Fe / nitrogen co-doped carbon microspheres, preparation method thereof and application of Fe / nitrogen co-doped carbon microspheres in uric acid detection

Abstract

The invention discloses Fe / nitrogen co-doped carbon microspheres as well as a preparation method and application thereof in uric acid detection, and belongs to the technical field of analytical chemistry. The preparation method comprises the following steps: dissolving a carbon source in water, stirring, carrying out hydrothermal reaction, and washing and drying an obtained solid product to obtain carbon microspheres; the preparation method comprises the following steps: putting the carbon microspheres, ferric salt and a nitrogen source into water, stirring and drying, heating, cooling, washing and drying to obtain the Fe / nitrogen co-doped carbon microspheres. The linear response range of the prepared carbon microspheres to uric acid reaches 14-454 M. In an actual urine test, the adding standard recovery rate is stabilized at 95.5-102.2%, the relative standard deviation is lower than 2.5%, high-sensitivity and high-selectivity detection of uric acid can be realized, and a new thought is provided for developing a low-cost and high-performance enzyme-free biosensor. The uric acid detection method does not need biological enzyme participation, has the advantages of low cost, good stability, simplicity and convenience in operation and the like, and provides a new technical scheme for clinical uric acid detection.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to an Fe / nitrogen co-doped carbon microsphere, its preparation method, and its application in uric acid detection. Background Technology

[0002] Uric acid (UA) is the final product of purine metabolism in the human body. As an important biomarker, its concentration level is closely related to the diagnosis and monitoring of various diseases. Under normal physiological conditions, the concentration of uric acid in human blood is maintained in the range of 120-460 μM, while the concentration of uric acid in urine is 1.4-4.4 mM. When uric acid metabolism is imbalanced, it leads to hyperuricemia, which is a direct cause of gout, kidney stones, and renal dysfunction. Numerous clinical studies in recent years have shown that abnormal uric acid levels are also significantly correlated with chronic diseases such as cardiovascular disease, metabolic syndrome, and type 2 diabetes. Therefore, developing rapid, accurate, reliable, and cost-effective uric acid detection methods can serve as an early screening and warning tool for various human diseases.

[0003] Currently, commonly used methods for uric acid detection mainly include enzymatic methods, chromatography, electrochemical methods, capillary electrophoresis, fluorescence spectroscopy, and chemiluminescence. Enzymatic methods offer high selectivity, but the inherent instability of biological enzymes, high preparation and purification costs, stringent storage conditions (requiring low temperature and protection from light), and sensitivity to pH and temperature limit their application in resource-constrained environments. Chromatographic methods (such as high-performance liquid chromatography), while highly accurate and specific, require expensive equipment, specialized operators, and complex sample pretreatment procedures, making them unsuitable for rapid on-site detection. Electrochemical methods have attracted attention due to their high sensitivity, rapid response, and potential for instrument miniaturization; however, electrodes are susceptible to contamination and passivation by complex matrices in biological samples, resulting in poor reproducibility, and often require complex electrode modification processes. Capillary electrophoresis, fluorescence spectroscopy, and chemiluminescence methods suffer from complex sample pretreatment, complex detection systems, and poor selectivity.

[0004] In recent years, enzyme-free colorimetric sensing technology has shown great promise in the field of bioanalysis due to its advantages such as simple operation, low cost, and the ability to perform semi-quantitative analysis with the naked eye. These methods typically utilize the catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to produce a color, which is then removed by the reducing properties of the target analyte, allowing for quantitative detection via a UV-Vis spectrophotometer or a smartphone camera. To date, researchers have developed various materials with good activity for enzyme-free uric acid detection, including noble metal nanoparticles (such as Au and Ag), metal oxides (such as Co3O4 and MnO2), metal-organic frameworks (MOFs), and polymetallic oxomolybdates. However, these materials still face many challenges: noble metal nanomaterials are expensive and prone to aggregation; some metal-based materials may leach metal ions, causing biotoxicity and environmental problems; and the preparation processes of most materials are relatively cumbersome. Therefore, there is an urgent need to develop a new material that is low-cost, stable, and environmentally friendly to achieve enzyme-free uric acid detection. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides Fe / nitrogen co-doped carbon microspheres, their preparation method, and their application in uric acid detection.

[0006] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of this invention: a method for preparing Fe / nitrogen co-doped carbon microspheres, comprising the following steps: (1) Dissolve the carbon source in water, stir, and then carry out a hydrothermal reaction. The resulting solid product is washed with water and dried to obtain carbon microspheres. (2) The carbon microspheres, iron salt and nitrogen source obtained in step (1) are placed in water, stirred and dried, then heated, cooled, washed and dried to obtain the Fe / nitrogen co-doped carbon microspheres.

[0007] As a preferred embodiment of the present invention, in step (1), the carbon source includes soluble starch, and the ratio of the carbon source to water is 1 g: (8-12) mL; the stirring temperature is 70-90℃ and the time is 20-40 min; the hydrothermal reaction temperature is 180-220℃ and the time is 10-14 h; the drying is specifically vacuum drying at 70-90℃ for 4-6 h.

[0008] As a preferred embodiment of the present invention, in step (2), the iron salt is a trivalent iron salt, including one or more of ferric chloride, ferric sulfate, and ferric nitrate; the nitrogen source includes urea; the ratio of carbon microspheres, ferric chloride, urea, and water is 2g:1g:2g:(30-50 mL); the stirring time is 20-30 h; and the heating is carried out under an inert atmosphere at a rate of 5℃ / min. -1 Heat to 600–800℃ and hold for 1.5–2.5 h; after washing, vacuum dry at 50–70℃.

[0009] The second technical solution of the present invention: Fe / nitrogen co-doped carbon microspheres prepared according to the preparation method described above.

[0010] The third technical solution of the present invention: an enzyme-free colorimetric sensing platform for uric acid detection, containing the Fe / nitrogen co-doped carbon microspheres described above.

[0011] The fourth technical solution of the present invention: an enzyme-free colorimetric detection method for uric acid, using the Fe / nitrogen co-doped carbon microspheres or the enzyme-free colorimetric sensing platform described above for detection.

[0012] As a preferred embodiment of the present invention, the enzyme-free colorimetric detection method for uric acid includes the following steps: (a) Mix NaAc-HAc buffer, Fe / nitrogen co-doped carbon microspheres and TMB colorimetric solution, react them, and then filter to obtain the filtrate; (b) Add uric acid standard solutions of different concentrations to the filtrate obtained in step (a), react, and after the reaction, use an ultraviolet spectrophotometer to scan the absorption spectrum of the reaction system in the wavelength range of 400 to 800 nm. Finally, take the absorbance value at 652 nm and plot a standard working curve with the uric acid concentration. (c) Replace the uric acid standard solution in step (b) with the uric acid sample to be tested, and repeat steps (a) to (b) to obtain the uric acid concentration through the standard working curve.

[0013] As a preferred embodiment of the present invention, in step (a), the concentration of the NaAc-HAc buffer solution is 0.2 M, the pH is 4.0, and the concentration of the TMB chromogenic solution is 8 mmol / L; the volume ratio of the NaAc-HAc buffer solution, Fe / nitrogen co-doped carbon microspheres, and TMB chromogenic solution is 2.5 mL:3 mg:20 μL; the reaction temperature is 35°C and the time is 2 min; in step (b), the concentration range of the uric acid standard solution is 14–454 μmol / L, the reaction temperature is 25–35°C, and the time is 5 min.

[0014] Fifth technical solution of the present invention: An application of the Fe / nitrogen co-doped carbon microspheres described above in the preparation of an enzyme-free colorimetric sensing platform for the detection of uric acid in isolated urine.

[0015] As a preferred embodiment of the present invention, the isolated urine is diluted to a uric acid concentration range of 14–454 μmol / L before detection.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) In this invention, carbon microspheres are first prepared using a carbon source via a hydrothermal method, and then Fe / nitrogen co-doped carbon microspheres (Fe-N-CMSs) with porous structure, high specific surface area, and abundant active sites are prepared by co-pyrolysis of iron salt, nitrogen source, and carbon microspheres. This material can efficiently catalyze the generation of dissolved oxygen under mild conditions. O - 2 and 1 O2, O - 2 and 1 O2 oxidizes TMB to form blue oxTMB, which uric acid can reduce to colorless oxTMB, enabling the visualization and control of the colorimetric signal, resulting in a decrease in absorbance at 652 nm. The concentration of UA is then determined based on the decrease in absorbance.

[0017] (2) The Fe-N-CMSs prepared in this invention exhibit a linear response range of 14-454 μM (R²=0.9951) to uric acid. In actual urine tests, the spiked recovery rate remained stable at 95.5%-102.2% (n=3), with a relative standard deviation (RSD) of less than 2.5%, indicating that the material maintains high catalytic selectivity in complex biological matrices, and the detection process is minimally affected by matrix interference, exhibiting high sensitivity and accuracy, thus meeting the clinical analytical requirements for biological sample detection. Therefore, the Fe-N-CMSs of this invention can achieve highly sensitive and selective detection of uric acid, providing a new approach for developing low-cost, high-performance enzyme-free biosensors and promoting their practical application in the field of health monitoring.

[0018] (3) The uric acid detection method of the present invention does not require the participation of biological enzymes, and has the advantages of low cost, good stability and simple operation, providing a new technical solution for clinical uric acid detection. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1The IR spectrum of Fe-N-CMSs prepared in Example 1; Figure 2 The XRD pattern of Fe-N-CMSs prepared in Example 1; Figure 3 In the middle (a), (b), (c), and (d), respectively, are the XPS spectra of C 1s, Fe 2p, N 1s, and O1s of Fe-N-CMSs prepared in Example 1; Figure 4 In the middle (a) and (b), SEM images of Fe-N-CMSs prepared in Example 1 are shown at different magnifications. Figure 5 The adsorption isotherm of Fe-N-CMSs prepared in Example 1; Figure 6 The pore size distribution diagram of Fe-N-CMSs prepared in Example 1 is shown. Figure 7 The figure shows the results of the catalytic activity study of Fe-N-CMSs prepared in Example 1; Figure 8 The figure shows the effect of reaction time on the color development of the Fe-N-CMSs-catalyzed TMB system. Figure 9 The figure shows the effect of Fe-N-CMS dosage on the color development of the Fe-N-CMS catalyzed TMB system. Figure 10 The figure shows the effect of reaction temperature on the color development of the Fe-N-CMSs-catalyzed TMB system. Figure 11 The figure shows the effect of pH on the color development of the Fe-N-CMSs-catalyzed TMB system. Figure 12 The UV-Vis absorption spectra of Fe-N-CMSs-TMB at different concentrations of UA (14–454 μmol / L) are shown. Figure 13 This is the UA detection standard curve; Figure 14 Figure showing the selectivity results of the Fe-N-CMSs catalytic TMB system; Figure 15 Figure 1 shows the results of the stability study of the Fe-N-CMSs catalyzed TMB system. (a) and (b) show the colorimetric reaction results of four batches of Fe-N-CMSs materials prepared at the same time and the same batch of materials placed for different times, respectively. Figure 16 The experimental spectra of Fe-N-CMSs catalytic mechanism are shown. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0022] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] The raw materials used in the following examples are all commercially available and conventional, and are not particularly limited; room temperature refers to 25–35°C. These will not be repeated below.

[0027] Example 1 The preparation of Fe-N-CMSs is as follows: Fe-N-CMSs were prepared by hydrothermal and pyrolysis methods: 5.0 g of soluble starch was dissolved in 50 mL of deionized water and magnetically stirred in an 80 °C water bath for 30 minutes. The mixture was then poured into a high-pressure hydrothermal reactor and hydrothermally reacted in a 200 °C oven for 12 hours. The solid product was washed alternately with anhydrous ethanol and deionized water until neutral, and then dried in a vacuum oven at 80 °C for 5 hours to obtain carbon microspheres.

[0028] 5 g of carbon microspheres, 2.5 g of FeCl3·6H2O, and 5 g of urea were placed in 100 mL of purified water and magnetically stirred for 24 h, then dried in an oven at 80 °C. Finally, the product was transferred to a tube furnace and dried at 5 °C·min under a nitrogen atmosphere. -1 The temperature was increased to 700°C at a rising rate and held at this temperature for 2 hours, then cooled to room temperature. The resulting product was washed with deionized water and dried under vacuum at 60°C to obtain Fe-N-CMSs.

[0029] Structural characterization of Fe-N-CMSs 1. FT-IR analysis was performed on Fe-N-CMSs using KBr pellets. Figure 1 As shown, it can be seen at 3430cm -1 The broad peak at 1580 cm⁻¹ is the absorption peak of the stretching vibration of -OH. -1 The absorption peak for the stretching vibration of CN is at 1143 cm⁻¹. -1 The absorption peak at 686 cm⁻¹ is the stretching vibration absorption peak of C-OH. -1 The absorption peak at this point is the stretching vibration peak of Fe-O, indicating that Fe and N have been successfully incorporated into the carbon microsphere structure.

[0030] 2. Fe-N-CMS XRD patterns are shown below. Figure 2 As shown, Fe-N-CMSs exhibit a broad peak at approximately 26° 2θ, corresponding to the typical characteristics of amorphous carbon. Fe-N-CMSs also show diffraction peaks at 18°, 30°, 35°, 43°, 53°, 57°, and 62° 2θ, matching the standard Fe3O4 diffraction peaks. The corresponding crystal planes are (111), (220), (311), (400), (422), (511), and (440), consistent with the diffraction card in PDF#19-0629, verifying that it is an inverse spinel structure Fe3O4. The results indicate that the successfully prepared Fe-N-CMSs have Fe3O4 loaded on their surface. The sharp characteristic peaks at approximately 45° and 65° 2θ are consistent with Fe... 0 The positions of the characteristic diffraction peaks match. Furthermore, the sharp characteristic peak at approximately 26° corresponds to the characteristic diffraction peaks of graphitic carbon.

[0031] 3. XPS spectra of C 1s, Fe 2p, N 1s, and O 1s for Fe-N-CMSs are shown below. Figure 3As shown in (a), (b), (c), and (d), in the C 1s high-resolution spectrum, three peaks can be distinguished. The peak at 284.98 eV is the hydroxyl group (–OH), which is relatively strong. The peaks at 285.28 and 286.53 eV are COC and CN, respectively. In the N 1s high-resolution spectrum, there are pyridine nitrogen at 398.63 eV, pyrrolidine nitrogen at 400.43 eV, and graphitic nitrogen at 403.88 eV. In the O 1s high-resolution spectrum, three peaks can also be distinguished. The peak at 532.43 eV is the hydroxyl group (–OH), the peak at 531.23 eV is (Fe-O), and the relatively weak peak at 533.68 eV is a monocoordinate oxygen (C=O). Finally, in the Fe 2p high-resolution spectrum, the peak at 718.59 eV represents Fe. 0 The peak at 724.74 eV in 2p3 / 2 represents Fe. 2+ The peaks at 713.47 and 729.99 eV in 2p3 / 2 represent Fe, respectively. 3+ 2p1 / 2 and Fe 3+ 2p3 / 2.

[0032] 4. SEM images of Fe-N-CMSs at different magnifications are shown below. Figure 4 As shown in the figure, the Fe-N-CMSs samples exhibit a spherical shape, and Fe3O4 particles can be observed adhering to the surface of the Fe-N-CMSs.

[0033] 5. Adsorption isotherms and pore size distribution diagrams of Fe-N-CMSs are shown below. Figure 5 and Figure 6 As shown, the specific surface area, average pore size, and total pore volume of Fe-N-CMSs are 147.590 m² / g, 3.384 nm, and 0.4718 e cc / g, respectively, and Fe-N-CMSs exhibit mesoporous characteristics in the range of approximately 3 to 15 nm.

[0034] Effect verification 1. Catalytic activity of Fe-N-CMSs Experiment on Fe-N-CMSs catalyzing TMB: Five centrifuge tubes were filled with 2.5 mL of 0.2 M NaAc-HAc buffer solution (pH=4.0). Centrifuge tube 1 contained 200 μL H2O2; centrifuge tube 2 contained 200 μL H2O2 and 100 μL TMB; centrifuge tube 3 contained 100 μL TMB solution and 10 mg Fe-N-CMSs; centrifuge tube 4 contained 200 μL H2O2, 100 μL TMB, and 10 mg Fe-N-CMSs; and centrifuge tube 5 contained 200 μL of 200 mg·L⁻¹ TMB solution. -1UA solution, 100 μL TMB, and 10 mg Fe-N-CMSs were sonicated for 60 s and then reacted at 30 ℃ for 10 min. The test solution in the centrifuge tube was filtered through a 0.22 μm filter membrane, and the absorbance of the filtrate was measured at 652 nm using UV-Vis.

[0035] The results are as follows Figure 7 As shown in the figure, both the Fe-N-CMSs+TMB and Fe-N-CMSs+TMB+H2O2 systems exhibit significant absorption peaks at 652 nm, and the solution appears blue, indicating that Fe-N-CMSs can oxidize TMB even without H2O2. When only TMB and H2O2 are present in the system, no absorption peak or blue solution is observed at 652 nm, indicating that no oxidation reaction occurs. When UA is added to the Fe-N-CMSs+TMB system, the color fades significantly to become colorless, indicating that UA can reduce the oxidized TMB. These results demonstrate that Fe-N-CMSs possesses good catalytic activity, and the Fe-N-CMSs+TMB system can be used for UA detection.

[0036] 2. Effect of reaction time on Fe-N-CMSs catalyzed TMB system Five centrifuge tubes were labeled I to V. 2.5 mL of 0.2 M NaAc-HAc buffer solution (pH=4.0), 20 μL of 8 mM TMB solution, and 5 mg of Fe-N-CMSs were added to each tube. The tubes were then incubated in a 30℃ water bath at 140 rpm for 2 min, 5 min, 7 min, 10 min, and 12 min, respectively. The solutions were then filtered through a 0.22 μm filter membrane. The absorbance of the filtrate was measured using UV-Vis at 652 nm.

[0037] The results are as follows Figure 8 As shown in the figure, the labels on the test tubes indicate the reaction times. The absorbance of the reaction system first increases and then decreases with reaction time. This is because as time extends, TMB is over-oxidized, resulting in a decrease in absorbance and a change in solution color from blue to yellow-green. At a reaction time of 2 minutes, the absorbance is close to 1. An absorbance higher than 1 is not conducive to the reduction of low-concentration uric acid; therefore, a reaction time of 2 minutes was chosen.

[0038] 3. Effect of Fe-N-CMS dosage on Fe-N-CMS catalytic TMB system First, five centrifuge tubes were labeled I through V. In each tube, 2.5 mL of 0.2 M NaAc-HAc buffer (pH 4.0) and 20 μL of 8 mM TMB solution were added sequentially. Next, 1 mg, 3 mg, 5 mg, 7 mg, and 10 mg of Fe-N-CMSs were added to each tube. Finally, the tubes were placed in a 30°C water bath with a shaker. The shaker was set to 140 rpm and shaken for 2 minutes. Finally, the solutions in the centrifuge tubes were filtered through a 0.22 μm filter, and the absorbance was measured at 652 nm using a UV-Vis spectrometer.

[0039] The results are as follows Figure 9 As shown in the figure, the amount of Fe-N-CMSs added is marked on the test tube. The figure shows that the absorbance of the system increases with the increase of Fe-N-CMSs dosage. When the Fe-N-CMSs dosage is higher than 3 mg, the absorbance is higher than 1, and excessive oxidation occurs. Therefore, a dosage of 3 mg of Fe-N-CMSs was chosen.

[0040] 4. Effect of temperature on the catalytic activity of Fe-N-CMSs Five centrifuge tubes were labeled I through V. Each tube contained 2.5 mL of 0.2 M NaAc-HAc buffer solution (pH 4.0), 20 μL of 8 mM TMB solution, and 5 mg of Fe-N-CMSs. The tubes were placed in a water bath with a constant temperature shaker (T = 25–45 °C, with a 5 °C interval) and reacted for 2 min at 140 rpm. The centrifuge tubes were then filtered through a 0.22 μm filter membrane, and the absorbance of the filtrate was measured using UV-Vis at 652 nm.

[0041] The results are as follows Figure 10 As shown in the figure, the labels on the test tubes indicate the reaction temperatures. The figure shows that the absorbance is 0.787 at a reaction temperature of 35℃. As the reaction temperature continues to increase, the absorbance changes little, but the system becomes slightly greenish. Therefore, a reaction temperature of 35℃ was chosen.

[0042] 5. Effect of pH on the catalytic activity of Fe-N-CMSs Add 2.5 mL of 0.2 M NaAc-HAc buffer solution (pH = 3.0–7.0) at different pH values ​​to five centrifuge tubes, followed by 20 μL of 8 mM TMB solution and 3 mg of Fe-N-CMSs. Place the centrifuge tubes on a 35°C water bath shaker at 140 rpm for 2 min. Then, filter the solution in the centrifuge tubes through a 0.22 μm filter membrane, and measure the absorbance of the filtrate at 652 nm using UV-Vis.

[0043] The results are as follows Figure 11 As shown in the figure, the catalytic effect is optimal in a solution environment with a pH of around 4. Within the pH range of 5-7, the absorbance decreases as the pH increases, and the absorbance is highest at pH 4. Therefore, the pH value should be controlled at 4.

[0044] 6. UA Detection First, prepare UA standard solutions with concentrations ranging from 14 to 454 μmol / L. Then, mix 2.5 mL of 0.2 M NaAc-HAc buffer (pH=4.0), 3 mg of Fe-N-CMSs, and 20 μL of 8 mmol / L TMB colorimetric solution in a centrifuge tube. Place the mixture in a 35°C water bath at 140 rpm for 2 minutes. Filter the solution from the centrifuge tube using a 0.22 μm filter membrane. Add UA solutions of different concentrations to the filtered solution, mix well, and incubate at room temperature for 5 minutes. After the reaction is complete, scan the absorption spectrum of the reaction system in the wavelength range of 400 to 800 nm using a UV spectrophotometer. Finally, take the absorbance value at 652 nm and plot a standard curve against the UA concentration. The UV-Vis absorption spectra of Fe-N-CMSs-TMB at different UA concentrations (14–454 μmol / L) are shown below. Figure 12 As shown in the figure, the absorbance value gradually decreases with increasing UA concentration. The UA detection standard curve is shown below. Figure 13 As shown. When the UA concentration is in the range of 14–454 μmol / L, its linear equation is y = 2.02788x + 0.00283, and the correlation coefficient (R) is... 2 The absorbance was 0.9951, and the detection limit was 4.0 μmol / L. Where A0 is the absorbance of the system without UA, and A... n The absorbance of the system after adding UA solution is shown. The above results indicate that within a certain concentration range, the UA concentration is related to (A0-A...). n The relationship between A0 and A0 is linear.

[0045] 7. Selective Research Based on the potential interference of substances in urine on the Fe-N-CMSs / TMB colorimetric system, its selectivity was investigated, and the results are as follows: Figure 14 As shown in the figure, the concentration of UA is 454 μmol, and the concentrations of other substances are all 8 mM. The figure also shows that ascorbic acid (AA), urea (Urea), and medium-chain triglycerides (C...) are present in... 39 H 74 Substances such as O6, Trig, cholesterol (Chol), glucose (Glu), KCl, and NaCl did not significantly interfere with the colorimetric system, indicating that the Fe-N-CMSs / TMB colorimetric system has strong selectivity.

[0046] 8. Stability of Fe-N-CMSs To investigate the stability of Fe-N-CMSs, four batches of Fe-N-CMSs materials were prepared simultaneously, and a colorimetric reaction was carried out under optimal conditions. The results are as follows: Figure 15 As shown in (a), there was no significant difference in the catalytic performance of the four batches of materials. The same batch of materials was stored for 5 weeks, and the colorimetric performance was measured weekly. The results are as follows: Figure 15 As shown in (b), the catalytic performance did not decrease significantly. These results indicate that the prepared Fe-N-CMSs possess good stability.

[0047] 9. Experiments on the catalytic mechanism of Fe-N-CMSs Add 2.5 mL of 0.2 M NaAc-HAc buffer (pH=4.0) and 3 mg of Fe-N-CMSs to three centrifuge tubes, respectively. Then add 200 μL of tert-butanol (TBA), sugar alcohol (FFA), and 4 mg of p-benzoquinone (BQ), i.e., add different scavenging agents, and observe their effects on the catalytic effect of Fe-N-CMSs. TBA can eliminate ·OH, and BQ can eliminate ·O2. - FFA can eliminate 1 O2 was added, and finally the colorimetric reagent TMB was added. The mixture was stirred and reacted for 2 min. The solution in the centrifuge tube was filtered through a 0.22 μm filter membrane. The absorbance of the filtrate was measured using UV-Vis at 652 nm. Results were obtained from... Figure 16 As shown in the figure, the catalytic effect of Fe-N-CMSs decreased significantly when p-benzoquinone (QB) and sugar alcohol (FFA) were added, while the catalytic effect did not change much after the addition of tert-butanol (TBA). This indicates that the catalytic effect of Fe-N-CMSs decreased significantly when p-benzoquinone (QB) and sugar alcohol (FFA) were added. - and 1 O2 participated in the catalysis of Fe-N-CMSs in the reaction system, in which 1 O2 is the most important active species.

[0048] 10. Actual sample testing Three volunteers provided urine samples, which were diluted 10 times with deionized water before uric acid content was measured. To verify the accuracy of UA detection in urine samples, UA spiked and recovered at different concentrations was performed on the urine samples.

[0049] The uric acid (UA) levels in the urine of three healthy adult volunteers were measured in parallel using a spiked recovery experiment. The data are shown in Table 1. The UA concentrations in the volunteers' urine were 3.80, 2.57, and 3.07 mmol / L, respectively, all within the normal physiological reference range (1.4–4.4 mmol / L). Method validation results showed that the spiked recoveries for the three samples were 95.5%, 102.2%, and 98.9%, respectively, with relative standard deviations (RSD) less than 2.5%. These data confirm that this method exhibits ideal accuracy and reproducibility in urinary uric acid detection, fully meeting the requirements for clinical biosample analysis.

[0050] Table 1. Results of actual sample testing and spiked recovery experiments In summary, the Fe-N-CMSs prepared in this invention can be used for rapid, sensitive, and selective detection of uric acid. This material is synthesized via a green and low-cost method, exhibiting excellent catalytic activity and stability. Under optimal conditions, the detection system demonstrates a broad linear range, low detection limit, and strong anti-interference ability. Mechanistic studies indicate that this material catalyzes the generation of O2 from dissolved oxygen. - and 1 O2 drives the colorimetric reaction of TMB. Uric acid triggers the signal conversion by reducing the blue TMBox to colorless TMB. Accurate and reliable detection results based on urine samples validate the broad application prospects of this method in the field of health monitoring.

[0051] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing Fe / nitrogen co-doped carbon microspheres, characterized in that, Includes the following steps: (1) Dissolve the carbon source in water, stir, and then carry out a hydrothermal reaction. The resulting solid product is washed with water and dried to obtain carbon microspheres. (2) The carbon microspheres, iron salt and nitrogen source obtained in step (1) are placed in water, stirred and dried, then heated, cooled, washed and dried to obtain the Fe / nitrogen co-doped carbon microspheres.

2. The preparation method according to claim 1, characterized in that, In step (1), the carbon source includes soluble starch, and the ratio of the carbon source to water is 1 g: (8-12) mL; the stirring temperature is 70-90℃ and the time is 20-40 min; the hydrothermal reaction temperature is 180-220℃ and the time is 10-14 h; the drying is specifically vacuum drying at 70-90℃ for 4-6 h.

3. The preparation method according to claim 1, characterized in that, In step (2), the iron salt is a trivalent iron salt, including one or more of ferric chloride, ferric sulfate, and ferric nitrate; the nitrogen source includes urea; the ratio of carbon microspheres, ferric chloride, urea, and water is 2 g:1 g:2 g:(30-50 mL); the stirring time is 20-30 h; the heating is carried out under an inert atmosphere, with the temperature increased to 600-800℃ at 5℃ / min-1 and held for 1.5-2.5 h; after washing, it is vacuum dried at 50-70℃.

4. Fe / nitrogen co-doped carbon microspheres prepared by the preparation method according to any one of claims 1 to 3.

5. An enzyme-free colorimetric sensing platform for uric acid detection, characterized in that, Contains the Fe / nitrogen co-doped carbon microspheres as described in claim 4.

6. A non-enzyme-free colorimetric method for the detection of uric acid, characterized in that, The detection was performed using the Fe / nitrogen co-doped carbon microspheres as described in claim 4 or the enzyme-free colorimetric sensing platform as described in claim 5.

7. The enzyme-free colorimetric detection method for uric acid according to claim 6, characterized in that, Includes the following steps: (a) Mix NaAc-HAc buffer, Fe / nitrogen co-doped carbon microspheres and TMB colorimetric solution, react them, and then filter to obtain the filtrate; (b) Add uric acid standard solutions of different concentrations to the filtrate obtained in step (a), react, and after the reaction, use an ultraviolet spectrophotometer to scan the absorption spectrum of the reaction system in the wavelength range of 400 to 800 nm. Finally, take the absorbance value at 652 nm and plot a standard working curve with the uric acid concentration. (c) Replace the uric acid standard solution in step (b) with the uric acid sample to be tested, and repeat steps (a) to (b) to obtain the uric acid concentration through the standard working curve.

8. The enzyme-free colorimetric detection method for uric acid according to claim 7, characterized in that, In step (a), the concentration of the NaAc-HAc buffer is 0.2 M, the pH is 4.0, and the concentration of the TMB chromogenic solution is 8 mmol / L; the volume ratio of the NaAc-HAc buffer, Fe / nitrogen co-doped carbon microspheres, and TMB chromogenic solution is 2.5 mL:3 mg:20 μL; the reaction temperature is 35℃ and the time is 2 min; in step (b), the concentration range of the uric acid standard solution is 14–454 μmol / L, the reaction temperature is 25–35℃, and the time is 5 min.

9. The application of the Fe / nitrogen co-doped carbon microspheres according to claim 4 in the preparation of an enzyme-free colorimetric sensing platform for the detection of uric acid in isolated urine.

10. The application according to claim 9, characterized in that, Before testing, the isolated urine was diluted to a uric acid concentration range of 14–454 μmol / L.

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