Reagent for detecting uric acid and preparation method thereof
By combining bisborate molecular clamps and sulfonated quantum dots, the problems of easily affected enzyme activity and insufficient selectivity in uric acid detection have been solved, achieving high-precision, long-term stable uric acid detection, suitable for point-of-care testing of complex biological samples.
Patent Information
- Application Number
- CN202511625606.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-07
- Publication Date
- 2026-02-06
AI Technical Summary
Existing uric acid detection technologies suffer from problems such as susceptibility of enzyme active sites to influence, poor batch-to-batch stability, insufficient selectivity, and signal baseline drift, making it difficult to achieve high-precision and long-shelf-life detection in complex biological samples.
A reversible covalent capture of uric acid cis-diol structure was achieved using a bis-boronic acid molecular clamp. The interfacial charge distribution was optimized by combining sulfonated quantum dots and zwitterionic surfactants. Ascorbic acid oxidase and Prussian blue nanoparticles were also introduced to form a liquid detection system with multiple anti-interference mechanisms and signal amplification functions.
It achieves highly selective capture of uric acid, enhances signal transduction sensitivity and stability, effectively inhibits nanoparticle aggregation, and constructs a high-precision, long-term stable detection system suitable for the point-of-care detection of complex biological samples.
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Figure CN121472371A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biological detection technology, specifically relating to a reagent for uric acid detection and its preparation method. Background Technology
[0002] Existing uric acid detection technologies mainly include enzymatic and electrochemical methods, but both methods have certain limitations in practical applications. In traditional enzymatic detection, the immobilization technology of uric acid oxidase has long relied on traditional processes such as physical adsorption, chemical loading, or gel embedding. Although these methods can achieve preliminary enzyme immobilization, they cannot effectively protect the enzyme active site from the effects of temperature fluctuations, changes in solution ionic strength, and oxidation in the storage environment. This leads to conformational distortion of the enzyme protein and shedding of cofactors under room temperature storage conditions, significantly reducing the batch-to-batch stability and clinical reproducibility of the test reagents. At the same time, high concentrations of reducing substances such as ascorbic acid and bilirubin in complex biological matrices can easily compete with uric acid for enzyme catalytic sites. Conventional anti-interference strategies are mostly limited to adding masking agents or constructing double-layer membrane filtration structures. This passive defense mechanism not only increases the complexity of the reagent system but also introduces new sources of interference, causing a nonlinear decrease in detection sensitivity.
[0003] In the field of non-enzymatic detection technologies, although researchers have attempted to construct optical or electrochemical sensors using novel nanomaterials such as noble metal nanoparticles, two-dimensional transition metal carbides, and metal-organic frameworks, the design logic of existing materials is mostly limited to the development of a single physicochemical mechanism. For example, it relies on the localized surface plasmon resonance effect of nanoparticles, the electron transfer properties of conductive polymers, or the size sieving effect of molecularly imprinted materials. This single-dimensional mechanism results in insufficient selectivity of sensors when facing uric acid structural analogs. Furthermore, the sensing interface is susceptible to non-specific adsorption of biomolecules when in contact with viscous samples such as blood and saliva, leading to signal baseline drift. These deep-seated contradictions severely restrict the clinical application of existing detection technologies. Summary of the Invention
[0004] To address the above issues, this invention provides a uric acid detection reagent and its preparation method. It achieves reversible covalent capture of uric acid in a cis-diol structure through precise construction of a diboronic acid molecular clamp, enhances the π-π interaction signal response through sulfonation modification, optimizes the interfacial charge distribution by introducing a zwitterionic surfactant, and adds ascorbic acid oxidase and Prussian blue nanoparticles to form a liquid detection system with multiple anti-interference mechanisms and signal amplification functions. This aims to overcome the technical bottlenecks of insufficient specificity and poor stability in traditional detection methods, and to develop a high-precision, long-shelf-life uric acid detection reagent suitable for complex biological samples.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a reagent for uric acid detection, the reagent comprising the following raw materials in parts by weight: component A 10-18 parts, component B 20-36 parts, component C 1-3 parts, surfactant 0.5 parts, oxidase solution 0.5 parts, Prussian blue dispersion 1.5 parts, and pH 7.4 phosphate buffer 40-67 parts.
[0006] Further, component A is prepared from the following raw materials in parts by weight: 10 parts of 4-bromophenylboronic acid, 7 parts of pinacol diboronic acid, 0.05 parts of nickel catalyst, 20 parts of triethylamine, 50 parts of THF (tetrahydrofuran), and 30 parts of pH 5.8 phosphate buffer.
[0007] Furthermore, component B is prepared from the following raw materials in parts by weight: 2 parts graphite powder, 15 parts ammonium persulfate, 10 parts 10% (volume ratio) phosphoric acid aqueous solution, 4 parts sodium p-toluenesulfonate, 1 part sodium bicarbonate and 47 parts deionized water.
[0008] Furthermore, component C is prepared from the following raw materials in parts by weight: 2 parts urea, 0.5 parts cyanuric acid, and 15 parts deionized water.
[0009] Furthermore, the surfactant is selected from any one of Tween 80, Triton X-100, and rhamnolipid.
[0010] Furthermore, the oxidase solution is a 50 mg / mL ascorbic acid oxidase solution.
[0011] Furthermore, the concentration of the Prussian blue dispersion is 0.1 mM.
[0012] Furthermore, the nickel catalyst is NiCl2 (dppf), where dppf is the ligand bis(diphenylphosphine)ferrocene, and the mass ratio of NiCl2 to dppf is 1:1.
[0013] The preparation method of component A includes the following steps: M1: Weigh 50 parts of THF, purge with nitrogen for 30 min to deoxidize, then weigh 0.05 parts of nickel catalyst and mix, stir at 60℃ and 100 rpm for 1 h to obtain catalyst solution; M2: Weigh 10 parts of 4-bromophenylboronic acid and 7 parts of pinacol diboronic acid and place them in a three-necked flask. Slowly add the catalyst solution, then weigh 20 parts of triethylamine and add them to the flask. React under nitrogen atmosphere, 100 rpm, 65℃ in the dark for 12 h to obtain the crude product. M3: After the crude product is cooled to room temperature, it is filtered under reduced pressure to remove the filter residue and obtain the filtrate. The filtrate is subjected to silica gel column chromatography and the fraction with Rf=0.3-0.4 is collected, which is the product fraction. The silica gel is 200-300 mesh, and the eluent is ethyl acetate:petroleum ether in a volume ratio of 1:5. The product fraction is dried by rotary evaporation to obtain the crystals. M4: Weigh 30 portions of pH 5.8 phosphate buffer to dissolve the crystals. The mass ratio of pH 5.8 phosphate buffer to crystals is 30:1. After complete dissolution, obtain component A. Store component A at 4℃ protected from light.
[0014] The preparation method of component B includes the following steps: N1: Weigh 15 parts of ammonium persulfate and 10 parts of 10% phosphoric acid aqueous solution and mix them in an ice bath. Weigh 2 parts of graphite powder and add them while stirring. Transfer the mixture to an acid-resistant microwave reaction vessel, heat it to 100°C at 800 W, keep it for 5 min, and then cool it down quickly after the reaction is complete to obtain a suspension. N2: Centrifuge the suspension at 8000 rpm for 10 min, discard the supernatant, collect the precipitate, wash the precipitate repeatedly with deionized water and centrifuge 3 times, dialyze it with a 3 kDa dialysis bag for 24 h and then freeze dry to obtain quantum dot powder; N3: Weigh 4 parts of sodium p-toluenesulfonate and 1 part of sodium bicarbonate and dissolve them in deionized water, wherein the mass ratio of sodium bicarbonate to deionized water is 1:50. Disperse quantum dot powder in the solution, heat in an oil bath at 120℃ and reflux for 2 h, cool, centrifuge at 10000 rpm for 15 min, discard the supernatant, wash the precipitate with anhydrous ethanol, disperse it in 47 parts of deionized water, and sonicate at 300 W for 20 min to obtain component B. Component B is stored at 4℃ protected from light.
[0015] The preparation method of component C includes the following steps: S1: Weigh 2 parts of urea, 0.5 parts of cyanuric acid and 15 parts of deionized water, stir and mix well, disperse by ultrasonication at 300 W for 30 min, transfer to high pressure reactor and react at 120℃ for 2 h, and obtain flocculent suspension after natural cooling; S2: The flocculent suspension is filtered to obtain a precipitate. The precipitate is dried and ground to obtain component C.
[0016] This invention also provides a method for preparing a reagent for uric acid detection, specifically including the following steps: Step 1: Weigh 10-18 parts of component A, 20-36 parts of component B, 1-3 parts of component C and 40-67 parts of pH 7.4 phosphate buffer, mix them together, then weigh 0.5 parts of surfactant and add them to the mixture. Vortex for 3 minutes to obtain the initial mixture. Step 2: Weigh 0.5 parts of oxidase solution and 1.5 parts of Prussian blue dispersion and add them to the initial mixture. Sonicate at 200 W for 10 min in an ice bath, then filter through a 0.22 μm filter membrane to obtain the reagent for uric acid detection. Store at 4℃ protected from light.
[0017] The beneficial effects achieved by this invention are as follows: This invention provides a uric acid detection reagent based on the precise design of a bisboronic acid molecular clamp. Utilizing the dynamic and reversible covalent bonding between the boric acid group and the cis-diol structure of uric acid, it achieves highly selective capture of target molecules. The π-π stacking effect and surface charge regulation of sulfonated quantum dots significantly enhance the sensitivity and stability of signal transduction. The topological sieving function of the supramolecular framework selectively excludes structural analogs such as ascorbic acid through a hydrogen bond network, further improving the specificity of the detection system. The introduction of zwitterionic surfactants optimizes the charge distribution at the colloidal interface, effectively inhibiting nanoparticle aggregation. The synergistic effect of ascorbic acid oxidase and Prussian blue nanoparticles constructs a dual anti-interference mechanism: the former eliminates interference from oxidizing substances through enzymatic reactions, while the latter amplifies the electrochemical signal through electron transfer effects. The functional complementarity and dynamic balance of each component in the liquid system not only retain the reversible advantage of boric acid-diol binding, but also overcome the interference of complex sample matrices through multi-level barrier effects of physical sieving and chemical catalysis. Ultimately, a detection system with high-precision identification, long-term stability and rapid response is formed, which breaks through the performance limitations of traditional detection reagents and provides a reliable solution for clinical point-of-care testing. Attached Figure Description
[0018] Figure 1 The Fourier transform infrared spectroscopy results are shown for component A before and after binding with uric acid. Figure 2 The absorbance results are those obtained by binding uric acid with the reagents for uric acid detection prepared in Component A, Example 2 and Comparative Example 1; Figure 3 The X-ray diffraction characterization results are for component B; Figure 4 The fluorescence quenching detection results are obtained after the uric acid detection reagents prepared in Component B, Example 4 and Comparative Example 2 bind with uric acid. Figure 5 The interference rate detection results are for the uric acid detection reagents prepared by component C, Example 6 and Comparative Example 3 after binding with uric acid. Figure 6 The results of stability studies on the uric acid detection reagents prepared in Examples 2, 4, 6 and Comparative Examples 1-3 are presented. Detailed Implementation
[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.
[0021] Unless otherwise specified, all methods used in the following examples are conventional. Unless otherwise specified, all materials used in the following examples and comparative examples are new materials purchased from the market. The concentration of ascorbic acid oxidase solution used is 50 mg / mL, the concentration of Prussian blue dispersion is 0.1 mM, and the nickel catalyst is NiCl2 (dppf).
[0022] In the following examples and comparative examples, component A was prepared from the following raw materials in parts by weight: 10 parts of 4-bromophenylboronic acid, 7 parts of pinacol diboronic acid, 0.05 parts of NiCl2 (dppf), 20 parts of triethylamine, 50 parts of THF, and 30 parts of pH 5.8 phosphate buffer. The preparation method of component A is as follows: M1: Weigh 50 parts of THF, purge with nitrogen for 30 min to remove oxygen, then weigh 0.05 parts of NiCl2 (dppf) and mix. Stir at 60℃ and 100 rpm for 1 h to obtain a catalyst solution. M2: Weigh 10 parts of 4-bromophenylboronic acid and 7 parts of pinacol diboronic acid and place them in a three-necked flask. Slowly add the catalyst solution, then weigh 20 parts of triethylamine and add them to the flask. React under nitrogen atmosphere, 100 rpm, 65℃ in the dark for 12 h to obtain the crude product. M3: After the crude product is cooled to room temperature, it is filtered under reduced pressure to remove the filter residue and obtain the filtrate. The filtrate is subjected to silica gel column chromatography and the fraction with Rf=0.3-0.4 is collected, which is the product fraction. The silica gel is 200-300 mesh, and the eluent is ethyl acetate:petroleum ether in a volume ratio of 1:5. The product fraction is dried by rotary evaporation to obtain the crystals. M4: Weigh 30 portions of pH 5.8 phosphate buffer to dissolve the crystals. The mass ratio of pH 5.8 phosphate buffer to crystals is 30:1. After complete dissolution, obtain component A. Store component A at 4℃ protected from light.
[0023] Component B is prepared from the following raw materials in parts by weight: 2 parts graphite powder, 15 parts ammonium persulfate, 10 parts 10% phosphoric acid aqueous solution, 4 parts sodium p-toluenesulfonate, 1 part sodium bicarbonate, and 47 parts deionized water. The preparation method of component B is as follows: N1: Weigh 15 parts of ammonium persulfate and 10 parts of 10% phosphoric acid aqueous solution and mix them in an ice bath. Weigh 2 parts of graphite powder and add them while stirring. Transfer the mixture to an acid-resistant microwave reaction vessel, heat it to 100°C at 800 W, keep it for 5 min, and then cool it down quickly after the reaction is complete to obtain a suspension. N2: Centrifuge the suspension at 8000 rpm for 10 min, discard the supernatant, collect the precipitate, wash the precipitate repeatedly with deionized water and centrifuge 3 times, dialyze it with a 3 kDa dialysis bag for 24 h and then freeze dry to obtain quantum dot powder; N3: Weigh 4 parts of sodium p-toluenesulfonate and 1 part of sodium bicarbonate and dissolve them in deionized water, wherein the mass ratio of sodium bicarbonate to deionized water is 1:50. Disperse quantum dot powder in the solution, heat in an oil bath at 120℃ and reflux for 2 h, cool, centrifuge at 10000 rpm for 15 min, discard the supernatant, wash the precipitate with anhydrous ethanol, disperse it in 47 parts of deionized water, and sonicate at 300 W for 20 min to obtain component B. Component B is stored at 4℃ protected from light.
[0024] Component C is prepared from the following raw materials in parts by weight: 2 parts urea, 0.5 parts cyanuric acid and 15 parts deionized water; The preparation method of component C is as follows: S1: Weigh 2 parts of urea, 0.5 parts of cyanuric acid and 15 parts of deionized water, stir and mix well, disperse by ultrasonication at 300 W for 30 min, transfer to high pressure reactor and react at 120℃ for 2 h, and obtain flocculent suspension after natural cooling; S2: The flocculent suspension is filtered to obtain a precipitate. The precipitate is dried and ground to obtain component C.
[0025] Example 1: This example provides a reagent for uric acid detection, which includes the following raw materials in parts by weight: 10 parts of component A, 20 parts of component B, 1 part of component C, 0.5 parts of Tween 80, 0.5 parts of ascorbic acid oxidase solution, 1.5 parts of Prussian blue dispersion, and 67 parts of pH 7.4 phosphate buffer. The preparation methods of components A, B, and C are as described above.
[0026] This embodiment also provides a method for preparing a reagent for uric acid detection, which specifically includes the following steps: Step 1: Weigh 10 parts of component A, 20 parts of component B, 1 part of component C and 67 parts of pH 7.4 phosphate buffer and mix them. Then weigh 0.5 parts of Tween 80 and add them to the mixture. Vortex for 3 minutes to obtain the initial mixture. Step 2: Weigh 0.5 parts of ascorbic acid oxidase solution and 1.5 parts of Prussian blue dispersion and add them to the initial mixture. Sonicate at 200 W for 10 min in an ice bath, then filter through a 0.22 μm filter membrane to obtain the reagent for uric acid detection. Store at 4℃ protected from light.
[0027] Example 2: This example provides a reagent for uric acid detection, which includes the following raw materials in parts by weight: 18 parts of component A, 36 parts of component B, 3 parts of component C, 0.5 parts of Tween 80, 0.5 parts of ascorbic acid oxidase solution, 1.5 parts of Prussian blue dispersion, and 40 parts of pH 7.4 phosphate buffer. The preparation methods of components A, B, and C are as described above.
[0028] This embodiment also provides a method for preparing a reagent for uric acid detection, which specifically includes the following steps: Step 1: Weigh 18 parts of component A, 36 parts of component B, 3 parts of component C and 40 parts of pH 7.4 phosphate buffer and mix them. Then weigh 0.5 parts of Tween 80 and add them to the mixture. Vortex for 3 minutes to obtain the initial mixture. Step 2: Weigh 0.5 parts of ascorbic acid oxidase solution and 1.5 parts of Prussian blue dispersion and add them to the initial mixture. Sonicate at 200 W for 10 min in an ice bath, then filter through a 0.22 μm filter membrane to obtain the reagent for uric acid detection. Store at 4℃ protected from light.
[0029] Example 3: This example provides a reagent for uric acid detection, which includes the following raw materials in parts by weight: 12 parts of component A, 24 parts of component B, 2 parts of component C, 0.5 parts of Triton X-100, 0.5 parts of ascorbic acid oxidase solution, 1.5 parts of Prussian blue dispersion, and 60 parts of pH 7.4 phosphate buffer. The preparation methods of components A, B, and C are as described above.
[0030] This embodiment also provides a method for preparing a reagent for uric acid detection, which specifically includes the following steps: Step 1: Weigh 12 parts of component A, 24 parts of component B, 2 parts of component C and 60 parts of pH 7.4 phosphate buffer and mix them. Then weigh 0.5 parts of Triton X-100 and add them to the mixture. Vortex for 3 min to obtain the initial mixture. Step 2: Weigh 0.5 parts of ascorbic acid oxidase solution and 1.5 parts of Prussian blue dispersion and add them to the initial mixture. Sonicate at 200 W for 10 min in an ice bath, then filter through a 0.22 μm filter membrane to obtain the reagent for uric acid detection. Store at 4℃ protected from light.
[0031] Example 4: This example provides a reagent for uric acid detection, which includes the following raw materials in parts by weight: 18 parts of component A, 36 parts of component B, 3 parts of component C, 0.5 parts of Triton X-100, 0.5 parts of ascorbic acid oxidase solution, 1.5 parts of Prussian blue dispersion, and 40 parts of pH 7.4 phosphate buffer. The preparation methods of components A, B, and C are as described above.
[0032] This embodiment also provides a method for preparing a reagent for uric acid detection, which specifically includes the following steps: Step 1: Weigh 18 parts of component A, 36 parts of component B, 3 parts of component C and 40 parts of pH 7.4 phosphate buffer and mix them. Then weigh 0.5 parts of Triton X-100 and add them to the mixture. Vortex for 3 min to obtain the initial mixture. Step 2: Weigh 0.5 parts of ascorbic acid oxidase solution and 1.5 parts of Prussian blue dispersion and add them to the initial mixture. Sonicate at 200 W for 10 min in an ice bath, then filter through a 0.22 μm filter membrane to obtain the reagent for uric acid detection. Store at 4℃ protected from light.
[0033] Example 5: This example provides a reagent for uric acid detection, which includes the following raw materials in parts by weight: 10 parts of component A, 20 parts of component B, 1 part of component C, 0.5 parts of rhamnolipid, 0.5 parts of ascorbic acid oxidase solution, 1.5 parts of Prussian blue dispersion, and 67 parts of pH 7.4 phosphate buffer. The preparation methods of components A, B, and C are as described above.
[0034] This embodiment also provides a method for preparing a reagent for uric acid detection, which specifically includes the following steps: Step 1: Weigh 10 parts of component A, 20 parts of component B, 1 part of component C and 67 parts of pH 7.4 phosphate buffer and mix them. Then weigh 0.5 parts of rhamnolipin and add it to the mixture. Vortex for 3 min to obtain the initial mixture. Step 2: Weigh 0.5 parts of ascorbic acid oxidase solution and 1.5 parts of Prussian blue dispersion and add them to the initial mixture. Sonicate at 200 W for 10 min in an ice bath, then filter through a 0.22 μm filter membrane to obtain the reagent for uric acid detection. Store at 4℃ protected from light.
[0035] Example 6: This example provides a reagent for uric acid detection, which includes the following raw materials in parts by weight: 14 parts of component A, 28 parts of component B, 2 parts of component C, 0.5 parts of rhamnolipid, 0.5 parts of ascorbic acid oxidase solution, 1.5 parts of Prussian blue dispersion, and 54 parts of pH 7.4 phosphate buffer. The preparation methods of components A, B, and C are as described above.
[0036] This embodiment also provides a method for preparing a reagent for uric acid detection, which specifically includes the following steps: Step 1: Weigh 14 parts of component A, 28 parts of component B, 2 parts of component C and 54 parts of pH 7.4 phosphate buffer and mix them. Then weigh 0.5 parts of rhamnolipin and add it to the mixture. Vortex for 3 min to obtain the initial mixture. Step 2: Weigh 0.5 parts of ascorbic acid oxidase solution and 1.5 parts of Prussian blue dispersion and add them to the initial mixture. Sonicate at 200 W for 10 min in an ice bath, then filter through a 0.22 μm filter membrane to obtain the reagent for uric acid detection. Store at 4℃ protected from light.
[0037] The difference between Comparative Example 1 and Example 2 is that component A was replaced with an equal weight of pH 7.4 phosphate buffer solution, while the rest of the preparation was the same as in Example 2.
[0038] The difference between Comparative Example 2 and Example 4 is that component B was replaced with an equal weight of pH 7.4 phosphate buffer solution, while the rest of the preparation was the same as in Example 4.
[0039] The difference between Comparative Example 3 and Example 6 is that component C was replaced with an equal weight of pH 7.4 phosphate buffer solution, while the rest of the preparation was the same as in Example 6.
[0040] Comparative Example 4 is a commercially available uric acid (UA) content detection kit (enzyme colorimetric method).
[0041] Diboronic acid molecular clamp FTIR (Fourier Transform Infrared Spectroscopy) investigation 0.1 mM uric acid standard solution (pH 7.4 PBS) was mixed with the prepared component A at a mass ratio of 1:10 by vortexing. The mixture was incubated in a 37℃ constant temperature water bath for 30 min and then lyophilized to obtain component A + uric acid powder. Using the lyophilized powder of component A as a control group, the changes in infrared spectra before and after uric acid binding were detected using the KBr pellet method. The results are shown in the figure. Figure 1 .
[0042] Investigation of the binding ability of diboronic acid molecular clamps 0.1 mM uric acid standard solution was mixed with component A, the uric acid detection reagent prepared in Example 2, and the uric acid detection reagent prepared in Comparative Example 1 (calculated based on the mass of component A contained in the reagents, the mass ratio of uric acid standard solution to component A was 1:10), to obtain control group 1, Example 2 group, and Comparative Example 1 group. After vortexing and mixing, the mixture was incubated in a 37°C constant temperature water bath for 30 min. An equal volume of PBS was used to replace the uric acid standard solution and mixed with component A as a blank group. Using a UV-Vis spectrophotometer, the sample was loaded into a quartz cuvette (optical path 1 cm), and the wavelength range of 200-400 nm was scanned. The absorbance at 293 nm was recorded. The results are shown in the figure. Figure 2 .
[0043] Sulfonated quantum dots XRD (X-ray diffraction) characterization Component B obtained was freeze-dried, and its structure was investigated by XRD. Radiation source: Cu Kα (λ=1.5406Å), voltage 40 kV, current 40 mA; scanning range: 5-80° (2θ), step size 0.02°, scanning speed 4° / min; detector: D / teX Ultra linear array detector, Solar slit 0.5°. Detection results are shown in […]. Figure 3 .
[0044] Investigation of signal response of sulfonated quantum dots 0.1, 1, 5, and 10 mM gradient uric acid standard solutions were prepared and mixed with component B, the uric acid detection reagent prepared in Example 4, and the uric acid detection reagent prepared in Comparative Example 2, respectively (calculated based on the mass of component B contained in the reagents; the mass ratio of uric acid standard solution to component B was 1:1). After vortexing and standing for 10 min, control group 2, Example 4 group, and Comparative Example 2 group were obtained. Using a fluorescence spectrometer, with an excitation wavelength of 360 nm, an emission wavelength of 450 nm, and a slit width of 5 nm, the fluorescence intensity (F) of each sample was measured. PBS at pH 7.4 was used instead of uric acid solution as a blank (F0). The fluorescence quenching rate was calculated as: quenching rate (%) = (F0 - F) / F0 × 100%. The results are shown in [Figure number missing]. Figure 4 .
[0045] Investigation of the anti-interference ability of supramolecular framework A 5 mM uric acid standard solution was mixed with 10 mM ascorbic acid, glucose, and lactate interferon at a 1:1 volume ratio. Then, the prepared component C, the uric acid detection reagent prepared in Example 6, and the uric acid detection reagent prepared in Comparative Example 3 were added respectively (calculated based on the mass of component C contained in the reagents; the mass ratio of uric acid standard solution to component C was 1:1). The mixture was incubated at 37°C for 20 min. The actual uric acid concentration of control group 3, Example 6 group, and Comparative Example 3 group was determined using the direct velocimetric method (DPV). The recovery rate (%) was calculated as: Detected concentration / Theoretical concentration × 100%, and the interference rate (%) was calculated as: |100% - Recovery rate|. The results are shown in [Figure number missing]. Figure 5 .
[0046] Reagent detection sensitivity study Five μL of the uric acid detection reagents prepared in Examples 1-6 and Comparative Examples 1-3 were drop-coated onto the surface of a glassy carbon electrode. After drying at room temperature, the electrode was polished sequentially with 0.3 μm and 0.05 μm alumina polishing powders, rinsed with ultrapure water, and set aside for use. Using an electrochemical workstation, a gradient concentration of uric acid samples was loaded into the three-electrode system. The scanning range was set to -0.2 to 0.6 V, the pulse amplitude to 50 mV, and the step size to 5 mV. The oxidation peak current value (I) was recorded. A linear equation was fitted with the current value as the ordinate and the uric acid concentration as the abscissa, and the detection limit was calculated. The results are shown in Table 1.
[0047] Reagent stability study The uric acid detection reagents prepared in Examples 2, 4, 6, and Comparative Examples 1-3 were aliquoted and stored in a 37°C incubator. A batch was taken out weekly, allowed to return to room temperature, and then the current response was detected (same as the DPV method). Retention rate (%) = I t / I0×100%, where I0 is the initial current, I t The current after cycle t is given, and the results are shown in [the original text]. Figure 6 .
[0048] Stability study of uric acid content Standard curves were established using 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and 20 mM uric acid standard solutions. 0.1, 1, and 10 mM standard solutions were mixed with the uric acid detection reagent prepared in Example 4 and the commercially available uric acid detection kit in Comparative Example 4. The recovery rate, repeatability, ascorbic acid interference rate, and batch-to-batch RSD were then measured. The results are shown in Table 2.
[0049] Figure 1 FTIR results showed that component A, after binding with uric acid, reached a depth of 1342 cm⁻¹. -1 The stretching vibration peak of the free boric acid group at 1665 cm⁻¹ changed from a double peak to a single peak and exhibited a red shift, indicating that the boric acid group formed a BOC covalent bond with cis-diol of uric acid. The decrease in electron cloud density led to a decrease in vibrational frequency. -1 The characteristic absorption peak of the ketone C=O form in the newly added uric acid molecule is 1605 cm⁻¹. -1 and 810 cm -1 The characteristic peak shifts, while the benzene ring structure and substituent positions of component A are retained, indicating that uric acid specifically binds only to boric acid molecules.
[0050] Figure 2The results showed that the uric acid detection reagent prepared in Example 2 had an absorbance of 0.85±0.03 at 293 nm after binding with uric acid, which was significantly higher than that of Comparative Example 1. This indicates that the diboronic acid molecular clamp (component A) can achieve specific capture. The high proportion of component A provides more binding sites, and the absorbance is increased by 23%, indicating that the binding ability is positively correlated with the content of component A. Comparative Example 1 had no characteristic absorption peak due to the lack of component A, which verifies the necessity of the diboronic acid molecular clamp.
[0051] Figure 3 XRD results show that the characteristic peak near 25° is a broadening peak after quantum dot sulfonation, indicating successful sulfonation modification and the presence of corresponding diffraction peaks. Sulfonation leads to the amorphization of some quantum dots.
[0052] Figure 4 The results showed that sulfonated quantum dots (component B) bind to uric acid through π-π interactions, inducing fluorescence quenching. High content of component B enhances signal amplification. In contrast, Comparative Example 2, lacking component B, relied only on a weak response from Prussian blue, resulting in a significant decrease in sensitivity.
[0053] Figure 5 The results showed that the ascorbic acid interference rate in Example 6 was 1.8%, while the interference rate in Comparative Example 3 (without component C) was 23.5%. This indicates that component C can selectively capture small molecule interfering substances such as ascorbic acid, but has no adsorption on uric acid. In Comparative Example 3, due to the absence of component C, the interfering substances easily interfered with the detection signal, thus verifying the core role of supramolecular sieving.
[0054] Table 1. Methodological Investigation of Reagents for Uric Acid Detection
[0055] Table 1 shows that the uric acid detection reagent prepared in Example 4 has the best linear range, and R 2 It has the largest detection limit, good linearity, and low detection limit, making it suitable for detecting samples with extremely low uric acid levels.
[0056] Figure 6 The results showed that the high component A / component B ratio and the surfactant inhibiting component degradation resulted in good stability of the uric acid detection reagents prepared in Examples 2, 4 and 6 within 4 weeks.
[0057] Table 2 Stability of uric acid content in detection
[0058] Table 2 shows that the uric acid detection reagent prepared in Example 4 had a recovery rate of 98.5±1.3% (10 mM), an anti-interference rate of 1.8%, and an inter-batch RSD of 1.8%, all of which were superior to commercially available reagents (recovery rate 82.1±5.8%, RSD=6.3%). The dual anti-interference mechanism of molecular sieve (component C) and ascorbic acid oxidase, combined with the optimized immobilization ratio of component A / component B, achieved high accuracy (recovery rate > 98%) and high stability (RSD < 2%), meeting the needs of complex clinical sample detection.
[0059] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
[0060] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.
Claims
1. A reagent for uric acid detection, characterized in that, The reagent comprises the following raw materials in parts by weight: component A 10-18 parts, component B 20-36 parts, component C 1-3 parts, surfactant 0.5 parts, oxidase solution 0.5 parts, Prussian blue dispersion 1.5 parts, and pH 7.4 phosphate buffer 40-67 parts; Component A is prepared from the following raw materials in parts by weight: 10 parts of 4-bromophenylboronic acid, 7 parts of pinacol diboronic acid, 0.05 parts of nickel catalyst, 20 parts of triethylamine, 50 parts of THF, and 30 parts of pH 5.8 phosphate buffer. The preparation method of component A includes the following steps: M1: THF is deoxidized and then mixed with a nickel catalyst to obtain a catalyst solution; M2: Mix 4-bromophenylboronic acid and pinacol diboronic acid, add catalyst solution and triethylamine to obtain crude product; M3: The crude product is purified and dried to obtain crystals; M4: The crystals were dissolved in a pH 5.8 phosphate buffer solution to obtain component A.
2. The uric acid detection reagent according to claim 1, characterized in that, Component B is prepared from the following raw materials in parts by weight: 2 parts graphite powder, 15 parts ammonium persulfate, 10 parts 10% phosphoric acid aqueous solution, 4 parts sodium p-toluenesulfonate, 1 part sodium bicarbonate, and 47 parts deionized water. The preparation method of component B includes the following steps: N1: Ammonium persulfate, 10% phosphoric acid aqueous solution and graphite powder are mixed and reacted with microwave to obtain a suspension; N2: The suspension was purified and dried to obtain quantum dot powder; N3: Sodium p-toluenesulfonate and sodium bicarbonate are dissolved in deionized water and then reacted with quantum dot powder to obtain component B.
3. The uric acid detection reagent according to claim 1, characterized in that, Component C is prepared from the following raw materials in parts by weight: 2 parts urea, 0.5 parts cyanuric acid and 15 parts deionized water; The preparation method of component C includes the following steps: S1: Mix urea, cyanuric acid and deionized water and react under high pressure to obtain a flocculent suspension; S2: The flocculent suspension was filtered to obtain component C.
4. The uric acid detection reagent according to claim 1, characterized in that, In step M3, the crude product purification process uses silica gel column chromatography and collects the fraction with Rf=0.3-0.
4.
5. The uric acid detection reagent according to claim 1, characterized in that, The nickel catalyst is NiCl2 with dppf as the ligand, and the mass ratio of NiCl2 to dppf is 1:
1.
6. A reagent for uric acid detection according to claim 1, characterized in that, The surfactant is selected from any one of Tween 80, Triton X-100, and rhamnolipid.
7. A method for preparing a uric acid detection reagent according to any one of claims 1-6, characterized in that, Specifically, the steps include the following: Step 1: Mix component A, component B, component C, pH 7.4 phosphate buffer, and surfactant to obtain the initial mixture; Step 2: Add oxidase solution and Prussian blue dispersion to the initial mixture to obtain the reagent for uric acid detection.