 4 μM, and 6 μM uric acid to each sample, respectively. Make up to 2 mL of the detection system with acetate-sodium acetate buffer (0.1 M, pH 4.0), sonicate to dissolve, and react in a 37°C water bath for 10 min. Perform three replicates for each experimental group. Measure the absorbance at 652 nm and calculate the UA2 concentration in the urine sample based on the standard curve plotted in Example 9.

[0084] The recovery rate is calculated using the formula R = (uric acid concentration after addition - uric acid concentration before addition) / added uric acid concentration × 100%. The relative standard deviation is calculated as the ratio of the standard deviation to the mean.

[0085] Table 6. Uric acid test results of actual samples

[0086]

[0087] The test results (Table 6) show that the recovery rate of uric acid ranged from 93.25% to 102.50%. The calculated relative standard deviations (RSDs) were all less than 5%.

[0088] The results above demonstrate that the analytical method exhibits good reproducibility and precision in the provided urine samples, with minimal data fluctuations and high reliability, meeting the basic precision requirements of routine quantitative analysis.

[0089] Example 13: Accuracy Analysis

[0090] First, urine samples were collected from five laboratory personnel (sample collection complied with voluntary and informed consent requirements). The urine samples were diluted 1000-fold with acetate-sodium acetate buffer (0.1M, pH 4.0). The five actual urine samples were analyzed according to the instructions of the UA colorimetric assay kit (purchased from Sangon Biotech, product number: D799285-0050). Simultaneously, five identical urine samples were analyzed according to the uric acid colorimetric assay system described in Example 10. Each experimental group was performed in triplicate. The results of both methods were statistically analyzed using EXCEL. The results (Table 7) show that the p-values ​​for all five groups were greater than 0.05, indicating good consistency between the two methods. This demonstrates that the uric acid colorimetric assay using the loaded dual nanozyme has significant practical application value.

[0091] Table 7 Comparison of LTDNs and reagent kit analysis results

[0092]

Claims

1. A nanoflower-based supported bi-nanoreactor, characterized in that, The supported double nanoscale enzyme is prepared by fixing a peroxidase-like nanoscale enzyme in a uricase nanoflower structure; the peroxidase-like nanoscale enzyme is prepared from Ce(NO3)3.6H2O, monohydrate citric acid and ferrous sulfate.

2. The supported bi-nanoreactor according to claim 1, wherein, The supported double nanoscale enzyme is prepared by the following steps: adding the uricase nanoflower and the peroxidase-like nanoscale enzyme into a buffer solution with pH 4-9 to form a reaction system, fully mixing, separating under an external magnetic field, repeatedly washing with the buffer solution with pH 4-9, pre-freezing in a-80℃ ultra-low temperature refrigerator for 8h, and then transferring to a freeze dryer for freeze-drying at-70℃ for 12h to obtain the supported double nanoscale enzyme.

3. The supported bi-nanoreactor according to claim 2, wherein, The buffer solution is 0.1M acetic acid-sodium acetate buffer solution with pH 4.

0.

4. The supported bi-nanoreactor according to claim 2, wherein, The mass ratio of the uricase nanoflower to the peroxidase-like nanoscale enzyme is 1:1-4; and the mixing time is 3-30min.

5. The supported bi-nanoreactor according to claim 1, wherein, The preparation method of the uricase nanoflower is as follows: adding uricase into a PBS buffer solution, then adding an aqueous solution of metal salt, incubating at 20-30℃ for 30-40h, centrifuging the suspension, washing the obtained precipitate with ultrapure water, pre-freezing the washed precipitate in a-80℃ ultra-low temperature refrigerator for 8h, and then transferring to a freeze dryer for freeze-drying at-70℃ for 12h to obtain the uricase nanoflower.

6. The supported bi-nanoreactor according to claim 5, wherein, The metal salt is one or more of CaCl2, MnCl2, ZnCl2, FeCl2 or FeCl3; the concentration of the aqueous solution of metal salt is 0.1-1mol / L, and the volume dosage is 0.1-1mL / g based on the mass of uricase; and the volume dosage of the PBS buffer solution is 1-10mL / mg based on the mass of uricase.

7. The supported bi-nanoreactor according to claim 1, wherein, The preparation method of the peroxidase-like nanoscale enzyme is as follows: (1) dissolving Ce(NO3)3.6H2O and monohydrate citric acid in ultrapure water, adjusting the pH to 9-10, transferring the solution into a high-pressure hydrothermal reactor with a polytetrafluoroethylene lining, and keeping the temperature at 150-200℃ for 4-8h; after the reaction, adding an appropriate amount of ethanol to induce the precipitate to form, collecting the precipitate by centrifugation, and washing with ultrapure water and ethanol in sequence to remove impurities; pre-freezing the washed precipitate in a-80℃ ultra-low temperature refrigerator for 8h, and then transferring to a freeze dryer for freeze-drying at-70℃ for 12h; after complete drying, grinding the product into fine powder, which is the peroxidase-like nanoscale enzyme, denoted as CeO2 QDs nanoscale enzyme; (2) dispersing the CeO2 QDs nanoscale enzyme in a 0.1-0.5mol / L NaOH aqueous solution, stirring vigorously at 60-100℃ for 20-50min, slowly adding a solution of ferrous sulfate and ultrapure water, stirring vigorously in air for 20-50min to generate black precipitate; collecting the precipitate by using a magnet, and washing with ultrapure water and ethanol in sequence until the washed precipitate is neutral; pre-freezing the obtained precipitate in a-80℃ ultra-low temperature refrigerator for 8h, and then transferring to a freeze dryer for freeze-drying at-70℃ for 12h to obtain the peroxidase-like nanoscale enzyme, denoted as Fe3O4@CeO2 QDs.

8. The supported double nanoszyme of claim 7, wherein, The mass ratio of the monohydrate citric acid to Ce(NO3)3·6H2O in step (1) is 1:1-3; the volume of the ultrapure water used for dissolution is 30-50 mL / g based on the mass of the monohydrate citric acid; the volume of the NaOH aqueous solution in step (2) is 100-300 mL / g based on the mass of the CeO2 QDs nanoscale enzyme; the amount of ferrous sulfate is 0.3-1 g / g based on the mass of the CeO2 QDs nanoscale enzyme; and the volume of the ultrapure water is 50-150 mL / g based on the mass of the CeO2 QDs nanoscale enzyme.

9. Use of the supported double nanoscale enzyme of claim 1 in detecting uric acid.

10. Use according to claim 9, wherein The method of the use is as follows: a uric acid colorimetric detection system is constructed using the supported double nanoscale enzyme, and the final concentration is composed of 5-50 μg / mL supported double nanoscale enzyme, 5-200 μM uric acid, 0.5-5 mM 3,3',5,5'-tetramethylbenzidine, 0.1 M acetic acid-sodium acetate buffer at pH 4.0-9.0; the reaction is carried out at 20-45 °C in a water bath for 5-30 min, the absorbance value at 652 nm is detected, and the concentration of uric acid is calculated according to the standard curve of the concentration of uric acid standard and the absorbance value.

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