Cerium-based nano-enzyme, preparation method, application and uric acid detection method

Abstract

The invention discloses a preparation method of cerium-based nano enzyme, which comprises the following steps: reacting cerium salt and zinc salt with 2-methylimidazole in methanol, then loading into a high-pressure reaction kettle to prepare a Ce-MOF precursor, then calcining the precursor in a tubular furnace to obtain powder, and finally pickling the powder to obtain the cerium-based nano enzyme. The prepared cerium-based nano-enzyme is used for uric acid detection, can replace natural enzyme for quantitative analysis, and has the advantages of high sensitivity, low detection limit and better selectivity and stability. The invention also provides a quantitative analysis method for detecting uric acid, which comprises the following steps: reacting uric acid with uricase to generate hydrogen peroxide, then catalyzing the hydrogen peroxide by the cerium-based nano-enzyme to oxidize a chromogenic substrate, and finally quantifying according to a color B value, absorbance or temperature.

Description

TECHNICAL FIELD

[0001] The present application relates to nano-enzyme and its application in the field of analytical detection, in particular to a cerium-based nano-enzyme Ce-PODs with peroxidase-like activity, a preparation method and application thereof, and a corresponding uric acid detection method. BACKGROUND

[0002] Uric acid (2,6,8-trihydroxy purine) is the end product of human purine metabolism, and its abnormal level is closely related to many diseases. For example, high concentration can cause hyperuricemia, and further cause gout, kidney dysfunction and cardiovascular and cerebrovascular diseases, while too low concentration may be related to increased oxidative stress and multiple sclerosis. In the detection method of uric acid, the traditional colorimetric method relies on the cascade reaction of uric acid oxidase (UOx) and peroxidase (such as HRP): UOx first catalyzes the oxidation of uric acid to generate H2O2, and then HRP catalyzes the oxidation of the color developing substrate (such as TMB) to produce color change. The system has the following disadvantages: natural enzymes have poor stability and are easily affected by environmental factors such as temperature and pH; the storage conditions of enzyme preparations are harsh (usually need to be stored at 4℃ in the dark); the production cost is high, and it is difficult to be applied on a large scale.

[0003] Nano-enzyme is a kind of simulated enzyme with the characteristics of nanomaterials and enzyme catalytic function, which has the advantages of strong stability, easy storage, wide applicable temperature range of catalytic activity, easy large-scale preparation, etc. Therefore, designing and synthesizing suitable nano-enzyme to replace natural enzyme for uric acid detection is expected to overcome the above-mentioned shortcomings of uric acid detection method. SUMMARY

[0004] An object of the present application is to provide a preparation method of cerium-based nano-enzyme, which can replace natural enzyme for quantitative analysis in uric acid detection, and has high sensitivity, low detection limit, and good selectivity and stability.

[0005] In order to achieve the above-mentioned object, the present application adopts the following technical solutions:

[0006] A preparation method of cerium-based nano-enzyme, which catalyzes hydrogen peroxide to oxidize color developing substrate in the quantitative analysis of uric acid, comprising the following steps:

[0007] Step S1: dissolve cerium salt and zinc salt in methanol according to the molar ratio of cerium element to zinc element of 1:2-100 to obtain solution A; add 2-methylimidazole into methanol to obtain solution B; mix solution A and solution B, and the molar ratio of cerium element to 2-methylimidazole is 1:20 to obtain solution C;

[0008] Step S2: load solution C into a high-pressure reaction kettle of a polytetrafluoroethylene substrate, react at 120-180℃ for 12-24h, and then separate the solid to obtain a Ce-MOF precursor;

[0009] Step S3: Put the Ce-MOF precursor into a tube furnace, heat to 800-900℃ under inert atmosphere, calcine for 2-4h, and obtain a powder;

[0010] Step S4: Add the powder into an acid solution, acid wash at 60-100℃ for 12-24h, wash with water until neutral, dry, and obtain a cerium-based nanoscale enzyme.

[0011] Preferably, the cerium salt is one of cerium nitrate or its hydrate, cerium sulfate or its hydrate, and cerium chloride or its hydrate; the zinc salt is one of zinc nitrate or its hydrate, zinc chloride or its hydrate, and zinc sulfate or its hydrate; the cerium salt and the zinc salt are dissolved in methanol in a cerium element to zinc element molar ratio of 1:5; the acid solution is 2-3 mol / L hydrochloric acid or nitric acid, and the powder is added into the acid solution and acid washed at 80℃ for 24h.

[0012] Another object of the present application is to provide a cerium-based nanoscale enzyme prepared by the above method, which catalyzes hydrogen peroxide to oxidize a color developing substrate in the quantitative analysis of uric acid.

[0013] The present application also provides an application of the above cerium-based nanoscale enzyme, which is to use the cerium-based nanoscale enzyme to catalyze hydrogen peroxide to oxidize a color developing substrate into a colored substance after hydrogen peroxide is generated by the reaction of uric acid and uricase, and then detect the content of the uric acid according to the quantitative relationship between the color, absorbance or temperature of the colored substance and the uric acid.

[0014] Preferably, the color developing substrate is 3,3'5,5'-tetramethylbenzidine or o-phenylenediamine; after the colored substance is generated, the content of uric acid is detected by using the linear relationship between the change value of the B channel value of the mobile phone RGB mode of the uric acid solution relative to the reference solution and the concentration of uric acid, or by using the linear relationship between the change value of the absorbance of the uric acid solution relative to the reference solution and the concentration of uric acid, or by using the linear relationship between the change value of the temperature of the uric acid solution relative to the reference solution and the concentration of uric acid after the uric acid solution and the reference solution are irradiated by laser.

[0015] The present application also provides a uric acid detection method based on absorbance, comprising the following steps:

[0016] Step S1: add uricase to several different concentrations or different volumes of uric acid standard sample and uric acid to be measured, then react at 37 DEG C for 15 min, then add NaAc-HAc buffer solution, 3,3'5,5'-tetramethylbenzidine and cerium-based nanometer enzyme, and add water to a certain volume to obtain different concentrations of uric acid standard solution and uric acid to be measured, at this time, the pH value of the uric acid standard solution and the uric acid to be measured is 3.8, the concentration of 3,3'5,5'-tetramethylbenzidine is 0.48 mmol / L, the concentration of cerium-based nanometer enzyme is 15 μg / mL, the concentration of uric acid is in the range of 20-320 μmol / L, the concentration of uricase is 0.6 mg / mL, the operation temperature is 25-35 DEG C, and the cerium-based nanometer enzyme is the cerium-based nanometer enzyme prepared by the above method;

[0017] Step S2: the absorbance change value of the uric acid standard solution and the uric acid to be measured relative to the reference solution is measured at 652 nm, the concentration of uric acid is taken as the horizontal coordinate, the change value of the B value of the uric acid to be measured solution relative to the reference solution is taken as the vertical coordinate, a standard curve is drawn using the concentration and the change value of the B value of the uric acid standard solution, then the concentration of the uric acid to be measured solution is determined according to the change value of the B value of the uric acid to be measured solution relative to the reference solution and the standard curve, and the concentration of uric acid in the uric acid to be measured sample is determined.

[0018] The application also provides a method for detecting uric acid based on the RGB color B channel, characterized by comprising the following steps:

[0019] Step S1: mix 30 μL of cerium-based nanometer enzyme dispersion liquid with a concentration of 1 mg / mL, 150 μL of TMB solution with a concentration of 6.4 mmol / L, 500 μL of HAc-NaAc buffer solution with a concentration of 20 mmol / L, and an appropriate amount of agarose, and add deionized water to 2000 μL to obtain a standby solution; add 170 μL of the standby solution to each hole of a 96-hole plate, and after solidification at room temperature, a hydrogel reagent kit is obtained; take 10 μL of different concentrations of uric acid standard solution and uric acid to be measured solution, add 20 μL of uricase solution with a concentration of 6 mg / mL, and incubate at 37 DEG C for 15 min to obtain an enzyme reaction liquid, and then add 30 μL of the enzyme reaction liquid to the 96-hole hydrogel, and the gel turns blue; the cerium-based nanometer enzyme is the cerium-based nanometer enzyme of claim 3;

[0020] Step S2: an Android smart phone APP (fixed ISO / white balance) is used to take a gel image; Image J is used to extract the B value of each hole, and a standard curve is drawn using the concentration and the change value of the B value of the uric acid standard solution, with the concentration of uric acid as the horizontal coordinate and the change value of the B value of the uric acid to be measured solution relative to the reference solution as the vertical coordinate; then the concentration of the uric acid to be measured solution is determined according to the change value of the B value of the uric acid to be measured solution relative to the reference solution and the standard curve.

[0021] The application also provides a temperature-based uric acid detection method, characterized by comprising the following steps:

[0022] Step S1: uricase is added to uric acid standard samples and uric acid to-be-tested samples of several different concentrations or different volumes, then reacted at 37 DEG C for 15 min, then NaAc-HAc buffer solution, 3,3'5,5'-tetramethylbenzidine and cerium-based nanoscale enzyme are added, and water is added to a certain volume to obtain uric acid standard solutions and uric acid to-be-tested solutions of different concentrations, at this time, the pH value of the uric acid standard solution and the uric acid to-be-tested solution is 3.8, the concentration of 3,3'5,5'-tetramethylbenzidine is 0.48 mmol / L, the concentration of cerium-based nanoscale enzyme is 15 μg / mL, the concentration of uricase is 0.6 mg / mL, the operation temperature is 25-35 DEG C, and the cerium-based nanoscale enzyme is the cerium-based nanoscale enzyme prepared by the above method;

[0023] Step S2: the uric acid standard solution and the uric acid to-be-tested solution and a reference solution are irradiated with an 808 nm laser for 3 min, a handheld temperature measurement thermal imager is used to measure the temperature change value, the concentration of uric acid is used as the abscissa, the temperature change value of the uric acid standard solution relative to the reference solution is used as the ordinate, a standard curve is drawn by using the concentration and the temperature change value of the uric acid standard solution, then the concentration of the uric acid to-be-tested solution is determined according to the temperature change value of the uric acid to-be-tested solution relative to the reference solution and the standard curve, and the concentration of uric acid in the uric acid to-be-tested sample is determined.

[0024] The application has the beneficial effects that Ce 3+ is introduced in the precursor synthesis stage, 3+ Ce 3+ is reduced in situ and fixed in the nitrogen-doped carbon skeleton during the high-temperature carbonization process in a nitrogen atmosphere, forming dispersed active sites, and Ce 3+ exists in the form of metal in the final product, and the content reaches 1.96 wt % (EDS result). The synthesis process not only retains the high specific surface structure of the dodecahedron, but also significantly accelerates the charge transfer step in the TMB-H2O2 catalytic cycle through Ce 4+ / Ce 4+ , thereby giving the cerium-based nanoscale enzyme material a peroxidase-like activity better than Ce-MOF. At the same time, the material has low synthesis cost, stable performance and easy storage; for uric acid quantitative analysis, the method has high sensitivity, low detection limit (2.60 μmol / L), good selectivity, and wide temperature and pH value applicability. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1Characterization maps of the material prepared in the application: (a) scanning electron microscope image of Ce-MOF; (b) scanning electron microscope image of Ce-PODs; (c) transmission electron microscope image of Ce-PODs; (d), (e) EDS characterization map of Ce-PODs;

[0026] Figure 2 Contrast diagram of peroxidase-like activity of cerium-based nanoszyme in the application: (a) absorption curves of three solutions of Ce-PODs+TMB+H2O2, Ce-PODs+TMB, TMB+H2O2; (b) absorption curves of three solutions of Ce-PODs+OPD+H2O2, Ce-PODs+OPD, OPD+H2O2; (c) sample photos of three solutions of Ce-PODs+TMB+H2O2, Ce-PODs+TMB, TMB+H2O2 (from left to right, top, middle and bottom) and Ce-PODs+OPD+H2O2, Ce-PODs+OPD, OPD+H2O2 (from left to right, top, middle and bottom);

[0027] Figure 3 Steady-state kinetics experiment of cerium-based nanoszyme in the application: (a) Michaelis-Menten diagram of Ce-MOF, Ce-PODs (TMB as substrate); (b) Lineweaver-Burk diagram of Ce-MOF, Ce-PODs (TMB as substrate); (c) Michaelis-Menten diagram of Ce-MOF, Ce-PODs (H2O2 as substrate); (d) Lineweaver-Burk diagram of Ce-MOF, Ce-PODs (H2O2 as substrate);

[0028] Figure 4 Stability result diagram of cerium-based nanoszyme in the application;

[0029] Figure 5 Influence diagram of different pH value buffer solution on experimental results in the application;

[0030] Figure 6 Curve diagram of absorbance change value (ΔA=A-A0) with reaction temperature in the application;

[0031] Figure 7 Curve diagram of absorbance change value (ΔA=A-A0) with TMB concentration in the application;

[0032] Figure 8 Curve diagram of absorbance change value (ΔA=A-A0) with nanoszyme concentration in the application;

[0033] Figure 9The graph of the change value (ΔA=A-A0) of absorbance of the application versus the concentration of uricase;

[0034] Figure 10 The graph of the change value (ΔA=A-A0) of absorbance of the application versus the concentration of uric acid;

[0035] Figure 11 The specific detection graph of the cerium-based nanozyme of the application;

[0036] Figure 12 The graph of the change value (ΔB=B-B0) of B of the application versus the concentration of uric acid;

[0037] Figure 13 The Figure 12 The photos of the standard samples corresponding to the detection;

[0038] Figure 14 The graph of the change value (ΔT=T1-T0) of T of the application versus the concentration of uric acid. DETAILED DESCRIPTION

[0039] The application will be described in detail below in conjunction with the drawings and examples.

[0040] Example 1:

[0041] Step S1: 1.190 g (4.0 mmol) of zinc nitrate hexahydrate and 0.347 g (0.8 mmol) of cerium nitrate hexahydrate were added to 30 mL of methanol to obtain solution A; 1.314 g (16.0 mmol) of 2-methylimidazole was added to 15 mL of methanol to obtain solution B; solution A and solution B were respectively ultrasonically treated for 5 min, then mixed and stirred at room temperature for 3 min to obtain solution C.

[0042] Step S2: solution C was transferred into a 100 mL stainless steel autoclave lined with polytetrafluoroethylene, and reacted at 120℃ for 24 h; after the reaction was completed, the product was washed with methanol and deionized water for 3 times respectively, the centrifugal speed was 4500 rmp, the centrifugal time was 5 min, and then the product was dried at 60℃ for 3 h to obtain a light yellow solid, i.e. the Ce-MOF precursor.

[0043] Step S3: the above Ce-MOF precursor was placed in a tube furnace, nitrogen was introduced, the nitrogen flow rate was controlled at 5 m 3 / h, after the air in the tube furnace was discharged by introducing nitrogen for 10 min, the temperature of the tube furnace was increased to 900℃ at a rate of 5℃ / min, and the temperature was kept for 2 h to obtain a powder.

[0044] Step S4: The obtained solid powder was reacted in 3 mol / L hydrochloric acid solution at 80 °C for 24 h, washed with deionized water for 3 times, and dried at 60 °C to obtain a black solid, which was cerium-based nanoszyme (Ce-PODs).

[0045] The above Ce-MOF precursor and cerium-based nanoszyme were characterized by scanning electron microscopy (SEM), and the Ce-PODs were characterized by transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS), and the results are shown in Figure 1 The SEM image shows that the Ce-MOF maintains the typical ZIF-8 rhombohedron morphology, with clear edges and corners, smooth surface, and no visible pores; the overall framework of Ce-PODs shrinks after high-temperature carbonization and acid washing, still retaining the outline of the dodecahedron, but the surface is rough and wrinkled, and the edges are blunt. The TEM image further confirms the formation of the dodecahedron structure of Ce-PODs, without embedding large cerium and zinc nanoparticles in the amorphous carbon framework. The dodecahedron structure of Ce-PODs can maintain a high specific surface area, and the surface wrinkles / micropores can increase the number of active sites, shorten the substrate diffusion path, and reduce the mass transfer resistance.

[0046] Example 2 Performance test of cerium-based nanoszyme

[0047] 1. Determination of cerium-based nanoszyme peroxidase-like activity

[0048] The cerium-based nanoszyme prepared in Example 1 was added to deionized water to prepare a dispersion solution with a concentration of 1 mg / mL for standby.

[0049] (1) TMB was used as a chromogenic substrate, and the following three solutions were prepared and the absorption curve was made:

[0050] Ce-PODs+TMB+H2O2 solution: 100 μL of 20 mmol / L NaAc-HAc buffer solution with pH of 3.8, 30 μL of 6.4 mmol / L 3,3'5,5'-tetramethylbenzidine (abbreviated as TMB), and 10 μL of 0.8 mol / L H2O2 were added to deionized water, then 6 μL of 1 mg / mL cerium-based nanoszyme dispersion solution was added, and the rest was supplemented with deionized water to a final volume of 400 μL. After standing at 25 °C for 20 min, the multifunctional microplate was detected to make the absorption curve.

[0051] Ce-PODs+TMB solution: 100 μL of NaAc-HAc buffer solution with a concentration of 20 mmol / L and a pH of 3.8, 30 μL of 3,3'5,5'-tetramethylbenzidine with a concentration of 6.4 mmol / L, and then 6 μL of a dispersion of cerium-based nanoscale enzyme with a concentration of 1 mg / mL were added in deionized water, and the rest was made up with deionized water to a final volume of 400 μL. After being placed at 25 °C for 20 min, a multifunctional microplate was used for detection, and an absorption curve was drawn.

[0052] TMB+ H2O2 solution: 100 μL of NaAc-HAc buffer solution with a concentration of 20 mmol / L and a pH of 3.8, 30 μL of 3,3'5,5'-tetramethylbenzidine with a concentration of 6.4 mmol / L, and 10 μL of 0.8 mol / L H2O2 were added in deionized water, and the rest was made up with deionized water to a final volume of 400 μL. After being placed at 25 °C for 20 min, a multifunctional microplate was used for detection, and an absorption curve was drawn.

[0053] The above absorption curves are shown in Figure 2 (a) the upper, middle and lower rows of the figure, and the samples are shown in Figure 2 (c) the upper, middle and lower rows of the left column.

[0054] (2) OPD was used as a chromogenic substrate, and the following three solutions were prepared and absorption curves were drawn:

[0055] Ce-PODs+OPD+ H2O2 solution: 100 μL of NaAc-HAc buffer solution with a concentration of 20 mmol / L and a pH of 3.8, 30 μL of o-phenylenediamine (OPD) with a concentration of 6.4 mmol / L, and 10 μL of 0.8 mol / L H2O2 were added in deionized water, and the rest was made up with deionized water to a final volume of 400 μL. After being placed at 25 °C for 20 min, a multifunctional microplate was used for detection, and an absorption curve was drawn.

[0056] Ce-PODs+OPD solution: 100 μL of NaAc-HAc buffer solution with a concentration of 20 mmol / L and a pH of 3.8, 30 μL of o-phenylenediamine with a concentration of 6.4 mmol / L, and then 6 μL of a dispersion of cerium-based nanoscale enzyme with a concentration of 1 mg / mL were added in deionized water, and the rest was made up with deionized water to a final volume of 400 μL. After being placed at 25 °C for 20 min, a multifunctional microplate was used for detection, and an absorption curve was drawn.

[0057] OPD + H2O2 solution: 100 μL of NaAc-HAc buffer solution with a concentration of 20 mmol / L and a pH of 3.8, 30 μL of o-phenylenediamine with a concentration of 6.4 mmol / L, and 10 μL of H2O2 with a concentration of 0.8 mol / L were added to deionized water, and the rest was made up with deionized water to a final volume of 400 μL. After being placed at 25 °C for 20 min, the absorption curve was drawn by using a multifunctional microplate detector.

[0058] The above absorption curves are as shown in Figure 2 (b) The samples are shown in the upper, middle, and lower rows in sequence. Figure 2 (c) The samples are shown in the upper, middle, and lower rows in the right column in sequence.

[0059] As can be seen from Figure 2 (a) and (c), the color of the Ce-PODs + TMB + H2O2 solution is significantly darker than that of the TMB + H2O2 solution and the Ce-PODs + TMB solution, and the absorbance of the former at 652 nm is significantly greater than that of the latter two, indicating that Ce-PODs can catalyze H2O2 to produce reactive oxygen species and rapidly oxidize TMB to develop color, thereby proving that the cerium-based nanoscale enzyme has excellent peroxidase-like activity; similarly, as can be seen from Figure 2 (b) and (c), the color of the Ce-PODs + OPD + H2O2 solution is significantly darker than that of the OPD + H2O2 solution and the Ce-PODs + OPD solution, and the absorbance of the former at 450 nm is significantly greater than that of the latter two, indicating that Ce-PODs can catalyze H2O2 to produce reactive oxygen species and rapidly oxidize OPD to develop color, thereby also proving that the cerium-based nanoscale enzyme has excellent peroxidase-like activity.

[0060] 2. Steady-state kinetic experiment of cerium-based nanoscale enzyme

[0061] The affinity and catalytic rate of the cerium-based nanoscale enzyme for TMB and H2O2 were tested, and the steady-state kinetic study was carried out by changing the concentration of one of the substances while keeping the concentration of the other substance unchanged, and the results are shown in Figure 3 As can be seen from Figure 3 , within a suitable concentration range of the substrate, the catalytic reaction of the cerium-based nanoscale enzyme is a typical Michaelis-Menten kinetic model, and the kinetic constants Km and maximum reaction rate Vmax can be obtained by fitting the Lineweaver-Burk equation. The kinetic parameters of Ce-PODs obtained by experiments are as follows: for TMB, Km = 0.214 mmol / L and Vmax = 9.02 × 10 -6 mol / (L·s); for H2O2, Km = 0.873 mmol / L and Vmax = 1.27 × 10 -3mol / (L s). The kinetic parameters of Ce-MOF were obtained: for TMB, Km = 0.230 mmol / L and Vmax = 1.73 × 10 -6 mol / (L s); for H2O2, Km = 1.25 mmol / L, Vmax = 0.366 × 10 -3 mol / (L s). Affinity (Km): the Km of Ce-PODs for TMB / H2O2 is smaller than that of Ce-MOF, the smaller the Km, the tighter the enzyme binds to the substrate, and the higher the catalytic efficiency; maximum reaction rate (Vmax): the Vmax of Ce-PODs for TMB / H2O2 is larger than that of Ce-MOF, and the larger the Vmax, the stronger the ability to convert substrates per unit time. The above results show that the peroxidase-like activity of cerium-based nanoszyme is superior to Ce-MOF, and has good performance.

[0062] 3. Stability experiment of cerium-based nanoszyme

[0063] The stability of cerium-based nanoszyme (Ce-PODs) during long-term storage was experimentally investigated, including the effects of different synthesis batches and room temperature light-avoiding storage conditions on catalytic activity, and the results are shown in Figure 4 a. As can be seen from Figure 4 a, the peroxidase-like activity of cerium-based nanoszyme (Ce-PODs) remained relatively stable after five weeks of storage, indicating that the material can effectively maintain its original performance during long-term storage. The batch stability was further evaluated by measuring the absorbance of the Ce-PODs / TMB system (100 μL of 20 mmol / L NaAc-HAc buffer solution with pH of 3.8, 30 μL of 6.4 mmol / L 3,3'5,5'-tetramethylbenzidine, and 10 μL of 0.8 mol / L H2O2 were added, then 6 μL of 1 mg / mL cerium-based nanoszyme dispersion was added, and the rest was made up with deionized water to a final volume of 400 μL. The absorbance at 652 nm was measured after 20 min at 25 °C. ), and the results are shown in Figure 4 b. As can be seen from Figure 4 b, the activity difference of the six consecutive synthesis batches is very small, indicating good batch reproducibility. Therefore, Ce-PODs has stable repeatability and long-term storage potential in practical applications, and is suitable for batch production and on-site detection.

[0064] Example 6: Conditions for cerium-based nanoszyme (Ce-PODs) for uric acid detection

[0065] 1. Test of buffer solution pH

[0066] Preparation of solution:

[0067] 20 μL of uric acid with a concentration of 5 mmol / L and 40 μL of uricase with a concentration of 5 mg / mL were added into a centrifuge tube, and incubated at 37 °C for 15 min for generating H2O2, then 100 μL of NaAc-HAc buffer solution with a concentration of 20 mmol / L and a pH in the range of 3.2-5.8, 30 μL of TMB with a concentration of 6.4 mmol / L, 6 μL of Ce-PODs nanoscale enzyme solution with a concentration of 1 mg / mL were added, and the rest was supplemented with deionized water to a final volume of 400 μL, and reacted for 25 min at 25 ℃, and the absorbance of the reaction system at 652 nm was A, and a solution with a uric acid concentration of 0 was prepared as a reference solution in the same way, and the absorbance thereof corresponded to A0. The pH was taken as the abscissa, and the change value ΔA (A-A0) of the absorbance of the system at 652 nm was taken as the ordinate, and a curve of the change of the absorbance with the pH was obtained, as shown in Figure 5 , the ΔA of the system reached a peak at pH 3.8; as the pH value continued to rise, ΔA showed a downward trend. Therefore, the HAc-NaAc buffer solution with pH 3.8 was finally selected as the optimal acidity condition for the subsequent uric acid detection experiment.

[0068] 2. Test of reaction temperature

[0069] 20 μL of uric acid with a concentration of 5 mmol / L and 40 μL of uricase with a concentration of 5 mg / mL were added into a centrifuge tube, and incubated at 37 °C for 15 min for generating H2O2, then 100 μL of NaAc-HAc buffer solution with a concentration of 20 mmol / L and a pH of 3.8, 30 μL of TMB with a concentration of 6.4 mmol / L, 6 μL of Ce-PODs nanoscale enzyme solution with a concentration of 1 mg / mL were added, and the rest was supplemented with deionized water to a final volume of 400 μL, and reacted for 25 min at a temperature in the range of 25-55 ℃, and the absorbance of the system at 652 nm corresponded to A, and the absorbance of the solution with a uric acid concentration of 0 corresponded to A0. The ΔA (A-A0) of the system at 652 nm was taken as the ordinate, and the temperature change was taken as the abscissa, as shown in Figure 6 , in the range of 25-35 ℃, the temperature fluctuation had a smaller influence, the uric acid detection signal slightly increased with the increase of the temperature, ΔA reached a maximum at 35 ℃, and then slightly decreased, which indicated that the method of the application could be applied in a relatively wide temperature range, and could meet the requirements of no additional temperature control in the scene of point-of-care testing (POCT). Based on the experimental results and actual application requirements, 25 ℃ was finally selected as the optimal reaction temperature for the subsequent experiments.

[0070] 3. Test of TMB concentration

[0071] 20 μL of uric acid with a concentration of 5 mmol / L and 40 μL of uricase with a concentration of 5 mg / mL were added to a centrifuge tube, and incubated at 37 °C for 15 min to produce H2O2, then 100 μL of NaAc-HAc buffer solution with a concentration of 20 mmol / L and pH of 3.8, 30 μL of TMB solution with a concentration of 0-24 mmol / L, 6 μL of Ce-PODs nanoscale enzyme solution with a concentration of 1 mg / mL, and the rest was filled with deionized water, with a final volume of 400 μL, and reacted at 25 °C for 25 min. At this time, the absorbance of the system at 652 nm corresponds to A, and the absorbance of the solution with a uric acid concentration of 0 corresponds to A0. The curve of ΔA (A-A0) of the system at 652 nm as the ordinate and the TMB concentration as the abscissa, see Figure 7 , presents a typical Michaelis-Menten trend: reaching a maximum at a final concentration of 0.48 mmol / L, and then slightly decreasing, so that 0.48 mmol / L is finally selected as the optimal TMB concentration for subsequent uric acid detection experiments.

[0072] 4. Test of Ce-PODs nanoscale enzyme concentration

[0073] 20 μL of uric acid with a concentration of 5 mmol / L and 40 μL of uricase with a concentration of 5 mg / mL were added to a centrifuge tube, and incubated at 37 °C for 15 min to produce H2O2, then 100 μL of NaAc-HAc buffer solution with a concentration of 20 mmol / L and pH of 3.8, 30 μL of TMB solution with a concentration of 6.4 mmol / L, 6 μL of Ce-PODs nanoscale enzyme solution with a concentration of 0.3-2.3 mg / mL, and the rest was filled with deionized water, with a final volume of 400 μL, and reacted at 25 °C for 25 min. At this time, the absorbance of the system at 652 nm corresponds to A, and the absorbance of the solution with a uric acid concentration of 0 corresponds to A0. The curve of ΔA (A-A0) of the system at 652 nm as the ordinate and the Ce-PODs nanoscale enzyme concentration as the abscissa, see Figure 8 , reaches a maximum at a final concentration of 15 μg / mL, and then slightly decreases, so that 15 μg / mL is finally selected as the optimal Ce-PODs nanoscale enzyme concentration for subsequent uric acid detection experiments.

[0074] 5. Reaction of uric acid and uricase

[0075] The 20 μL of 5 mmol / L uric acid and 40 μL of uricase with different concentrations were added to the centrifuge tube and incubated at 37°C for 15 min to produce H2O2, followed by adding 100 μL of 20 mmol / L NaAc-HAc buffer solution with pH 3.8, 30 μL of 6.4 mmol / L TMB solution and 6 μL of 1 mg / mL Ce-PODs nanoscale enzyme solution, and the rest was filled with deionized water, with a final volume of 400 μL, and reacted at 25°C for 25 min. At this time, the absorbance of the system at 652 nm corresponds to A, and the absorbance of the solution with a uric acid concentration of 0 corresponds to A0. The ΔA of the system at 652 nm (A-A0) was taken as the vertical coordinate, and the uricase concentration was taken as the horizontal coordinate. The curve of ΔA changing with the uricase concentration is as shown in Figure 9 When the final concentration of uricase was 0.6 mg / mL, ΔA reached the maximum. Finally, 0.6 mg / mL of uricase was selected as the optimal concentration for the subsequent reaction.

[0076] 6. Determination of standard curve

[0077] By changing the concentration of uric acid (UA, purchased from China Macklin Biochemical Technology Co., Ltd.), the detection was carried out, and the final concentrations of TMB, uricase and Ce-PODs nanoscale enzyme solution were kept at 0.48 mmol / L, 0.6 mg / mL and 15 μg / mL, respectively. The change value ΔA of the absorbance of the detection system at 652 nm was detected. As shown in Figure 10 , in the range of 20-320 μmol / L, the uric acid concentration had a good linear relationship with the absorbance, ΔA = 0.0012 × C UA + 0.0552, R 2 = 0.9937, the detection limit was 2.60 μmol / L (3σ / k, n = 11), C UA was the UA concentration, and ΔA was the change value of absorbance (A-A0).

[0078] 7. Specificity test

[0079] Uric acid, NaCl, CaCl2, MgCl2, KCl, Na2SO4, (NH4)2SO4, urea, creatine and creatinine (all provided by China Macklin Biochemical Technology Co., Ltd.) were respectively prepared into solutions with a final concentration of 0.25 mmol / L. When preparing each solution, 6 μL of 1 mg / mL cerium-based nanoscale enzyme dispersion prepared in Example 1, 100 μL of 20 mmol / L, pH 3.8 HAc-NaAc buffer solution, TMB and uricase were taken, and deionized water was added to a total volume of 400 μL. The final concentration of TMB was 0.48 mmol / L, and the final concentration of uricase was 0.6 mg / mL. The reaction was carried out at 25 °C for 25 min, and the detection was carried out using a multifunctional microplate detector. The results are shown in Figure 11 Compared with uric acid, the absorbance of other interfering substances is very small, indicating that the method has good selectivity for the detection of uric acid.

[0080] 8. Visual detection

[0081] 30 μL of 1 mg / mL cerium-based nanoscale enzyme (Ce-PODs) dispersion, 150 μL of 6.4 mmol / L TMB solution, 500 μL of 20 mmol / L HAc-NaAc buffer solution and agarose were mixed, and deionized water was added to a total volume of 2000 μL. 170 μL of the above mixture was added to each well of a 96-well plate to prepare a hydrogel kit. 10 μL of different concentrations of uric acid and 20 μL of 6 mg / mL uricase were added to a centrifuge tube and incubated at 37 °C for 15 min to generate hydrogen peroxide (H2O2). The above 30 μL enzyme reaction solution was added to the hydrogel well of the 96-well plate, and Ce-PODs catalyzed H2O2 to generate ·OH free radicals. TMB in the well was oxidized by ·OH to generate oxTMB, so that the color of the gel changed from colorless to blue Figure 13 ). With the help of a smart phone application program based on the Android platform, the B value of the hydrogel well plate was extracted using the RGB channel algorithm. A calibration curve between the concentration of uric acid and the B value was established, as shown in FIG. 12, ΔB = 0.7657 × C UA +15.1273, R 2 = 0.9955, C UA is the concentration of UA, and ΔB is the change value of B (ΔB = B-B0, B0 is the B value when the concentration of uric acid is 0), which can realize the quantitative detection of uric acid. This method can realize the portable and visual detection of uric acid, and is suitable for on-site analysis of actual samples in different scenarios.

[0082] 9. Photothermal detection

[0083] The 20 μL of 5 mmol / L uric acid (UA) and 40 μL of 6 mg / mL uricase (UOx) were incubated at 37 °C for 15 min to generate hydrogen peroxide (H2O2); then 100 μL of 20 mmol / L HAc-NaAc solution with pH of 3.8, 6 μL of 1 mg / mL Ce-PODs and 30 μL of 6.4 mmol / L TMB solution were added, and the volume was made up to 400 μL with deionized water, and reacted at 25 °C for 25 min. The 808 nm laser was irradiated for 3 min, and the temperature value was measured by a handheld temperature measurement thermal imager. The T value was recorded, and the standard curve between the uric acid concentration and T was established, as shown in FIG. 14, ΔT = 0.1303 × C + 0.9903, R = 0.9954, C is the UA concentration, and ΔT is the temperature change value (ΔT = T-T0). The quantitative detection of uric acid can be achieved. UA + 0.9903, R 2 = 0.9954, C UA is the UA concentration, and ΔT is the temperature change value (ΔT = T-T0). The quantitative detection of uric acid can be achieved.

[0084] Example 7 Actual sample test

[0085] In order to evaluate the feasibility of the method in actual sample analysis, the urine sample was diluted 10 times and then determined, and the standard addition recovery experiment was carried out by using the absorbance method and the color B value method, in order to investigate the accuracy of the two methods established for detecting in urine samples. The results are shown in Table 1. As shown in Table 1, the relative standard deviation of the detection value of the absorbance method is ≤1.25 % (n=3), and the recovery rate is 98.13-105.08 %. The relative standard deviation of the detection value of the color B value method is ≤2.19 % (n=3), and the recovery rate is 91.16-104.29 %. The above results show that the colorimetric sensor based on cerium-based nanoscale enzyme has good accuracy and good application value in the detection method for uric acid in urine.

[0086] The test process is as follows:

[0087] 1. 20 μL of diluted urine sample and 40 μL of uricase with a concentration of 6 mg / mL were added to a centrifuge tube, and incubated at 37°C for 15 min to produce H2O2, followed by the addition of 100 μL of NaAc-HAc buffer solution with a concentration of 20 mmol / L and pH of 3.8, 30 μL of TMB with a concentration of 6.4 mmol / L, 6 μL of Ce-PODs nanoscale enzyme solution with a concentration of 1 mg / mL, and the rest was made up with deionized water, with a final volume of 400 μL, and reacted at 25°C for 25 min. At this time, the absorbance of the system at 652 nm corresponds to A, and the absorbance of the solution with a concentration of 0 of uric acid solution corresponds to A0. The standard curve was drawn using the concentration and absorbance change value of the uric acid standard solution, and then the concentration of the uric acid test solution was determined according to the absorbance change value of the uric acid test solution and the standard curve, and the concentration of uric acid in the uric acid test sample was determined.

[0088] 2. 10 μL of diluted urine sample and 20 μL of uricase with a concentration of 6 mg / mL were added to a centrifuge tube, and incubated at 37°C for 15 min, 30 μL of enzyme reaction solution was added dropwise to the hydrogel kit, and the B value of the hydrogel hole plate was extracted by means of the RGB channel algorithm of the Android platform smart phone application. The standard curve was drawn using the concentration and B change value of the uric acid standard solution, and then the concentration of the uric acid test solution was determined according to the B change value of the uric acid test solution and the standard curve, and the concentration of uric acid in the uric acid test sample was determined.

[0089] Table 1. Recovery rate of standard addition in human urine system

[0090]

Claims

1. A method for preparing a cerium-based nanozyme, wherein the cerium-based nanozyme catalyzes the oxidation of a chromogenic substrate by hydrogen peroxide in the quantitative analysis of uric acid, characterized in that... Includes the following steps: Step S1: Dissolve cerium salt and zinc salt in methanol at a molar ratio of cerium to zinc of 1:2~100 to obtain solution A; add 2-methylimidazole to methanol to obtain solution B; mix solution A and solution B with a molar ratio of cerium to 2-methylimidazole of 1:20 to obtain solution C. Step S2: The solution C is placed into a high-pressure reactor with a polytetrafluoroethylene substrate and reacted at 120-180 °C for 12-24 h. The solid is then separated to obtain the Ce-MOF precursor. Step S3: Place the Ce-MOF precursor in a tube furnace, heat it to 800-900℃ under an inert atmosphere, and calcine it for 2-4 h to obtain powder; Step S4: Add the powder to an acid solution and acid wash at 60~100℃ for 12~24 h, wash with water until neutral, and dry to obtain cerium-based nanozyme.

2. The method for preparing cerium-based nanozymes as described in claim 1, characterized in that, The cerium salt is one of cerium nitrate or its hydrate, cerium sulfate or its hydrate, or cerium chloride or its hydrate; the zinc salt is one of zinc nitrate or its hydrate, zinc chloride or its hydrate, or zinc sulfate or its hydrate; the cerium salt and zinc salt are dissolved in methanol at a molar ratio of cerium to zinc of 1:5; the acid solution is 2-3 mol / L hydrochloric acid or nitric acid; the powder is added to the acid solution and acid-washed at 80°C for 24 h.

3. A cerium-based nanozyme, wherein the cerium-based nanozyme catalyzes the oxidation of a chromogenic substrate by hydrogen peroxide in the quantitative analysis of uric acid, characterized in that... It is prepared by the method described in claim 1 or 2.

4. The application of the cerium-based nanozyme as described in claim 3, wherein after uric acid reacts with uricase to produce hydrogen peroxide, the cerium-based nanozyme catalyzes the hydrogen peroxide to produce reactive oxygen species, thereby oxidizing the chromogenic substrate into a colored substance, and then the content of uric acid is detected based on the quantitative relationship between the color, absorbance, or temperature of the colored substance and the uric acid.

5. The application as described in claim 4, characterized in that, The chromogenic substrate is 3,3',5,5'-tetramethylbenzidine or o-phenylenediamine. After the colored substance is generated, the uric acid content is detected by the linear relationship between the change in the B channel value of the uric acid solution relative to the reference solution and the uric acid concentration, or by the linear relationship between the change in the absorbance of the uric acid solution relative to the reference solution and the uric acid concentration, or by the linear relationship between the temperature change of the uric acid solution relative to the reference solution and the uric acid concentration after laser irradiation of the uric acid solution and the reference solution.

6. A method for detecting uric acid based on absorbance, characterized in that... Includes the following steps: Step S1: Uricase is added to several uric acid standard samples and uric acid test samples of different concentrations or volumes, and then reacted at 37℃ for 15 min. Then, NaAc-HAc buffer solution, 3,3'5,5'-tetramethylbenzidine and cerium-based nanozyme are added, and water is added to a certain volume to obtain uric acid standard solutions and uric acid test solutions of different concentrations. At this point, the pH value of the uric acid standard solutions and uric acid test solutions is 3.8, the concentration of 3,3'5,5'-tetramethylbenzidine is 0.48 mmol / L, the concentration of cerium-based nanozyme is 15 μg / mL, the concentration of uric acid is in the range of 20-320 μmol / L, the concentration of uricase is 0.6 mg / mL, and the operating temperature is 25–35 ℃. The cerium-based nanozyme is the cerium-based nanozyme described in claim 3. Step S2: Measure the absorbance changes of the uric acid standard solution and the uric acid test solution relative to the reference solution at 652 nm. Plot a standard curve using the uric acid concentration as the abscissa and the absorbance change as the ordinate, and then determine the concentration of the uric acid test solution and the concentration of uric acid in the uric acid test sample based on the absorbance change and the standard curve.

7. A method for detecting uric acid based on the B channel of RGB color, characterized in that... Includes the following steps: Step S1: Mix 30 μL of 1 mg / mL cerium-based nanozyme dispersion, 150 μL of 6.4 mmol / L TMB solution, 500 μL of 20 mmol / L HAc-NaAc buffer, and an appropriate amount of agarose. Adjust the volume to 2000 µL with deionized water to obtain the ready-to-use solution. Add 170 µL of the ready-to-use solution to each well of a 96-well plate. After solidification at room temperature, obtain a hydrogel kit. Take 10 µL of uric acid standard solutions and uric acid test solutions of different concentrations, add 20 µL of 6 mg / mL uricase solution, and incubate at 37°C for 15 min to obtain the enzyme reaction solution. Add 30 µL of the enzyme reaction solution to the wells of the 96-well hydrogel. The gel turns blue. The cerium-based nanozyme is the cerium-based nanozyme described in claim 3. Step S2: Use an Android smartphone APP to capture gel images; Image J extracts the B value of each well, plots a standard curve with uric acid concentration as the x-axis and the change in the B value of the uric acid test solution relative to the B value of the reference solution as the y-axis, and uses the concentration of the uric acid standard solution and the change in the B value to plot the standard curve. Then, the concentration of the uric acid test solution is determined based on the change in the B value of the uric acid test solution relative to the B value of the reference solution and the standard curve.

8. A temperature-based method for detecting uric acid, characterized in that... Includes the following steps: Step S1: Uricase is added to several uric acid standard samples and uric acid test samples of different concentrations or volumes, and then reacted at 37℃ for 15 min. Then, NaAc-HAc buffer solution, 3,3'5,5'-tetramethylbenzidine and cerium-based nanozyme are added, and water is added to a certain volume to obtain uric acid standard solutions and uric acid test solutions of different concentrations. At this point, the pH value of the uric acid standard solutions and uric acid test solutions is 3.8, the concentration of 3,3'5,5'-tetramethylbenzidine is 0.48 mmol / L, the concentration of cerium-based nanozyme is 15 μg / mL, the concentration of uricase solution is 0.6 mg / mL, and the operating temperature is 25–35 ℃. The cerium-based nanozyme is the cerium-based nanozyme described in claim 3. Step S2: Irradiate the uric acid standard solution, the uric acid test solution, and the reference solution with an 808 nm laser for 3 minutes. Measure the temperature change using a handheld thermal imager. Plot a standard curve using uric acid concentration on the x-axis and the temperature change of the uric acid standard solution relative to the reference solution on the y-axis. Then, determine the concentration of the uric acid test solution based on the temperature change of the uric acid test solution relative to the reference solution and the standard curve, and determine the concentration of uric acid in the uric acid test sample.

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