A colorimetric method for detecting uric acid and its application

Abstract

This invention provides a colorimetric detection method for uric acid and its application. Based on the synergistic effect of a defect-engineered manganese metal-organic framework material and an organic compound containing a primary amine group to enhance the sensitivity of uric acid colorimetric detection, a uric acid oxidation catalytic reaction is performed. The supernatant after the reaction can increase the response value of the "4-aminoantipyrine-sodium p-aminobenzenesulfonate" colorimetric system by 6-10 times. During the colorimetric process, no free hydrogen peroxide is generated by the manganese metal-organic framework material during uric acid oxidation. No additional peroxidase is required in the colorimetric reaction. The linear range of this detection method is 0.1-7.5 mg / dL (R0). 2 The detection limit is 0.08 mg / dL (P / N=23), and the cross-reactivity response to 15 blood interfering substances is <1.45%, making it suitable for routine biochemical detection of serum samples.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a colorimetric detection method for uric acid and its application. Background Technology

[0002] Traditional uric acid detection mainly relies on the uricase-peroxidase coupling method (Uricase-POD method). This method involves uricase specifically catalyzing the oxidation of uric acid to generate hydrogen peroxide (H2O2), which is then catalyzed by peroxidase (such as horseradish peroxidase HRP) to oxidize the chromogenic substrate (such as 3,3',5,5'-tetramethylbenzidine TMB, o-phenylenediamine OPD, etc.) to produce a detectable colorimetric signal. Despite its good specificity, this method faces three major bottlenecks in clinical application: ① Enzyme stability defects: Uricase activity decreases significantly when stored at 4℃, and the effective shelf life is usually no more than 3 months, leading to increased batch-to-batch variability and reduced reproducibility of test results; ② Difficulty in cost control: Enzyme preparations (uricase + peroxidase) account for more than 60% of the total test cost and require cold chain transportation and storage throughout the process, significantly raising the implementation threshold for primary healthcare institutions; ③ High operational complexity: Precise control of the dual-enzyme cascade reaction conditions (temperature 37±0.5℃, pH 7.8±0.2) is required, and an additional peroxidase catalytic colorimetric step is added, increasing the risk of operational errors.

[0003] Metal-organic frameworks (MOFs), as emerging nanozymes, have shown potential in addressing the stability and cost issues of natural enzymes due to their tunable pore structure, high specific surface area, and room-temperature synthesis characteristics. For example, iron-based MOFs (such as MIL-100(Fe)) can achieve uric acid oxidation through biomimetic catalysis, avoiding the high cost of uricase. However, existing MOF nanozymes still have fundamental limitations in uric acid detection: ① H2O2 pathway dependence: The catalytic process still requires the generation of H2O2 intermediates, necessitating indirect quantification of uric acid through a colorimetric reaction catalyzed by peroxidase, failing to overcome the inherent defects of enzymatic methods; ② Insufficient anti-interference performance: High concentrations of reducing substances in blood easily compete with the colorimetric system for reaction, leading to increased false positive rates and deterioration of the specificity of clinical sample detection; ③ Limited catalytic efficiency: The low accessibility of active sites and high mass transfer resistance of MOFs result in slow uric acid oxidation kinetics, making it difficult to meet the high-sensitivity detection requirements of trace uric acid samples (such as serum from children / nephropathy patients), with detection limits generally higher than 0.5 mg / dL.

[0004] In summary, developing a non-natural enzyme-catalyzed uric acid detection method that combines H2O2-free pathway (direct catalytic colorimetric development), high anti-interference ability (tolerance to complex matrices), and ultra-high sensitivity (detection limit ≤0.1mg / dL) is a key innovative direction to overcome the clinical translation bottleneck of existing uric acid detection technologies. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a highly sensitive colorimetric detection method for uric acid.

[0006] Another technical problem to be solved by the present invention is to provide an application of the above-mentioned highly sensitive colorimetric detection method for uric acid.

[0007] Another technical problem to be solved by the present invention is to provide a uric acid detection kit that uses the above-described highly sensitive colorimetric uric acid detection method.

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

[0009] A highly sensitive colorimetric method for detecting uric acid, the specific steps of which are as follows:

[0010] (1) The uric acid sample to be tested, the defect-engineered manganese metal-organic framework material, and the organic compound solution containing primary amine groups are added to the reaction system in sequence, and after thorough mixing, the uric acid oxidation reaction is carried out;

[0011] (2) After the oxidation reaction is completed, the defective engineered manganese metal-organic framework material is removed by centrifugation, and the supernatant is collected;

[0012] (3) Dissolve 4-aminoantipyrine and phenolic chromogen in phosphate buffer solution to prepare a colorimetric solution;

[0013] (4) Mix the supernatant obtained in step (2) with the colorimetric solution obtained in step (3) to carry out the colorimetric reaction;

[0014] (5) Measure the absorbance of the mixed solution after the colorimetric reaction, and calculate the concentration of uric acid in the sample to be tested based on the absorbance.

[0015] In the above-mentioned highly sensitive colorimetric detection method for uric acid, the introduction of the organic compound containing a primary amine group can increase the colorimetric response value by more than 6 times; the linear range of the method is 0.1-7.5 mg / dL, and the linear correlation coefficient R0 is [missing value]. 2 It has a value of ≥0.99, a cross-reactivity rate of less than 1.45% with 15 blood interfering substances, and no peroxidase is involved throughout the process.

[0016] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the defect-engineered manganese metal-organic framework material is an aqueous solution of manganese MOFs material. The preparation method of manganese MOFs material can be found in Chinese Patent CN120005219B (publication date: August 29, 2025, "Manganese Metal-Organic Framework Material with Oxidase Properties and its Preparation Method and Application").

[0017] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the preparation of the defect-engineered manganese metal-organic framework material further includes a post-processing step based on the above preparation method: repeatedly washing the obtained manganese metal-organic framework material with ultrapure water by shaking; after washing 3 times, starting from the 4th wash, centrifuging to collect the supernatant, the centrifugation conditions are 4200 rpm and the centrifugation time is 2 min.

[0018] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, in step (1), the final concentration of uric acid sample in each 1 mL reaction system is 0.1-7.5 mg / dL, the final content of defect-engineered manganese metal-organic framework material is 100 μg, and the final concentration of organic compound containing primary amine group is 10 mM.

[0019] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the organic compound containing a primary amine group is an amino acid or an alkylamine.

[0020] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the amino acid is arginine, and when its concentration is 5-15 mM, the response value is increased by 6-10 times, and the signal-to-noise ratio (P / N) is 16-23.

[0021] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the amino acid is arginine, and when its concentration is 10 mM, the response value is increased by 10 times, and the signal-to-noise ratio (P / N) is 23.

[0022] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the alkylamine is methylamine, and when its concentration is 5-15 mM, the response value is increased by 6-8 times, and the signal-to-noise ratio (P / N) is 16-20.

[0023] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the reaction time for uric acid oxidation in step (1) is 2-5 min.

[0024] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the reaction time for uric acid oxidation in step (1) is 3 minutes.

[0025] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the centrifugation procedure in step (2) is a rotation speed of 12000-14000 rpm and a centrifugation time of 2-5 min.

[0026] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the centrifugation procedure in step (2) is a rotation speed of 14000 rpm and a centrifugation time of 2 min.

[0027] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the colorimetric solution in step (3) contains 1-3 mM 4-aminoantipyrine and 6-18 mM phenolic chromogen.

[0028] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the colorimetric solution in step (3) contains 2 mM 4-aminoantipyrine and 12 mM phenolic chromogen (the final concentration ratio of 4-aminoantipyrine to phenolic chromogen is 1:6).

[0029] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the phenolic chromogen in step (3) is sodium p-aminobenzenesulfonate, which together with 4-aminoantipyrine forms a quinone imine dye.

[0030] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the concentration of the phosphate buffer solution in step (3) is 0.1M and the pH is 7.2.

[0031] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the volume ratio of the supernatant to the colorimetric solution in step (4) is 5:1.

[0032] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the reaction temperature in step (4) is 37 degrees Celsius and the reaction time is 15-30 minutes.

[0033] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the reaction temperature in step (4) is 37 degrees Celsius and the reaction time is 20 minutes.

[0034] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the wavelength range for measuring the absorbance after the colorimetric reaction in step (5) is 480-520 nm.

[0035] Preferably, in the above-mentioned highly sensitive colorimetric detection method for uric acid, the wavelength for measuring the absorbance after the colorimetric reaction in step (5) is 510 nm.

[0036] The application of the above-mentioned highly sensitive colorimetric detection method for uric acid in the detection of human serum samples.

[0037] The application of the above-mentioned highly sensitive colorimetric detection method for uric acid in uric acid sample detection.

[0038] Preferably, in the above application, the uric acid detection method has a linear range of 0.1-7.5 mg / dL for the detection of standard uric acid solutions (R0). 2 ≥0.99).

[0039] Preferably, in the above application, the uric acid detection method is applied to 15 blood interfering substances with a cross-reactivity of <1.45%, wherein the 15 blood interfering substances are D-glucose, inositol, uracil nucleoside, adenine sulfate, folic acid, biotin, vitamin B1, sodium chloride, galactose, ribose, sucrose, sorbitol, urea, cholesterol, and triglycerides.

[0040] Preferably, in the above application, the detection limit of the uric acid detection method is 0.08 mg / dL (P / N=23).

[0041] Application of the above-mentioned highly sensitive colorimetric detection method for uric acid in the detection kit.

[0042] A uric acid detection kit comprises: a defect-engineered manganese metal-organic framework material; an organic compound solution containing a primary amine (preferably an arginine solution) with a final concentration of 5-15 mM; and a chromogenic solution containing 4-aminoantipyrine and phenolic chromogens in a buffer solution.

[0043] The organic compound solution containing primary amines, in synergy with defect-engineered manganese metal-organic framework materials, improves detection sensitivity by 6-10 times.

[0044] The beneficial effects of this invention are:

[0045] The aforementioned colorimetric detection method for uric acid, through material design and process optimization, leverages the synergistic effect of defect-engineered manganese metal-organic framework materials and organic compounds containing primary amine groups to enhance the sensitivity of uric acid colorimetric detection. Material characterization confirms that the defect-engineered manganese metal-organic framework material possesses near-high-density atomic-level defects, enabling it to synergistically catalyze the uric acid oxidation reaction with organic compounds containing primary amine groups (preferably arginine). The supernatant after the reaction can increase the response value of the "4-aminoantipyrine-sodium p-aminobenzenesulfonate" colorimetric system by 6-10 times. This colorimetric process differs from the traditional hydrogen peroxide colorimetric pathway; the manganese metal-organic framework material does not generate free hydrogen peroxide during uric acid oxidation, and no additional peroxidase (such as horseradish peroxidase) is required in the colorimetric reaction. The linear range of this detection method is 0.1-7.5 mg / dL (R0). 2 The detection limit is 0.08 mg / dL (P / N=23), with a cross-reactivity of <1.45% to 15 blood interfering substances, making it suitable for routine biochemical testing of serum samples. Specifically: First, non-enzymatic catalytic materials replace traditional biological enzyme systems, completely solving the preservation and cost problems caused by insufficient enzyme stability, while avoiding the need for complex multi-enzyme reaction condition control; second, a direct oxidation colorimetric pathway is established, completely eliminating dependence on hydrogen peroxide intermediates and eliminating detection errors introduced by peroxidase at the source; third, the introduction of a primary amine-containing organic compound synergist significantly enhances the colorimetric signal intensity, and combined with a solid-liquid separation step, efficiently eliminates interference from complex matrices, greatly improving detection accuracy; fourth, the overall process is simple and efficient, with smooth transitions between reaction and colorimetric steps, meeting the requirement for rapid detection within 30 minutes; finally, this method has excellent clinical applicability and can be stably applied to the detection of various biological samples, providing a reliable and easily promoted solution for uric acid analysis. Attached Figure Description

[0046] Figure 1 SEM images of the defective engineered manganese-based material in the high-sensitivity colorimetric detection system for uric acid constructed in this invention: (a) is a 100μm scale SEM image, and (b) is a 5μm scale SEM image.

[0047] Figure 2 Characterization images of the defect-engineered manganese-based material in the high-sensitivity colorimetric detection system for uric acid constructed in this invention: (a) is a Raman spectrum, and (b) is an X-ray diffraction pattern.

[0048] Figure 3 The HPLC result shows that the defective engineered manganese-based material in the high-sensitivity colorimetric detection system for uric acid constructed in this invention did not produce hydrogen peroxide when applied to the oxidation of uric acid.

[0049] Figure 4 The effects of different compounds on a colorimetric detection system for uric acid based on defect-engineered manganese-based materials.

[0050] Figure 5 Thermographs showing the synergistic effect of different amounts of defect-engineered manganese-based materials (Mn-MOF-Ox) and different concentrations of arginine.

[0051] Figure 6 The following graphs illustrate the specificity and accuracy evaluation of the highly sensitive colorimetric uric acid detection system constructed in this invention: (a) shows the specificity of the colorimetric uric acid detection system, and (b) shows the accuracy of the colorimetric uric acid detection system.

[0052] Figure 7 The diagram shows the detection range of the highly sensitive colorimetric uric acid detection system constructed in this invention. Detailed Implementation

[0053] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0054] Example 1: Preparation of Defect-Enhanced Manganese Metal-Organic Framework Materials

[0055] The preparation method of the manganese metal-organic framework base material (hereinafter referred to as "basic manganese MOFs") is described in Chinese Patent CN120005219B. According to the method described in the embodiments of that patent, manganese MOF precursors are obtained through a solvothermal synthesis technique using 1,3,5-pyromellitic tricarboxylic acid as an organic ligand, manganese acetate tetrahydrate as a manganese source, and water-ethanol solution as a solvent. The manganese MOF precursor is then post-modified with an oxidant, which is a mixed solution of an alkali metal hydroxide aqueous solution and H2O2. The ratio of the manganese MOF precursor to the alkali metal hydroxide is (400–600) mg:(0.5–2) mmol (equivalent to a concentration of 4–6 mg / mL:5–20 μmol / mL), resulting in a manganese metal-organic framework material with oxidase properties. After all the steps of the above-described basic manganese MOF preparation method are completed, the following post-processing is performed to obtain the defect-engineered manganese metal-organic framework material (hereinafter referred to as "defective manganese MOFs") used in this invention:

[0056] The above-mentioned basic manganese MOFs material was repeatedly washed with ultrapure water by shaking. After washing three times, starting from the fourth wash, the supernatant was collected by centrifugation at 4200 r / min for 2 min to obtain the defect-engineered manganese metal-organic framework material.

[0057] Example 2 Morphology and structural characterization of defect-engineered manganese metal-organic framework materials

[0058] The morphology of the material obtained in Example 1 was characterized using scanning electron microscopy, and the results are shown in the figure. Figure 1 .Depend on Figure 1 It can be seen that the material has an irregular morphology and a large number of defects on its surface. Figure 1 (a) is a scanning electron microscope image under a 100 μm scale, and (b) is a scanning electron microscope image under a 5 μm scale.

[0059] The structure of the material obtained in Example 1 was characterized by Raman spectroscopy and X-ray diffraction, and the results are shown in the figure. Figure 2 Raman spectroscopy ( Figure 2 As shown in (a), compared with the manganese metal-organic framework based material with a crystalline structure (the material prepared in Chinese Patent CN120005219B, denoted as Mn-MOF), the defect-engineered manganese metal-organic framework material obtained in Example 1 (denoted as Mn-MOF-Ox) exhibits only one characteristic peak with a low peak intensity, indicating that its structure is disordered, containing only metal-oxygen bonds, and exhibiting amorphous characteristics. X-ray diffraction pattern ( Figure 2As shown in (b), compared with the standard cards of Mn-MOF and C4H6MnO4·4H2O, Mn-MOF-Ox has a lower baseline bulge and a lower main peak intensity, which further confirms that it has obvious amorphous characteristics, high density of surface defects and metal vacancies, forming abundant coordination unsaturated sites.

[0060] Example 3: High-performance liquid chromatography verification of uric acid oxidation products

[0061] The product of uric acid oxidation by the defect-engineered manganese metal-organic framework material in Example 1 was verified by high performance liquid chromatography (HPLC). Chromatographic conditions: TTOSOX C18 column (250 mM × 4.6 mM, particle size 5 μm), column temperature 37 °C, mobile phase 90:10 (methanol to triethylamine phosphate solution, with 8 mL H3PO4 added to 1000 mL pure water and pH adjusted to 2.0 with triethylamine), flow rate 0.3 mL / min, injection volume 20 μL, and detection at 210 nm using a diode array detector.

[0062] Sample solution preparation: Take 5 mg of the defect-engineered manganese metal-organic framework material prepared in Example 1, mix it with 2 mL of uric acid solution (500 μmol / L, pH 6), and react with shaking at room temperature for 1 hour. Then, centrifuge at 14,000 r / min for 2 minutes and take the supernatant for testing.

[0063] Preparation of standard solutions: Allantoin solution (500 μmol / L, pH 6), hydrogen peroxide solution (500 μmol / L, pH 7), and uric acid solution (500 μmol / L, pH 6) were respectively taken, shaken at room temperature for 1 hour, centrifuged at 14,000 r / min for 2 minutes, and the supernatant was collected for testing. Three parallel samples were prepared for each solution.

[0064] Test results are shown Figure 3 The results showed that allantoin standard eluted at a retention time of 6.0 min, hydrogen peroxide standard at 5.8 min, and uric acid standard at 10.7 min. Only peaks at retention times of 6.0 min and 10.7 min appeared in the sample solution, indicating that the manganese metal-organic framework material oxidized uric acid to produce only allantoin and not hydrogen peroxide (elution time 5.8 min).

[0065] Example 4: A Highly Sensitive Colorimetric Detection Method for Uric Acid Based on Defect-Engine-Enhanced Manganese Metal-Organic Framework Materials and Arginine

[0066] A colorimetric method for detecting uric acid concentration without the involvement of peroxidase, the specific steps of which are as follows:

[0067] In a 1 mL reaction system, using 100 mM phosphate buffer (pH=6) as the reaction medium, the sample to be tested, 100 μg of the defect-engineered manganese metal-organic framework material prepared in Example 1, and an organic compound containing a primary amine group were added sequentially. The organic compound containing a primary amine group used in this example was L-arginine, with a final concentration of 10 mM. The final concentration of uric acid in the system was adjusted to range from 0.1 to 7.5 mg / dL.

[0068] The above mixture was reacted at room temperature for 3 minutes to complete the uric acid oxidation process. After the reaction was completed, the reaction solution was centrifuged at 14,000 r / min for 2 minutes to remove the solid material and collect the supernatant.

[0069] Take 100 μL of the supernatant and mix it with 500 μL of phosphate chromogenic buffer containing 4-aminoantipyrine (2 mM) and sodium p-aminobenzenesulfonate (12 mM). Incubate at 37 °C for 20 minutes to develop the color. Measure the absorbance of the chromogenic solution at 510 nm and calculate the concentration of uric acid in the sample according to the pre-established standard curve.

[0070] The results show that the introduction of amino acid-based organic amines in the method described in this embodiment increases the colorimetric response value by more than 6 times, and no peroxidase is involved in the entire process.

[0071] Example 5: Synergistic activity of different compounds in the colorimetric detection of uric acid based on defect-engineered manganese metal-organic frameworks

[0072] A peroxidase-free colorimetric method for uric acid concentration detection was developed, and the synergistic activity of compounds containing different functional groups for uric acid colorimetric detection was systematically investigated. The specific steps are as follows:

[0073] In a 1 mL reaction system, using 100 mM phosphate buffer (pH=6) as the reaction medium, uric acid standard (final concentration 7 mg / dL), 100 μg of the defect-engineered manganese metal-organic framework material prepared in Example 1, and the organic amine compound to be tested were added sequentially. The following experimental groups were set up: blank control group (with an equal volume of PBS), L-aspartic acid, L-asparagine, L-glutamic acid, L-glutamine, oxaloacetic acid, L-alanine, L-serine, glycine, acetic acid, methylamine hydrochloride, ammonium sulfate, and ammonium chloride. The final concentrations of all amino acids, oxaloacetic acid, acetic acid, methylamine hydrochloride, ammonium sulfate, and ammonium chloride were all 10 mM.

[0074] The above mixture was reacted at room temperature for 3 minutes to complete the uric acid oxidation process. After the reaction was completed, the reaction solution was centrifuged at 14,000 r / min for 2 minutes to remove the solid material and collect the supernatant.

[0075] Take 100 μL of the supernatant and mix it with 500 μL of phosphate chromogenic buffer containing 4-aminoantipyrine (2 mM) and sodium p-aminobenzenesulfonate (12 mM). Incubate at 37 °C for 20 minutes to develop the color. Measure the absorbance of each chromogenic solution at 510 nm. The experimental results are shown in [Figure number missing]. Figure 4 .

[0076] The results showed that the blank control group (PBS) showed only weak color development. Neutral amino acids with free α-primary amine structures (alanine, glycine) and methylamine hydrochloride showed extremely strong synergistic effects, with color response values ​​significantly higher than the blank group. Acidic amino acids (aspartic acid, glutamic acid) showed only very weak synergistic effects, with color response values ​​close to those of the blank group. Although amide derivatives (asparagine, glutamine) retained the free α-primary amine structure, their side chains were amino groups bound by secondary amide bonds, showing only moderate synergistic activity, weaker than neutral α-primary amine amino acids without side chain modification. Oxaloacetic acid and acetic acid without amino groups, as well as inorganic ammonium salts (ammonium sulfate, ammonium chloride), showed no synergistic effect and even showed slight signal inhibition. Serine with hydroxyl groups showed extremely weak synergistic effects, indicating that hydroxyl groups have no significant synergistic activity. The above results confirm that the free α-primary amine structure is the core functional group for achieving colorimetric signal amplification, while acidic carboxyl groups, side-chain amide groups, and hydroxyl groups cannot effectively replace this function, and inorganic ammonium salts also have no synergistic activity.

[0077] Example 6: Optimization of a Highly Sensitive Colorimetric Detection Method for Uric Acid Based on Defect-Engineered Manganese Metal-Organic Framework Materials and Synergistic Organic Amines

[0078] Based on the method described in Example 4, the effects of the amount of manganese metal-organic framework material (0, 12.5, 25.0, 37.5, 50.0, 62.5, 75.0, 100, 125 μg) and the concentration of L-arginine (0, 2.5, 5.0, 7.5, 10, 15, 20, 30 mM) on the colorimetric response value (signal-to-noise ratio P / N value) were investigated, and a thermodynamic diagram was constructed.

[0079] See results Figure 5 In a 1 mL detection system, when the amount of defect-engineered manganese metal-organic framework material was 100 μg and the concentration of L-arginine was 10 mM, the response value of the detection system was good, and the signal-to-noise ratio reached P / N=23. This was used as the optimal condition for subsequent experiments.

[0080] Example 7 Evaluation of the specificity and accuracy of the detection method

[0081] The specificity and accuracy of the detection method described in Example 4 were evaluated using the optimized conditions of Example 6. The responses of uric acid and 15 common blood interfering substances (D-glucose, inositol, uracil nucleoside, adenine sulfate, folic acid, biotin, vitamin B1, sodium chloride, galactose, ribose, sucrose, sorbitol, urea, cholesterol, and triglycerides) were evaluated. For specificity evaluation, all 16 substances were used at a concentration of 7 mg / dL (0.42 mM). For accuracy evaluation, the 15 interfering substances were added simultaneously to the reaction solution at a 10-fold concentration (4.2 mM) with uric acid (0.42 mM), and the colorimetric response values ​​were measured. Three parallel samples were prepared for each interfering substance.

[0082] See results Figure 6 . Figure 6 As shown in (a), this method has high specificity for uric acid; Figure 6 As shown in Figure (b), the cross-reaction response values ​​of each interfering substance are all below 1.45%, indicating that the method has good accuracy.

[0083] Example 8: Evaluation of the linear range of the detection method

[0084] The linear range of the detection method described in Example 4 was evaluated using the optimized conditions of Example 6. The sensitivity and dynamic measurement range of the detection system were evaluated using uric acid standard solutions. Under the optimized conditions, the absorbance of uric acid standard solutions of different concentrations was measured.

[0085] See results Figure 7 As uric acid concentration increases, the absorbance at 510 nm gradually increases. Within the concentration range of 0.1-7.5 mg / dL, absorbance shows a good linear relationship with uric acid concentration, with a linear regression equation of y = 2.1130x + 0.0566 and a correlation coefficient R0. 2 =0.993.

[0086] Example 9: Evaluation of the blank limit and detection limit of the detection method

[0087] Using the optimized conditions of Example 6, the blank limit and detection limit of the detection method described in Example 4 were evaluated. Blank limit (LoB) evaluation: Ten blank samples (phosphate buffer) were taken and tested continuously for 3 days using two batches of reagents, with two replicates for each sample, resulting in 60 test results per batch. The test results did not follow a normal distribution, and non-parametric statistical methods were used. The test results were sorted from smallest to largest and labeled X1-X60. The probability of a test result exceeding LoB was preset to 5%. The ranking position corresponding to this 5% probability and the LoB calculation formula are as follows: LoB is the maximum value of the two batches of results.

[0088] Grade position = 0.5 + 60 × (1 - 5%) Equation (1)

[0089] LoB = 0.5 × (X58 + X57) Equation (2)

[0090] X 58 X is the value for the 58th position. 57 This is the value for the 57th position.

[0091] The 60 test results were arranged in descending order, and the rank position was calculated using formula (2) as 57.5. Formula 2 was used to calculate the LoB value, which was the median between the 57th and 58th ranked test results. Table 1 shows that the LoB value of the first batch of reagents was 0.0057 mg / dL, and the LoB value of the second batch was 0.0122 mg / dL. The largest LoB value was taken as the final calculation result. Considering all factors, the result was rounded to two decimal places, so the final LoB was determined to be 0.01 mg / dL.

[0092] Table 1 LoB Detection

[0093]

[0094] Limit of Detection (LoD) Assessment: Five low-concentration uric acid solution samples were collected, with concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 mg / dL, respectively. Two batches of reagents were used for continuous testing over three days. Each sample was tested in duplicate, with two replicates per batch, resulting in a total of 60 test results per batch. The formula is as follows:

[0095] LoD = LoB + Cp × SD L Equation (3)

[0096] Calculate the SD of 5 low-level samples L :

[0097] Equation (4)

[0098] In the formula, L represents the total number of test results for each batch, and SD L SD of the i-th low-level sample L , where n is the number of detection results for the i-th low-level sample, J is the number of low-level samples, J=5, L=45.

[0099] If the probability of a detection result lower than that of LoB in the LoD evaluation is set to 5%, then:

[0100] Equation (5)

[0101] In the formula, the value 1.645 represents the 95th percentile of the normal distribution when a=0.05. Calculate the LoD for each batch of reagents following the steps above, and take the maximum LoD from the two batches.

[0102] SD L The value is calculated according to Equation 4. The first batch of reagents SD L =0.1938, SD of the second batch of reagents L =0.0374. Assuming the probability of the test result being below the blank limit is 5%, the Cp value for both batches of reagents is calculated to be 1.6534 according to Equation 5. The LoD value, already evaluated in the above steps, is 0.0122 mg / dL. Therefore, the LoD of the 120 samples obtained (see Table 2) is calculated according to Equation 3. It can be seen that the LoD result of the first batch of reagents is 0.0758 mg / dL, and the LoD of the second batch of reagents is 0.0741 mg / dL. The larger LoD value is taken as the final calculation result. Taking all factors into consideration, the result is rounded to two decimal places, so the final LoD is determined to be 0.08 mg / dL.

[0103] Table 2 LoD Detection

[0104]

[0105] Example 10: Evaluation of the application of the detection method in serum samples

[0106] The ability of the detection method to quantitatively detect uric acid in serum samples was evaluated using the optimized conditions of Example 6. Different concentrations of uric acid (0 mg / dL, 2 mg / dL, 3 mg / dL, 4 mg / dL, 5 mg / dL, 6 mg / dL) were added to porcine serum. The baseline uric acid concentration was determined using a commercially available uric acid quantitative detection kit, and a standard curve was plotted using this method to determine the spiked concentration. Three replicates were performed for each sample, and the spiked recovery rate and intra-assay relative standard deviation (RSD) were calculated. The results are shown in Table 3.

[0107] Table 3 Spiked recovery experiment of uric acid in porcine serum

[0108]

[0109] As shown in Table 3, the spiked recovery rate was 96.53%–103.54%, and the intra-assay RSD was 1.26%–5.61%. These results indicate that the uric acid quantitative detection system established in this invention has good accuracy and precision and can be applied to the quantitative detection of uric acid in serum samples.

[0110] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A highly sensitive colorimetric method for detecting uric acid, characterized in that: The specific steps are as follows: (1) The uric acid sample to be tested, the defect-engineered manganese metal-organic framework material, and the organic compound solution containing primary amine groups are added to the reaction system in sequence, and after thorough mixing, the uric acid oxidation reaction is carried out; (2) After the oxidation reaction is completed, the defective engineered manganese metal-organic framework material is removed by centrifugation, and the supernatant is collected; (3) Dissolve 4-aminoantipyrine and phenolic chromogen in phosphate buffer solution to prepare a colorimetric solution; (4) Mix the supernatant obtained in step (2) with the colorimetric solution obtained in step (3) to carry out the colorimetric reaction; (5) Measure the absorbance of the mixed solution after the colorimetric reaction, and calculate the concentration of uric acid in the sample to be tested based on the absorbance.

2. The highly sensitive colorimetric detection method for uric acid according to claim 1, characterized in that: The defect-engineered manganese metal-organic framework material is an aqueous solution of manganese MOFs material. The treatment steps are as follows: the manganese MOFs material is repeatedly shaken and washed with ultrapure water; after washing 3 times, starting from the 4th wash, the supernatant is collected by centrifugation at 4200 rpm for 2 min.

3. The highly sensitive colorimetric detection method for uric acid according to claim 1, characterized in that: In step (1), the final concentration of uric acid sample in each 1 mL reaction system is 0.1-7.5 mg / dL, the final content of defect-engineered manganese metal-organic framework material is 100 μg, and the final concentration of organic compound containing primary amine group is 10 mM.

4. The highly sensitive colorimetric detection method for uric acid according to claim 1 or 3, characterized in that: The organic compound containing a primary amine group is an amino acid or an alkylamine.

5. The highly sensitive colorimetric detection method for uric acid according to claim 1, characterized in that: The colorimetric solution in step (3) contains 1-3 mM 4-aminoantipyrine and 6-18 mM phenolic chromogens.

6. The highly sensitive colorimetric detection method for uric acid according to claim 1, characterized in that: In step (4), the volume ratio of the supernatant to the colorimetric solution is 5:

1.

7. The application of the highly sensitive colorimetric detection method for uric acid according to any one of claims 1-6 in the detection of human serum samples.

8. The application of the highly sensitive colorimetric detection method for uric acid according to any one of claims 1-6 in the detection of uric acid samples.

9. Application in the detection kit of the highly sensitive colorimetric detection method for uric acid according to any one of claims 1-6.

10. A uric acid detection kit for detection using the method described in any one of claims 1-6, characterized in that: Includes: defect-engineered manganese metal-organic framework material; organic compound solution containing primary amine: final concentration of 5-15 mM; chromogenic solution: buffer solution containing 4-aminoantipyrine and phenolic chromogens.

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