Colorimetric and photothermal dual-mode uric acid instant detection method based on atp-enhanced peroxidase-like activity of platinum nanoszyme, reagent and application

Abstract

The present application relates to the present application belongs to the technical field of nanomaterials and medical chemical analysis, specifically relates to a kind of based on adenosine triphosphate enhanced peroxidase-like activity of platinum nanoscale enzyme's colorimetric and photothermal dual-mode uric acid instant detection method, reagent and application.The present application constructs the dual-mode POCT platform based on PtNZ / adenosine triphosphate sensitive, on-site detection of uric acid UA level.Compared with PtNZ, PtNZ / adenosine triphosphate shows excellent POD activity under neutral conditions, uric acid oxidase UAO hydrolyzes uric acid UA to generate H2O2, PtNZ / adenosine triphosphate catalyzes ABTS oxidation to ABTS + , which triggers the colorimetric response from colorless to green color change, and under the assistance of 808 nm NIR laser, photothermal signal is generated.The detection result of the present application to uric acid UA level not only maintains stronger consistency, and improves the reliability of detection result by self-verification mode.

Description

Technical Field

[0001] This invention relates to the fields of nanomaterials and medical chemical analysis technology, specifically to a colorimetric and photothermal dual-mode method, reagent, and application for the real-time detection of uric acid based on adenosine triphosphate-enhanced platinum nanozymes with peroxidase-like activity. Background Technology

[0002] Uric acid (UA) is a major metabolic product of exogenous xanthine (from fatty meat, animal organs, and seafood) and endogenous xanthine (from the oxidative breakdown of nucleic acids) and their derivatives in the human body. UA levels have been used as an important indicator for assessing health status. Common methods for detecting uric acid include colorimetric methods, fluorescence methods, chemiluminescence methods, high-performance liquid chromatography (HPLC), high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS), and gas chromatography-mass spectrometry (GC-MS).

[0003] Traditional chromatography and mass spectrometry methods rely on large instruments and require cumbersome sample pretreatment, failing to meet the requirements of accuracy, convenience, and low cost for point-of-care testing (POCT). POCT, with its miniaturized instruments, sensitive sensors, intelligent technology, and simplified operation, has been widely applied in food safety, environmental protection, and biosensing. In our previous research, we successfully measured biochemical indicators such as alkaline phosphatase, ascorbic acid, and cholinesterase using POCT. Therefore, it is feasible to design a suitable POCT platform to develop a method for detecting UA similar to routine glucose testing for daily UA monitoring.

[0004] Colorimetric detection of urobilinogen (UA) involves the oxidation of UA by urate oxidase (UOX) to allantoin and H₂O₂. Peroxidase (POD), in the presence of H₂O₂, catalyzes chromogenic substrates such as 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and 1,2-diaminobenzene (OPD) to generate colored oxidation products such as blue oxTMB, green oxABTS, and yellow oxOPD. This amplifies the catalytic reaction signal, and the UA content is determined by UV-Vis spectrophotometry. Natural POD exhibits strong substrate specificity and high catalytic efficiency, but its preparation and purification costs are high, it is difficult to store, and the demanding reaction conditions limit its application. Nanozymes possess unique advantages such as low cost, high stability, and tunable catalytic activity, making them promising alternatives to natural enzymes and applicable to a wide range of fields including biomedicine and the environment. Based on their catalytic properties, nanozymes can be broadly classified into peroxidase-like (POD-like), oxidase-like, hydrogen peroxide-like, superoxide dismutase, and glutathione peroxidase-like enzymes.

[0005] Nanozymes, as a class of nanomaterials with enzyme-mimicking activity, offer advantages over natural enzymes, including simple preparation, low cost, high stability, and tunable catalytic activity, and have been widely applied in fields such as biosensing, targeted therapy, and medical imaging. Particularly in biosensing, the cascade reaction between natural enzymes (such as glucose oxidase, uricase (UAO), and cholesterol oxidase) and peroxidase-like enzymes (POD-like enzymes) has successfully determined the levels of biomolecules such as glucose, uric acid (UA), and cholesterol. Natural enzymes oxidize substrates to produce the intermediate product H₂O₂, while POD-like enzymes catalyze the decomposition of H₂O₂ and oxidize chromophores / fluorophores for colorimetric / fluorescent / electrochemical assays. However, the pH-dependent nature of POD-like enzymes in acidic environments limits their application in physiological or alkaline environments. To overcome the pH limitation on POD-like enzyme activity, one approach is to reconstruct the optimal pH environment, but this two-step process may weaken its analytical performance. Another approach is to modify the size distribution and regulate crystallinity through surface modification (e.g., adenosine triphosphate (ATP)) or by forming complexes (e.g., Ru nanozymes supported on graphdiyne oxide), thereby directly improving catalytic activity. Therefore, enhancing the POD-like activity of nanozymes under suboptimal conditions remains an active research area.

[0006] In addition, single-mode POCT techniques based on various signals, such as color, temperature, pressure, and distance, have been developed. While single-mode methods exhibit excellent performance in quantitative analysis, they may lead to increased false positive or false negative results due to non-standardized protocols, operator differences, environmental interference, and background signals. To overcome these limitations, dual-mode or multi-mode systems such as colorimetric-fluorescence, colorimetric-photothermal, and electrochemical-chemiluminescence methods have been proposed. These not only combine the advantages of different technologies but also allow for cross-validation between different modes, improving the accuracy and reliability of disease diagnosis. With the development of photothermal reagents' excellent conversion efficiency and high-precision thermometers, photothermal methods offer a simpler and more suitable POCT platform due to their simple and direct temperature readout. Different types of photothermal reagents include organic compounds (e.g., oxidized 3,3,5,5-tetramethylbenzidine (oxTMB)) and oxidized 2,2-azidobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS). ·+ Inorganic materials (e.g., Cu) 2-x Ag x Photothermal methods, including nanocrystals and composite materials (e.g., chitosan-stabilized platinum nanoparticles), have been extensively studied for developing photothermal sensors. Therefore, photothermal methods offer an alternative technique for constructing dual-mode point-of-care testing (POCT) platforms for UA detection. Summary of the Invention

[0007] The main objective of this invention is to provide a colorimetric and photothermal dual-mode uric acid instant test strip based on adenosine triphosphate-enhanced platinum nanozymes with peroxidase-like activity, as well as its preparation method and application.

[0008] To achieve the above objectives, this invention provides a dual-mode real-time detection method for uric acid content based on adenosine triphosphate-enhanced platinum nanozymes, comprising the following steps: S1, Sample Pretreatment: Dilute 3.0 μL of serum sample 10-fold with 27.0 μL of 10 mM NaAc-HAc buffer (pH 8.0) to a final volume of 30 μL (Serious Diluent A). Add the diluent to solution B and incubate at 36-38°C for 15-25 min to obtain solution C. Solution B is prepared by diluting 30 μL of 1.3 × 10⁵ μL NaAc-HAc buffer. -1 The solution was prepared by adding UOX solution to 270 μL of NaAC-HAc solution with a concentration of 10 mM and pH=8.0. S2, Uric Acid Detection: A uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme was added to solution C and incubated at 36-38 °C for 15-25 min to obtain detection solution D; wherein, the uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme comprises 10 μL of solution with a concentration of 80.0 mg / mL. -1 Adenosine triphosphate-enhanced platinum nanozyme PtNZ solution, 12 μL of adenosine triphosphate solution with a concentration of 0.2 M, and 20 μL of ABTS solution with a concentration of 0.01 M; S31, Colorimetric instantaneous detection of uric acid: The color of a portion of the test solution D is compared with the colorimetric data of the reaction between the standard uric acid concentration and the uric acid test solution based on adenosine triphosphate enhanced platinum nanozyme by software to obtain uric acid detection data based on colorimetry. S32, uric acid detection based on photothermal method: 200 μL of the detection solution D was loaded into an EP tube and the real-time temperature T0 was recorded; the solution was exposed to an 808 nm wavelength, 2.0 W NIR laser lamp for 5 min and the real-time temperature T was recorded; by comparing the photothermal changes of the standard uric acid concentration with the uric acid detection solution based on adenosine triphosphate enhanced platinum nanozyme after reaction, the uric acid detection data based on photothermal method was obtained.

[0009] Preferably, before step S1, the method further includes: establishing colorimetric data after the reaction of a standard uric acid concentration with a uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme. 30 μL of uric acid UA solution A' of different concentrations was added to solution B, and then incubated at 36-38°C for 15-25 min to obtain solution C'; Uric acid detection solution based on adenosine triphosphate enhanced platinum nanozyme was added to solutions C' with different uric acid concentrations and incubated at 36-38 °C for 15-25 min to obtain detection solutions D' with different uric acid concentrations. The absorbance of the test solution at 417 nm and 735 nm was measured using a UV-Vis spectrometer to obtain the colorimetric data of the reaction between the standard uric acid concentration and the uric acid test solution based on adenosine triphosphate-enhanced platinum nanozyme.

[0010] Preferably, before step S1, the method further includes: establishing photothermal change data of a standard uric acid concentration reacting with a uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme. 200 μL of detection solution D with different uric acid concentrations was loaded into an EP tube, and the real-time temperature T0 was recorded. The solution was exposed to an 808 nm wavelength, 2.0 W power NIR laser lamp for 5 min, and the real-time temperature T was recorded. The photothermal change data of the standard uric acid concentration reacting with the uric acid detection solution based on adenosine triphosphate enhanced platinum nanozyme were obtained.

[0011] Preferably, the preparation method of the adenosine triphosphate enhanced platinum nanozyme PtNZ is as follows: 80-100 parts by mass of citric acid, 80-100 parts by mass of urea, 80-100 parts by mass of H2PtCl4, and 80-100 parts by mass of NaOH are added to 80-100 parts by mass of water, dispersed, and then reacted in a high-pressure reactor at 120-180°C for 8-12 h. After cooling, the obtained product is centrifuged, washed, and dried to obtain PtNZ.

[0012] Preferably, the mass ratio of citric acid, urea, H2PtCl4, NaOH and water is 1:1:1:1:1.

[0013] This invention also provides a uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme, comprising 10 parts by volume of a solution with a concentration of 80.0 mg / mL. -1 The solution is a mixture of adenosine triphosphate-enhanced platinum nanozyme PtNZ solution, 12 parts by volume of a 0.2 M adenosine triphosphate solution, and 20 parts by volume of a 0.01 M ABTS solution.

[0014] Preferably, the preparation method of the adenosine triphosphate enhanced platinum nanozyme PtNZ is as follows: 80-100 parts by mass of citric acid, 80-100 parts by mass of urea, 80-100 parts by mass of H2PtCl4, and 80-100 parts by mass of NaOH are added to 80-100 parts by mass of water, dispersed, and then reacted in a high-pressure reactor at 120-180 °C for 8-12 h. After cooling, the obtained product is centrifuged, washed, and dried to obtain PtNZ.

[0015] Preferably, the mass ratio of citric acid, urea, H2PtCl4, NaOH and water is 1:1:1:1:1.

[0016] This invention also provides an application of a uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme in the real-time detection of uric acid levels in a dual-mode system, comprising the following steps: S1, Sample Pretreatment: Dilute 3.0 μL of serum sample 10-fold with 27.0 μL of 10 mM NaAc-HAc buffer (pH 8.0) to a final volume of 30 μL (serum dilution A). Add the dilution to solution B and incubate at 36-38 °C for 15-25 min to obtain solution C. Solution B is prepared by diluting 30 μL of 1.3 × 10⁵ μL NaAc-HAc buffer. -1 The solution was prepared by adding UOX solution to 270 μL of NaAC-HAc solution with a concentration of 10 mM and pH=8.0. S2, Uric Acid Detection: A uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme was added to solution C and incubated at 36-38 °C for 15-25 min to obtain detection solution D; wherein, the uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme comprises 10 μL of solution with a concentration of 80.0 mg / mL. -1 Adenosine triphosphate-enhanced platinum nanozyme PtNZ solution, 12 μL of adenosine triphosphate solution with a concentration of 0.2 M, and 20 μL of ABTS solution with a concentration of 0.01 M; S31, Colorimetric instantaneous detection of uric acid: The color of a portion of the test solution D is compared with the colorimetric data of the reaction between the standard uric acid concentration and the uric acid test solution based on adenosine triphosphate enhanced platinum nanozyme by software to obtain uric acid detection data based on colorimetry. S32, uric acid detection based on photothermal method: 200 μL of the detection solution D was loaded into an EP tube and the real-time temperature T0 was recorded; the solution was exposed to an 808 nm wavelength, 2.0 W NIR laser lamp for 5 min and the real-time temperature T was recorded; by comparing the photothermal changes of the standard uric acid concentration with the uric acid detection solution based on adenosine triphosphate enhanced platinum nanozyme after reaction, the uric acid detection data based on photothermal method was obtained.

[0017] This application constructs a sensitive, on-site POCT platform based on PtNZ / adenosine triphosphate (ATP) for detecting uric acid (UA) levels. Compared to PtNZ, PtNZ / ATP exhibits superior POD activity under neutral conditions, which is attributed to ATP's effective promotion of H₂O₂ decomposition to generate -OH and O₂. - Uric acid oxidase (UAO) hydrolyzes uric acid (UA) to produce H₂O₂. PtNZ / adenosine triphosphate (ATP) catalyzes the oxidation of ABTS to ABTS. + It triggers a colorimetric response from colorless to green, and with the assistance of an 808nm NIR laser, generates a photothermal signal. The colorimetric-POCT mode for uric acid (UA) detection monitors the solution color change via a smartphone camera, while the photothermal-POCT mode measures the temperature change using a portable thermometer. This dual-mode method not only maintains strong consistency in uric acid (UA) level detection results but also improves the reliability of the results through self-validation. The accuracy and practicality of this method have been validated in clinical samples, demonstrating its sensitivity, speed, simplicity, and reliability. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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 the structures shown in these drawings without creative effort.

[0019] Figure 1 The graph shows the effect of the mass of K2PtCl4 on the POD-like activity of PtNZ.

[0020] Figure 2 This is the full XPS spectrum of PtNZ.

[0021] Figure 3 The images show the FT-IR and TEM spectra of PtNZ.

[0022] Figure 4 The UV-vis spectra of different reaction systems using ABTS, TMB, and OPD as substrates are shown at pH=4.

[0023] Figure 5 Comparison of POD-like and OXD-like activities of PtNZ at different concentrations.

[0024] Figure 6 Comparison of POD-like and OXD-like activities of PtNZ at different pH values.

[0025] Figure 7 The Michaelis-Menten curves of PtNZ at pH 4.0 with ABTS (A) and H2O2 (C) as substrates are shown. The Double-reciprocal plot of PtNZ at pH 4.0 with ABTS (B) and H2O2 (D) as substrates is also shown.

[0026] Figure 8 The relative POD-like activity of PtNZ at different temperatures.

[0027] Figure 9 The relative POD-like activity of PtNZ at different storage times.

[0028] Figure 10 The UV-vis spectra of different reaction systems using ABTS, TMB, and OPD as substrates are shown at pH=7.0.

[0029] Figure 11 Comparison of POD-like and OXD-like activities of PtNZ at different pH values.

[0030] Figure 12 To compare the POD-like activity of PtNZ before and after the addition of adenosine triphosphate at pH=7.0.

[0031] Figure 13 To investigate the effect of different adenosine triphosphate concentrations on the POD-like activity of PtNZ under pH=7.0 conditions.

[0032] Figure 14 In the diagram, (A) is the curve of the reaction rate versus the substrate ABTS concentration catalyzed by the POD-like activity of PtNZ / ATP; (B) is the Lineweaver-Burk curve of the oxidation of ABTS with different concentrations by H2O2 catalyzed by PtNZ / ATP; (C) is the curve of the reaction rate versus the substrate H2O2 concentration catalyzed by the POD-like activity of PtNZ / ATP; and (D) is the Lineweaver-Burk curve of the oxidation of ABTS with different concentrations by H2O2 catalyzed by PtNZ / ATP.

[0033] Figure 15 In the diagram, (A) is a graph showing the reaction rate of PtNZ's POD-like active catalysis against the concentration of the substrate ABTS; (B) is a Lineweaver-Burk curve showing the oxidation of ABTS by PtNZ with H2O2 at different concentrations; (C) is a graph showing the reaction rate of PtNZ's POD-like active catalysis against the concentration of the substrate H2O2; and (D) is a Lineweaver-Burk curve showing the oxidation of ABTS by PtNZ with H2O2 at different concentrations.

[0034] Figure 16 The effect of different temperatures on the POD-like activity of PtNZ / adenosine triphosphate.

[0035] Figure 17 The effect of different ROS scavengers on the UV-vis spectrum of the PtNZ / ABTS / H2O2 system at pH=4.0 was investigated.

[0036] Figure 18 ESR spectra of DMPO-OH adducts in the DMPO, PtNZ+DMPO and PtNZ+H2O2+DMPO systems.

[0037] Figure 19 In the DMPO, PtNZ+DMPO and PtNZ+H2O2+DMPO systems, DMPO-O2 - ESR spectrum of the adduct.

[0038] Figure 20The effect of different ROS scavengers on the UV-vis spectrum of the PtNZ / ABTS / H2O2 system at pH=7.0 was investigated.

[0039] Figure 21 In the systems PtNZ+H2O2+DMPO and PtNZ+adenosine triphosphate+H2O2+DMPO, DMPO- • ESR spectrum of OH adduct.

[0040] Figure 22 In the systems PtNZ+H2O2+DMPO and PtNZ+adenosine triphosphate+H2O2+DMPO, DMPO-O2 - ESR spectrum of the adduct.

[0041] Figure 23 This is a schematic diagram of the oxidation of ABTS by free radical catalysis of PtNZ / adenosine triphosphate.

[0042] Figure 24 To investigate the effect of different analogues on the POD-like activity of PtNZ at pH=7.0.

[0043] Figure 25 The particle size changes of PtNZ after incubation with different substances for 20 min are shown.

[0044] Figure 26 The absorbance of the PtNZ / ABTS / H2O2 system with adenosine triphosphate (ATP) at 0, 2 min and without ATP is given.

[0045] Figure 27 ABTS under white light conditions, with or without adenosine triphosphate + Solution stability.

[0046] Figure 28 The sensing mechanism and dual-mode detection of uric acid (UA) concentration.

[0047] Figure 29 The UV-vis spectra of different reaction systems at pH=8.0 are shown.

[0048] Figure 30 The NIR (808 nm) values ​​for different reaction system solutions represent the temperature changes after 5 min of irradiation.

[0049] Figure 31 The results of the optimization experiment for detecting uric acid UA in the uric acid oxidase UAO / PtNZ / ATP / ABTS system are as follows: (A) is the pH of the buffer solution, (B) is the concentration of PtNZ, (C) is the concentration of ATP, (D) is the incubation time, and (E) is the NIR (808 nm) illumination time.

[0050] Figure 32 In the figure, (A) shows the UV-vis spectra of the reaction between uric acid (UA) of different concentrations and the uricase (UAO / PtNZ / ATP / ABTS) system. (B) shows the absorbance at 417 nm and the uric acid (UA) concentration correction curves and the absorbance at 735 nm and the uric acid (UA) concentration correction curves. Figure 33 In the figure, (A) is a graph showing the linear relationship between the G / R ratio and the concentration of uric acid (UA) (the inset shows the graphs corresponding to reaction systems with different concentrations of uric acid UA), and (B) is a graph showing the linear relationship between ΔT and the logarithm of the concentration of uric acid UA.

[0051] Figure 34 In the study, (A) the selectivity of uric acid UA detection based on the colorimetric (top) / photothermal (bottom) method using the uric acid oxidase UAO / PtNZ / ATP / ABTS system; and (B) the effect of coexisting interfering substances on the colorimetric (top) / photothermal (bottom) method for uric acid UA detection.

[0052] Figure 35 To enable dual-mode detection of uric acid (UA) using colorimetric and photothermal-POCT methods.

[0053] Figure 36 To improve the reproducibility of dual-mode detection of uric acid (UA) using colorimetric and photothermal-POCT methods.

[0054] Figure 37 This study compares the results of colorimetric and photothermal-POCT dual-mode detection with those of a biochemical analyzer. Detailed Implementation

[0055] The technical solutions in this embodiment will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0056] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0057] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0058] Synthesis of PtNZ PtNZ was prepared using a one-step hydrothermal method. Citric acid (CA) (10 mg), urea (10 mg), H₂PtCl₄ (10 mg), and NaOH (10 mg) were added to 10 mL of ultrapure water. The mixture was sonicated for 10 min until completely dispersed. The solution was transferred to a polytetrafluoroethylene reactor, placed in a high-pressure reactor, and reacted at 150 °C for 10 h. After natural cooling, the product was centrifuged and washed three times with ultrapure water. Finally, the solid obtained by centrifugation was freeze-dried to obtain a dry black powder, which was PtNZ.

[0059] POD-like activity studies of PtNZ and PtNZ / adenosine triphosphate: The POD-like activity of PtNZ was determined using a steady-state kinetic method, with ABTS and H2O2 as substrates and a PtNZ concentration of 0.8 mg / mL. -1 The reaction was carried out in NaAC-HAc (10 mM, pH=4.0) buffer. First, the H₂O₂ concentration was fixed at 0.3 mM. Using ABTS (0.05 mM–0.30 mM) as the substrate, PtNZ was added, and the absorbance-time curve of the reaction system at 417 nm was recorded immediately afterward, with a time interval of 10 s and a recording time of 2 min. Similarly, after adding H₂O₂ (0.10 mM–0.35 mM) and PtNZ to a fixed concentration of ABTS (0.2 mM), the absorbance-time curve of the reaction system at 417 nm was recorded immediately. The reaction was performed according to the Michaelis-Menten equation. The equation of the Lineweaver-Burk curve is derived as follows: ,calculate and .in Represents the reaction rate. Indicates the maximum reaction rate. Represents substrate concentration. , where is the Michaelis constant. When testing the POD-like activity of PtNZ / ATP under neutral conditions (NaAC-HAc, 10 mM, pH=7.0), the concentration of ATP was fixed at 2.0 mM, and the testing method was the same as that for PtNZ.

[0060] Colorimetric and photothermal detection of uric acid (UA) based on PtNZ / ATP: Add 1.3 × 10⁻⁶ urate oxidase UOX to NaAC-HAc (300 μL, 10 mM, pH=8.0). 5 UL -130 μL of uric acid (UA) solution and 30 μL of uric acid (UA) solution of different concentrations were added, and the mixture was incubated at 37°C for 20 min. Then, PtNZ (80.0 mg / mL) was added sequentially. -1 Add 10 μL of adenosine triphosphate (0.2 M, 12 μL) and ABTS (0.01 M, 20 μL) to the above mixed solution and incubate the mixed solution at 37°C for 15 min.

[0061] The absorbance at 417 nm and 735 nm was measured using a UV-Vis spectrometer. Colorimetric data were obtained by reacting standard uric acid concentrations with a uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme.

[0062] The reaction solution (200 μL) was transferred into an EP tube (300 μL), and the temperature was recorded as T0. Subsequently, the solution was exposed to an NIR laser lamp (808 nm, 2.0 W) for 5 min, and a thermometer detector was quickly inserted into the solution to record the temperature (T), which was recorded as T-T0. The photothermal changes after the reaction of the standard uric acid concentration with the uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme were obtained, thus yielding uric acid detection data based on the photothermal method.

[0063] The uric acid UA method based on colorimetric POCT sensing: 100 μL of reaction solution was introduced into a 96-well plate, and images were captured using a smartphone (OPPO ACE2). The color signals were analyzed into R, G, and B values ​​using a free RGB Test application, and the G / R value was calculated.

[0064] The photothermal POCT-based uric acid UA method: The reaction solution (200 μL) was loaded into an EP tube (300 μL), and the temperature was recorded as T0. Subsequently, the solution was exposed to an NIR laser lamp (808 nm, 2.0 W) for 5 min, and a thermometer detector was quickly inserted into the solution to record the temperature (T), which was recorded as T-T0.

[0065] Detection of serum uric acid (UA) levels: To verify the practicality of the PtNZ / ATP sensing platform for detecting uric acid (UA), we measured the UA content in human serum samples. Serum samples were obtained from the Department of Laboratory Medicine, Shanxi Bethune Hospital. UA content was provided by the Department of Laboratory Medicine's biochemical analyzer. All experiments were conducted in accordance with the Declaration of Helsinki and institutional guidelines. This study was approved by the Ethics Committee of Shanxi Bethune Hospital (No. SBQLL-2023-010). All adult volunteers signed written informed consent forms. Serum samples (3.0 μL) were diluted 10-fold to 30 μL with NaAc-HAc buffer (pH 8.0, 10 mM, 27.0 μL) before use and added to a solution containing uricase (UOX) (1.3 × 10⁻⁶). 5 UL -1 The solution was incubated in 30 μL of NaAC-HAc (300 μL, 10 mM, pH=8.0) at 37 °C for 20 min. Then, PtNZ (80.0 mg / mL) was added. -1 The following reagents were added: 10 μL of adenosine triphosphate (0.2 M, 12 μL) and ABTS (0.01 M, 20 μL), and incubated at 37°C for 15 min. The serum uric acid (UA) content was then measured following the above experimental procedure.

[0066] Synthesis and characterization of PtNZ nanozymes: To investigate the effect of K2PtCl4 content on the POD-like activity of PtNZ, different amounts of K2PtCl4 (0~20 mg) were added, and the results were evaluated by measuring the UV-vis absorbance at 417 nm.

[0067] like Figure 1 As shown, Pt doping leads to an overall increase in the POD-like activity of PtNZ, while no POD-like activity was detected without Pt doping. Therefore, the amount of K2PtCl4 added was chosen to be 10 mg.

[0068] like Figure 3 As shown, transmission electron microscopy (TEM) images of PtNZ reveal a well-dispersed spherical morphology with a uniform diameter of 32.0 ± 2.0 nm, confirming their structural stability.

[0069] The corresponding energy-dispersive spectroscopy and elemental mapping images showed that Pt was distributed in PtNZ, confirming the successful doping of Pt. The crystal properties of PtNZ were characterized by XRD. The diffraction peaks with 2θ values ​​of 15°-25° corresponded to the (110) crystal plane of graphitic carbon, and the diffraction peaks with 2θ values ​​of 39.9°, 46.2°, and 67.1° corresponded to the (111), (200), and (220) crystal planes of Pt, respectively.

[0070] X-ray photoelectron spectroscopy (XPS) was used to investigate the chemical composition of the PtNZ surface. Figure 2 The XPS full spectrum of PtNZ shows distinct peaks at binding energies of 284.82 eV, 398.96 eV, 531.44 eV, and 73.5 eV, revealing the presence of C (41.17%), N (20.27%), O (26.54%), and Pt (12.01%). In the high-resolution 4f spectrum of Pt, the two peaks at the binding energies at these values ​​correspond to Pt orbitals. The HR-XPS spectrum of O1s shows peaks at 532.6 and 531.1 eV, corresponding to the binding energies of CO and C=O, respectively. The N1s spectrum is fitted with three peaks, corresponding to the binding energies of Graphitic N (400.4 eV), Pyrrolic N (399.5 eV), and Pyridinic N (398.5 eV). The deconvolution results of the C1s spectrum show four absorption peaks at 288.5 eV (C=O), 287.6 eV (CO), 285.9 eV (CN), and 284.7 eV (CC / C=C). The HR-XPS spectrum of Pt4f fits to six peaks, corresponding to Pt4f and C1s respectively. 6+ (77.9 eV, 74.6 eV), Pt 4+ (75.9 eV, 72.6 eV), Pt 0+ Binding energies of (71.2 eV, 73.8 eV).

[0071] Please refer to Figure 3 In the Fourier transform infrared spectrum, 3432 cm⁻¹ -1 (OH / NH), 2915 cm -1 (CH), 2895 cm -1 (CH), 1630 cm -1 (C=O / C=N), 1515 cm -1 (C=C), 1381 cm -1 (CN) and 1043 cm -1 The vibrational peak at (CC) further confirms the rich chemical structure of PtNZ.

[0072] Evaluation of POD-like activity of PtNZ and PtNZ / adenosine triphosphate: such as Figure 4As shown, after introducing PtNZ and H2O2 into solutions of the color source substrates ABTS, TMB, and OPD, respectively, the reaction system changed from colorless to the corresponding colored oxidation states oxABTS (λmax = 417 nm), oxTMB (λmax = 652 nm), and oxOPD (λmax = 450 nm), demonstrating that PtNZ possesses POD-like activity. In this study, ABTS and H2O2 were selected as substrates to evaluate the POD-like activity of PtNZ. Figure 5 As shown, the absorption signal intensity of oxABTS increases with increasing PtNZ concentration. However, under the same concentration conditions, when no H2O2 is added to the reaction system, the absorption signal intensity of oxABTS is very low, indicating that PtNZ has POD-like activity but no OXD-like enzyme activity. Figure 6 In this study, PtNZ exhibited pH dependence, showing high POD-like activity under acidic conditions (pH < 4.0). However, its activity decreased significantly within the pH range of 4.0–9.0, and was very weak under near-neutral conditions. Please refer to [reference needed]. Figure 7 The kinetic parameters of PtNZ were calculated using Lineweaver-Burk plots. When ABTS was used as the substrate, the Kt of PtNZ was... m It is 0.021 mM, V max 5.0 × 10 −8 Ms −1 When H2O2 is used as the substrate, K m It is 0.40 mM, V max It is 9.2 × 10 −8 Ms −1 Please refer to this. Figure 8 and Figure 9 The POD-like activity of PtNZ reaches its maximum within the temperature range of 30-40°C. During a 150-day storage period, the POD-like activity of PtNZ remains at approximately 80%, demonstrating the excellent stability of PtNZ.

[0073] like Figures 10-16As shown, under neutral conditions, the introduction of PtNZ and H2O2 into the ABTS, TMB, and OPD systems respectively resulted in weak UV absorption signals for the corresponding colored oxidation states. However, the addition of adenosine triphosphate (ATP) significantly enhanced the UV absorption signal and deepened the solution color, demonstrating that ATP can significantly enhance the POD-like activity of PtNZ. Introducing ATP into the PtNZ / ABTS / H2O2 system increased the absorbance of the PtNZ / H2O2 / substrate system, and the solution color at pH 7.0 was deeper than without ATP. This may be because increasing ATP concentration alters the surface area, electron density distribution, or active sites of PtNZ, significantly enhancing its POD-like activity. The kinetic parameters of PtNZ before and after the addition of ATP were tested at pH 7.

[0074] When ABTS is used as a substrate, the K of PtNZ / adenosine triphosphate m It is 0.21 mM, V max 3.6 × 10 −8 Ms −1 When H2O2 is used as the substrate, K m It is 0.46 mM, V max 4.2 × 10 −8 Ms −1 When no adenosine triphosphate is added, and ABTS is used as a substrate, the K of PtNZ is... m It is 1.41 mM, V max 3.4 × 10 −8 Ms −1 When H2O2 is used as the substrate, K m It is 2.1 mM, V max 3.7 × 10 −8 Ms −1 Compared to PtNZ alone, PtNZ / adenosine triphosphate (ATP) has a greater effect on the K+ of ABTS and H2O2. m These figures were reduced by approximately 85% and 78%, respectively. This indicates that the introduction of adenosine triphosphate (ATP) can significantly enhance the affinity of PtNZ for its substrate, while the lower K... m The values ​​indicate that PtNZ has a stronger affinity for the substrate than other POD-like enzymes at pH 7.0 (Table 1). Furthermore, the POD-like activity of PtNZ / ATP remains stable within the temperature range of 30–50 °C. We hypothesize that ATP significantly enhances the affinity of PtNZ for the substrate, strengthens the contact between PtNZ and the substrate, promotes electron transfer, and thus increases the POD-like activity of PtNZ.

[0075] Table 1 Comparison of POD-like enzyme kinetic parameters of PtNZ with those of previously reported materials To clarify the POD-like activity mechanism of PtNZ, we investigated the types of ROS generated during the reaction. EDTA, Trp, PBQ, and IPA were selected as oxygen vacancies (O2, 22, 32, 42, 54, 6 ... V Singlet oxygen () 1 O2), superoxide anion (O2) - It is a scavenger of hydroxyl radicals (OH) and hydroxyl radicals (OH).

[0076] like Figure 1 As shown, the absorbance of the PtNZ / H2O2 / ABTS system did not decrease significantly after the addition of EDTA and Trp, respectively. However, the absorbance decreased significantly upon the addition of PBQ and IPA. Therefore, OH and O2... - It plays an important role in the POD-like activity of PtNZ. We further confirmed the type of ROS by ESR, 5,5-dimethyl-1-pyrroline- N -oxide (DMPO) acts as a free radical scavenger. The ESR spectrum of the PtNZ / H₂O₂ / DMPO system shows a quadruple characteristic peak (intensity ratio = 1:2:2:1) of the DMPO-OH adduct. - The six characteristic peaks of the adduct indicate the presence of OH and O2. - It plays an important role in the POD-like activity of PtNZ.

[0077] The mechanism by which adenosine triphosphate (ATP) enhances the POD-like activity of PtNZ at pH 7.0 was investigated. First, the effect of ATP on ROS formation was tested. The absorption spectra of the PtNZ / ATP / H₂O₂ / ABTS system remained almost unchanged after the addition of EDTA and Trp. However, the absorption spectra significantly decreased after the addition of PBQ and IPA, indicating the influence of OH and O₂. - It participated in the reaction (Figure S18). In the ESR spectrum, after the addition of adenosine triphosphate to the PtNZ / H2O2 / DMPO system, • OH and O2 •− The intensity of the characteristic peak was significantly enhanced. This indicates that adenosine triphosphate (ATP) promotes... • OH and O2 •− The generation of these compounds enhances the POD-like activity of PtNZ. These results indicate that PtNZ / ATP catalyzes the decomposition of H2O2 to produce OH and O2. - This leads to the oxidation of ABTS to ABTS. •+ .

[0078] The effects of adenosine triphosphate analogs such as ADP, AMP, A, and Na2HPO4 on the POD-like activity of PtNZ. For example... Figures 24-27 As shown, at pH 7.0, the absorbance of the PtNZ / ABTS / H2O2 system significantly increased after the addition of adenosine triphosphate (ATP), ADP, or AMP. With the decrease of phosphate groups in ATP, ADP, and AMP, the enhancing effect gradually weakened, and the particle size of the reaction system also decreased. Introducing ATP with multiple phosphate groups into the PtNZ / ABTS / H2O2 system facilitates the adsorption of ATP on the PtNZ surface, leading to a decrease in the distance between PtNZ particles, an increase in the overall particle size, improved PtNZ stability, inhibition of decomposition, and a significant enhancement of PtNZ's POD-like activity. The effect of A (ADP) without phosphate groups on catalytic activity is negligible. The high concentration of PO4 produced by Na2HPO4... 3- The repulsive force and increased pH of the system may weaken the stability of PtNZ. For the PtNZ / ABTS / H2O2 system, the absorbance gradually increased after the addition of adenosine triphosphate (ATP), while without ATP, the absorbance increased slightly and then remained constant. When ATP was added at 2 min, the absorbance resumed its upward trend. ATP can also enhance the absorption of ABTS. •+ The photostability of PtNZ is improved, which is beneficial for further photothermal research. According to literature reports, adenosine triphosphate (ATP) may form a complex with PtNZ and participate in free radical reactions, accelerating the oxidation of ABTS. Therefore, ATP can promote the stability of PtNZ and increase the oxidation of OH and O2. - This increases the yield of PtNZ, thereby enhancing its POD-like activity and ultimately accelerating the reaction process.

[0079] Please refer to this as well. Figure 31 To achieve highly sensitive quantitative analysis of uric acid (UA), the experimental conditions for UA detection were optimized, including the pH of the buffer system, PtNZ concentration, adenosine triphosphate concentration, reaction incubation time, and near-infrared light exposure time.

[0080] Figure 31 In this study, the conditions for detecting uric acid UA in the uric acid oxidase UAO / PtNZ / ATP / ABTS system were optimized as follows: (A) pH of the buffer solution, (B) concentration of PtNZ, (C) concentration of ATP, (D) incubation time, and (E) NIR (808nm) illumination time.

[0081] Figure 32In the figure, (A) is the UV-vis spectrum of the reaction between uric acid UA of different concentrations and the uric acid oxidase UAO / PtNZ / adenosine triphosphate / ABTS system, and (B) is the absorbance at 417 nm and the uric acid UA concentration correction curve and the absorbance at 735 nm and the uric acid UA concentration correction curve.

[0082] Figure 33 In the figure, (A) is a graph showing the linear relationship between the G / R ratio and the concentration of uric acid (UA) (the inset shows the graphs corresponding to reaction systems with different concentrations of uric acid UA), and (B) is a graph showing the linear relationship between ΔT and the logarithm of the concentration of uric acid UA.

[0083] ΔT was used as the standard for optimizing detection conditions, where ΔT = T - T0, and T0 and T represent the temperatures of the system before and after near-infrared laser irradiation, respectively. The effect of buffer solution pH between 5.0 and 9.0 on the detection of uric acid (UA) was first investigated. When pH was less than 7.0, the activity of uricase (UOX) did not reach its maximum. When pH was greater than 7.0, ΔT tended to stabilize, reaching its maximum at pH 8.0. Therefore, the pH of the reaction system was set to 8.0. As the concentration of PtNZ gradually increased, ΔT gradually increased, reaching its maximum at a concentration of 2.0 mg / mL. -1 At this point, ΔT essentially reaches a plateau; therefore, the optimal concentration of PtNZ is 2.0 mg / mL. -1 As the concentration of adenosine triphosphate (ATP) increases, the POD-like activity of PtNZ is gradually enhanced, and ΔT increases. ΔT reaches its maximum at an ATP concentration of 6.0 mM, therefore 6.0 mM is the optimal concentration. With increasing reaction time, uric acid (UA) is fully hydrolyzed, generating more H₂O₂, resulting in a higher degree of ABTS oxidation. ΔT reaches its maximum at 15 min. With increasing NIR (808 nm) irradiation time, the reaction system absorbs more energy. ΔT reaches its maximum at a near-infrared irradiation time of 5 min; therefore, 5 min is selected as the optimal irradiation time for the system. In short, the uric acid oxidase (UOX) solution (1.3 × 10⁻⁶)… 5 UL -1 30 μL of uric acid (UA) solution and 30 μL of different concentrations of UA solution were added to NaAC-HAc solution (300 μL, 10 mM, pH 8.0) and incubated at 37°C for 20 min. Then, PtNZ (80.0 mg / mL) was added. -1 The NIR light was applied for 10 μL of adenosine triphosphate (0.2 M, 12 μL) and ABTS (0.01 M, 20 μL) and incubated at 37°C for 15 min. When the uric acid (UA) content was detected by photothermal method, the NIR light exposure time was 5 min.

[0084] Under optimal reaction conditions, as the uric acid (UA) concentration increased from 1.5 to 70.0 μM, the UV absorption signals at 417 nm and 735 nm gradually increased. The signal value at the maximum UV absorption at 417 nm showed a good linear relationship with the UA concentration (1.5–70.0 μM). y =0.0106 x +0.1921, R 2 =0.9991). The signal value at the maximum UV absorption of 735 nm showed a good linear relationship with the uric acid (UA) concentration (1.5-70.0 μM). y =0.0052 x +0.1045, R 2 =0.9997). The above results lay the foundation for the real-time detection of uric acid (UA) content using colorimetric and photothermal methods.

[0085] Table 2 Comparison of uric acid (UA) detection methods with previously reported methods. Choose cations commonly found in physiological environments such as blood (Na) + K + Ca 2+ Mg 2+ ), anion (Cl) - NO3 - SO4 2- Using small molecules (GLU, Chol, Crea) and large molecules (GOX, ALP) as interfering factors, the selectivity and anti-interference ability of the colorimetric / photothermal dual-mode sensing strategy for uric acid UA in the PtNZ / ATP / ABTS system were investigated.

[0086] Figure 34 In the study, (A) the selectivity of uric acid UA detection based on the colorimetric (top) / photothermal (bottom) method using the uric acid oxidase UAO / PtNZ / ATP / ABTS system; and (B) the effect of coexisting interfering substances on the colorimetric (top) / photothermal (bottom) method for uric acid UA detection.

[0087] Cation (Na) + K + Ca 2+ Mg 2+ The concentration of ) was 1.0 mM, and the anion (Cl) - NO3 - SO4 2- The concentration of the active ingredient was 1.0 mM, the concentration of small molecules (GLU, Chol, Crea) was 10 mM, and the concentration of large molecules (GOX, ALP) was 1.0 mg / mL.-1。

[0088] like Figure 34 As shown in Figure A, the system's response to the colorimetric / photothermal signal for uric acid (UA) detection is significantly higher than that of other interfering factors. Figure 34 In section B, after adding the interference factor, the sensing system caused minimal interference to the uric acid (UA) detection signal. These results indicate that the sensing system has high specificity for UA detection and can be effectively used for UA sensing. Next, the stability of the PtNZ / ATP / ABTS system after storage at -20°C for one month was investigated. In Figure S26, the PtNZ / ATP / ABTS system showed consistent colorimetric (RSD < 7.1%) and photothermal (RSD < 6.9%) signals in response to UA, indicating good stability. No significant differences in dual-mode signals were observed among the five different batches (colorimetric RSD < 4.3%, photothermal RSD < 4.6%), indicating good reproducibility for UA detection (Figure S27). Therefore, the robustness of the developed sensor provides a viable platform for the detection of UA in real-world samples.

[0089] To evaluate the performance of this colorimetric / photothermal-POCT dual-mode sensor in detecting uric acid (UA) in real samples, the concentration of UA in human serum samples (15 healthy adults and 5 patients with hyperuricemia) was measured. As shown in Table 3, the UA level in patients with hyperuricemia was higher than that in healthy adults. Furthermore, the results of the dual-mode method were consistent with those obtained by clinical laboratory biochemical analyzers, and the results of the colorimetric and photothermal-POCT methods were very similar to the results of UA detection in human serum samples. Figure 6 The RSDs (n=3) of the colorimetric method and the photothermal-POCT method were less than 9.10% and 9.90%, respectively. The relative errors between the actual uric acid (UA) levels and the dual-mode method were less than 8.28% and 6.93%, respectively. These results indicate that the PtNZ / ATP-based dual-mode method is reliable, accurate, and practical in detecting UA in real samples and can be used for on-site detection of UA in human serum samples.

[0090] Table 3. Results of dual-mode determination of uric acid (UA) levels in human serum samples using colorimetric and photothermal-POCT methods. In summary, this study constructed a sensitive, on-site POCT platform based on PtNZ / ATP for the detection of uric acid (UA) levels. Compared to PtNZ, PtNZ / ATP exhibited superior POD activity under neutral conditions, which is attributed to ATP's effective promotion of H₂O₂ decomposition to generate OH and O₂. -Uric acid oxidase (UAO) hydrolyzes uric acid (UA) to produce H₂O₂. PtNZ / adenosine triphosphate (ATP) catalyzes the oxidation of ABTS to ABTS. + It triggers a colorimetric response from colorless to green, and, with the assistance of an 808 nm NIR laser, generates a photothermal signal. The colorimetric-POCT mode for uric acid (UA) detection is achieved by monitoring the solution color change using a smartphone camera, while the photothermal-POCT mode is achieved by measuring temperature changes using a portable thermometer. The dual-mode method not only maintains strong consistency in the detection results of UA levels but also improves the reliability of the results through self-validation. The accuracy and practicality of this method have been validated in clinical samples. Therefore, the sensitive, rapid, simple, and reliable dual-mode colorimetric and photothermal method for detecting UA provides a potentially promising tool for the early diagnosis and screening of UA-related diseases in resource-limited areas.

[0091] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. Any equivalent structural or procedural transformations made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A method for real-time detection of uric acid content based on adenosine triphosphate-enhanced platinum nanozymes in a dual-mode approach, characterized in that, Including the following steps: S1, Sample Pretreatment: Dilute 3.0 μL of serum sample 10-fold with 27.0 μL of 10 mM NaAc-HAc buffer (pH 8.0) to a final volume of 30 μL (Serious Diluent A). Add the diluent to solution B and incubate at 36-38°C for 15-25 min to obtain solution C. Solution B is prepared by diluting 30 μL of 1.3 × 10⁵ μL NaAc-HAc buffer. -1 The solution was prepared by adding UOX solution to 270 μL of NaAC-HAc solution with a concentration of 10 mM and pH=8.

0. S2, Uric Acid Detection: A uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme was added to solution C and incubated at 36-38 °C for 15-25 min to obtain detection solution D; wherein, the uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme comprises 10 μL of solution with a concentration of 80.0 mg / mL. -1 Adenosine triphosphate-enhanced platinum nanozyme PtNZ solution, 12 μL of adenosine triphosphate solution with a concentration of 0.2 M, and 20 μL of ABTS solution with a concentration of 0.01 M; S31, Colorimetric instantaneous detection of uric acid: The color of a portion of the test solution D is compared with the colorimetric data of the reaction between the standard uric acid concentration and the uric acid test solution based on adenosine triphosphate enhanced platinum nanozyme by software to obtain uric acid detection data based on colorimetry. S32, uric acid detection based on photothermal method: 200 μL of the detection solution D was loaded into an EP tube and the real-time temperature T0 was recorded; the solution was exposed to an 808 nm wavelength, 2.0 W NIR laser lamp for 5 min and the real-time temperature T was recorded; by comparing the photothermal changes of the standard uric acid concentration with the uric acid detection solution based on adenosine triphosphate enhanced platinum nanozyme after reaction, the uric acid detection data based on photothermal method was obtained.

2. The method for real-time detection of uric acid content based on adenosine triphosphate-enhanced platinum nanozyme according to claim 1, characterized in that, Before step S1, the method further includes: establishing colorimetric data after the reaction of a standard uric acid concentration with a uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme. 30 μL of uric acid UA solution A' of different concentrations was added to solution B, and then incubated at 36-38°C for 15-25 min to obtain solution C'; Uric acid detection solution based on adenosine triphosphate enhanced platinum nanozyme was added to solutions C' with different uric acid concentrations and incubated at 36-38°C for 15-25 min to obtain detection solutions D' with different uric acid concentrations. The absorbance of the test solution at 417 nm and 735 nm was measured using a UV-Vis spectrometer to obtain the colorimetric data of the reaction between the standard uric acid concentration and the uric acid test solution based on adenosine triphosphate-enhanced platinum nanozyme.

3. The method for real-time detection of uric acid content based on adenosine triphosphate-enhanced platinum nanozyme according to claim 2, characterized in that, Before step S1, the method further includes: establishing photothermal change data after the reaction of a standard uric acid concentration with a uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme. 200 μL of detection solution D with different uric acid concentrations was loaded into an EP tube, and the real-time temperature T0 was recorded. The solution was exposed to an 808 nm wavelength and 2.0 W NIR laser lamp for 5 min, and the real-time temperature T was recorded. The photothermal change data of the standard uric acid concentration reacted with the uric acid detection solution based on adenosine triphosphate enhanced platinum nanozyme were obtained.

4. The method for real-time detection of uric acid content based on adenosine triphosphate-enhanced platinum nanozyme according to claim 1, characterized in that, The preparation method of the adenosine triphosphate enhanced platinum nanozyme PtNZ is as follows: 80-100 parts by mass of citric acid, 80-100 parts by mass of urea, 80-100 parts by mass of H2PtCl4, and 80-100 parts by mass of NaOH are added to 80-100 parts by mass of water. After dispersion, the mixture is reacted in a high-pressure reactor at 120-180 °C for 8-12 h. After cooling, the obtained product is centrifuged, washed, and dried to obtain PtNZ.

5. The method for real-time detection of uric acid content based on adenosine triphosphate-enhanced platinum nanozyme according to claim 4, characterized in that, The mass ratio of citric acid, urea, H2PtCl4, NaOH and water is 1:1:1:1:

1.

6. A uric acid detection reagent based on adenosine triphosphate-enhanced platinum nanozyme, characterized in that, The concentration, including 10 parts by volume, is 80.0 mg / mL. -1 The solution is a mixture of adenosine triphosphate-enhanced platinum nanozyme PtNZ solution, 12 parts by volume of a 0.2 M adenosine triphosphate solution, and 20 parts by volume of a 0.01 M ABTS solution.

7. The uric acid detection solution reagent based on adenosine triphosphate-enhanced platinum nanozyme according to claim 6, characterized in that, The preparation method of the adenosine triphosphate enhanced platinum nanozyme PtNZ is as follows: 80-100 parts by mass of citric acid, 80-100 parts by mass of urea, 80-100 parts by mass of H2PtCl4, and 80-100 parts by mass of NaOH are added to 80-100 parts by mass of water. After dispersion, the mixture is reacted in a high-pressure reactor at 120-180°C for 8-12 h. After cooling, the obtained product is centrifuged, washed, and dried to obtain PtNZ.

8. The uric acid detection solution reagent based on adenosine triphosphate-enhanced platinum nanozyme according to claim 7, characterized in that, The mass ratio of citric acid, urea, H2PtCl4, NaOH and water is 1:1:1:1:

1.

9. An application of a uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme in the local dual-mode detection of uric acid content, characterized in that, Including the following steps: S1, Sample Pretreatment: Dilute 3.0 μL of serum sample 10-fold with 27.0 μL of 10 mM NaAc-HAc buffer (pH 8.0) to a final volume of 30 μL (serum dilution A). Add the dilution to solution B and incubate at 36-38 °C for 15-25 min to obtain solution C. Solution B is prepared by diluting 30 μL of 1.3 × 10⁵ μL NaAc-HAc buffer. -1 The solution was prepared by adding UOX solution to 270 μL of NaAC-HAc solution with a concentration of 10 mM and pH=8.

0. S2, Uric Acid Detection: A uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme was added to solution C and incubated at 36-38 °C for 15-25 min to obtain detection solution D; wherein, the uric acid detection solution based on adenosine triphosphate-enhanced platinum nanozyme comprises 10 μL of solution with a concentration of 80.0 mg / mL. -1 Adenosine triphosphate-enhanced platinum nanozyme PtNZ solution, 12 μL of adenosine triphosphate solution with a concentration of 0.2 M, and 20 μL of ABTS solution with a concentration of 0.01 M; S31, Colorimetric instantaneous detection of uric acid: The color of a portion of the test solution D is compared with the colorimetric data of the reaction between the standard uric acid concentration and the uric acid test solution based on adenosine triphosphate enhanced platinum nanozyme by software to obtain uric acid detection data based on colorimetry. S32, uric acid detection based on photothermal method: 200 μL of the detection solution D was loaded into an EP tube and the real-time temperature T0 was recorded; the solution was exposed to an 808 nm wavelength, 2.0 W NIR laser lamp for 5 min and the real-time temperature T was recorded; by comparing the photothermal changes of the standard uric acid concentration with the uric acid detection solution based on adenosine triphosphate enhanced platinum nanozyme after reaction, the uric acid detection data based on photothermal method was obtained.

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