Electrochemical sensor for continuously monitoring uric acid as well as preparation method and application of electrochemical sensor

Abstract

The invention belongs to the technical field of electrochemical sensors, and particularly relates to an electrochemical sensor for continuously monitoring uric acid as well as a preparation method and application thereof. The sensor comprises a substrate electrode, a sensitive layer, an anti-interference layer and a diffusion limiting layer, the sensitive layer covers the surface of the substrate electrode and comprises a conductive metal complex for catalyzing oxidation of uric acid; the anti-interference layer covers the surface of the sensitive layer and is used for eliminating interference of ascorbic acid; the diffusion limiting layer covers the surface of the anti-interference layer and is used for controlling the uric acid diffusion rate; coating the surface of a working electrode area of the substrate electrode with a mixed solution of a conductive metal complex and a cross-linking agent, and forming a sensitive layer through a cross-linking reaction; and coating the anti-interference layer and the diffusion limiting layer on the surface of the sensitive layer layer by layer, and carrying out heat treatment and curing at 20-50 DEG C after each layer is coated, so as to obtain the electrochemical sensor. Compared with the prior art, the electrochemical sensor prepared by the invention can rapidly and continuously detect the concentration of uric acid in a liquid environment, and is simple in preparation steps and good in stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical sensors, and in particular relates to an electrochemical sensor for continuously monitoring uric acid, and a preparation method and application thereof. Background Art

[0002] Uric acid is the end product of purine metabolism in the human body. Humans lack the enzyme uricase, which breaks down the poorly water-soluble uric acid into the more water-soluble allantoin. However, some uric acid is still excreted in the urine through the kidneys. Under normal circumstances, the total daily uric acid production and excretion in the body is maintained in balance. When the body experiences excessive purine intake, abnormally elevated endogenous purine levels, or abnormal renal excretion, uric acid concentrations in the body can rise dramatically. In severe cases, uric acid can precipitate and form crystals in body fluids, leading to symptoms such as gout.

[0003] Currently, the market primarily uses laboratory equipment or point-of-care (POCT) products with disposable test strips to test blood uric acid concentrations. These methods require venous or fingertip blood collection, requiring multiple needle sticks from the patient, making continuous monitoring inconvenient. This is extremely inconvenient for daily monitoring or clinical testing of uric acid, as it is impossible to capture the changing trends of uric acid levels over time. Continuous uric acid monitoring sensors based on electrochemical technology remain to be developed.

[0004] Existing products for uric acid testing are primarily based on colorimetry and electrochemical methods. The principle of the colorimetric method is that uric acid, catalyzed by uricase, oxidizes to produce allantoin and H₂O₂. H₂O₂ reacts with 4-aminoantipyrine (4-AAP) and 3,5-dichloro-2-hydroxybenzenesulfonic acid (DHBS) under the catalysis of peroxidase to form a colored substance (quinoneimine compound). The color change is proportional to the UA content in the sample. The reaction equation is as follows: Uric acid + O2 + H2O → allantoin + CO2 + H2O2; H2O2+4-AAP+DHBS→colored compound+H2O; Please refer to: "Technical Review Guidelines for Registration of Uric Acid Determination Reagents (No. 74 of 2019)".

[0005] The electrochemical method works by converting uric acid into allantoin under the action of an electron mediator or urate oxidase. This reaction generates an electrical signal, which is proportional to the uric acid concentration in the blood. Uric acid testing products based on this principle perform a single test on the sample's uric acid level, requiring complex procedures and sampling, and are time-consuming.

[0006] Patent CN108303454A discloses a uric acid electrochemical sensor comprising a substrate layer; a wire layer and an electrode layer located on the substrate layer, the wire layer including a start electrode, and the electrode layer including a counter electrode and a working electrode; an insulating layer located on the wire layer and electrode layer, the insulating layer having a window formed therein to expose the working portions of the counter electrode and the working electrode, the window being in the form of a partially enclosed pattern with a sampling notch; a reagent layer covering the window; a double-sided adhesive layer; and a hydrophilic film layer located on the double-sided adhesive layer. However, the patent cannot be used multiple times for uric acid testing. If multiple measurements are required, multiple uses are required, making the method of use complex.

[0007] Currently, there are products on the market for continuous blood glucose monitoring, which can achieve continuous monitoring for approximately two weeks after wear without any complex operation. However, there are no products for continuous uric acid monitoring. Therefore, the present invention provides a method for constructing a continuous uric acid monitoring electrode. Summary of the Invention

[0008] (1) Technical issues to be resolved The purpose of the present invention is to solve at least one of the above problems and provide an electrochemical sensor for continuous monitoring of uric acid, its preparation method and application, so as to solve the problems in the prior art that uric acid detection only supports single detection and the detection operation is complicated and time-consuming, so as to achieve the effect of rapid and continuous detection of uric acid in a liquid environment, and the preparation steps are simple and the stability is good.

[0009] (2) Technical solution The purpose of the present invention is achieved through the following technical solutions: The electrochemical sensor for continuously monitoring uric acid provided by the present invention and its preparation method and application can simplify preparation and have better stability compared with enzyme electrodes.

[0010] One of the technical solutions of the present invention is to disclose an electrochemical sensor for continuously monitoring uric acid, the sensor comprising a base electrode, a sensitive layer, an anti-interference layer and a diffusion limiting layer; The sensitive layer covers the surface of the base electrode and contains a conductive metal complex that catalyzes the oxidation of uric acid; The anti-interference layer covers the surface of the sensitive layer and is used to eliminate the interference of ascorbic acid; The diffusion limiting layer covers the surface of the anti-interference layer and is used to control the diffusion rate of uric acid.

[0011] Furthermore, the conductive metal complex is prepared from a metal M and a polymer ligand, wherein the metal M includes any one of Os, Ir, and Ru; and the polymer ligand includes a monomer and a copolymer.

[0012] Furthermore, the monomer includes any one of poly-4-vinylpyridine and poly-2-vinylimidazole; The copolymer includes any one of poly(vinylimidazole-co-methacrylic acid), poly(vinylimidazole-co-hydroxyethyl methacrylate), and poly(vinylimidazole-co-hydroxypropyl methacrylate).

[0013] Furthermore, the metals including Os, Ir, and Ru all have good electrical conductivity and catalytic properties, which can effectively reduce the overpotential required for uric acid oxidation, thereby improving the sensitivity of detection; the nitrogen-containing groups in the polymer provide sites for cross-linking the material, allowing the material to be fixed with the epoxy cross-linker on the working electrode surface of the base electrode to form a stable sensitive film layer.

[0014] The metal and polymer ligands work together to enable the sensitive membrane layer to continuously monitor uric acid at a low voltage of +0.2 V. The metal provides catalytic activity, the polymer ligands provide cross-linking sites to form a stable membrane structure, and the additional ligands are used to optimize the electrochemical properties of the metal complex.

[0015] Furthermore, the preparation method of the conductive metal complex comprises the following steps: S1: subjecting the polymer monomer to a free radical polymerization reaction in the presence of an initiator to obtain a polymer ligand; S2: heating a soluble metal salt containing metal M and a nitrogen-containing bidentate ligand in a solvent to react to obtain a metal complex; S3: mixing the polymer ligand obtained in step S1 and the metal complex obtained in step S2 in a polar solvent, and heating the mixture to react to form the conductive metal complex.

[0016] Furthermore, the method for fixing the sensitive layer includes the following steps: preparing the above-mentioned conductive metal complex into an aqueous solution with a concentration of 50~200 mg / mL, adding 1%~10% of the epoxy crosslinking agent relative to the weight of the conductive metal complex to the solution; then uniformly coating the solution on the surface of the base electrode, cross-linking and curing to obtain a base electrode modified with the sensitive layer.

[0017] Furthermore, the epoxy crosslinking agent includes one or more of polyethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, neopentyl glycol diglycidyl ether, trimethylolpropane triglycidyl ether, 1,3-bis(3-glycidyloxypropyl)tetramethyldisiloxane, and polydimethylsiloxane diglycidyl ether.

[0018] Furthermore, the anti-interference layer is an ascorbic acid oxidase layer modified by a cross-linking agent; the cross-linking agent includes any one of an epoxy cross-linking agent and an aldehyde cross-linking agent.

[0019] Furthermore, the ascorbate oxidase in the anti-interference layer is fixed by the following steps: dissolving the ascorbate oxidase in a pH buffer solution at a concentration of 10-100 mg / ml, adding serum albumin accounting for 1%-10% of the enzyme mass to the enzyme solution; adding 1 wt%-10 wt% of an aldehyde- or epoxy-containing cross-linking agent, mixing, and coating the mixture on the surface of the sensitive membrane layer to form the anti-interference layer through a covalent cross-linking reaction.

[0020] After adding the anti-interference layer, the electrode can effectively avoid interference from ascorbic acid when detecting uric acid, thereby improving the accuracy of the test results. In contrast, electrodes without the anti-interference layer may be affected by ascorbic acid during the detection process because the detection voltage is greater than the oxidation potential of ascorbic acid, resulting in deviations in the test results.

[0021] Furthermore, the diffusion limiting layer is a hydrophilic polymer membrane layer.

[0022] Furthermore, when the diffusion limiting layer is a cross-linked hydrophilic polymer membrane layer, the membrane solution for preparing the diffusion limiting layer uses a mixture of 75%-100% ethanol and 25-0% water as a solvent, and the components include 5%-10% polyvinyl pyridine and 0.5%-3% epoxy cross-linking agent.

[0023] Furthermore, when the diffusion limiting layer is a cross-linked hydrophilic polymer membrane layer, the membrane solution for preparing the diffusion limiting layer uses a mixture of 80% ethanol and 20% water as a solvent, and the components include 12% hydrophilic polyurethane or polyvinyl pyridone and 4% epoxy crosslinking agent.

[0024] Furthermore, when the diffusion limiting layer is a non-crosslinked hydrophilic polymer membrane layer, the membrane solution for preparing the diffusion limiting layer uses tetrahydrofuran as a solvent, and the components include 5%-15% polyurethane.

[0025] Furthermore, the preparation method of the diffusion-limiting layer includes the following steps: dissolving the components in a solvent at a concentration of 50-200 mg / mL, adding a crosslinker and mixing, and then coating the membrane liquid of the diffusion-limiting layer on the surface of the anti-interference layer to form the diffusion-limiting layer through a covalent crosslinking reaction.

[0026] Furthermore, the diffusion limiting layer extends the linear range of uric acid detection to 100 μM-2000 μM.

[0027] Furthermore, the diffusion limiting layer has a thickness of 100-200 μm.

[0028] After adding the diffusion limiting layer, the electrode can obtain a wider linear detection range when detecting uric acid, thereby being able to more accurately detect higher concentrations of uric acid. However, the electrode without the diffusion limiting layer has a relatively narrow detection range and may not be able to accurately detect high concentrations of uric acid. Although the diffusion limiting layer reduces the sensitivity of uric acid detection, within an acceptable sensitivity range, the diffusion limiting layer can greatly improve the linear range of electrode detection. Specifically, the detection range of the electrode of the present invention can be extended to 100μM-2000μM, which is much larger than the detection range of fingertip blood detection products.

[0029] Furthermore, the base electrode is configured as a micro-flexible electrode, including a reference electrode, a working electrode and a counter electrode.

[0030] Furthermore, the reference electrode is any one of a solid Ag / AgCl electrode or a conductive polymer reference electrode; the working electrode is a carbon electrode, a platinum electrode or a gold electrode; and the counter electrode is a carbon electrode or a platinum electrode.

[0031] Furthermore, the base electrode can be prepared by any one of screen printing, MEMS processing, and inkjet printing.

[0032] Furthermore, the base electrode is prepared by screen printing in steps on a flexible substrate (such as polyimide PI or polyethylene terephthalate PET); the reference electrode is obtained by screen printing Ag / AgCl ink; screen printing carbon paste (including graphite, carbon nanotubes, or graphene paste) or platinum paste or gold paste; the counter electrode is obtained by screen printing carbon paste (including graphite, carbon nanotubes, or graphene paste) or platinum paste or gold paste.

[0033] The second technical solution of the present invention is to disclose a method for preparing an electrochemical sensor for continuously monitoring uric acid as described above, comprising the following steps: coating a mixed solution of a conductive metal complex and a cross-linking agent on the surface of the working electrode area of ​​the base electrode to form a sensitive layer through a cross-linking reaction; coating an anti-interference layer and a diffusion limiting layer on the surface of the sensitive layer layer by layer, and curing each layer by heat treatment at 20-50°C after coating to obtain the electrochemical sensor.

[0034] Furthermore, the above coating method includes any one of spot coating, dip coating, drop coating, and spin coating.

[0035] The third technical solution of the present invention is to disclose the application of the electrochemical sensor for continuously monitoring uric acid as described above in a liquid environment.

[0036] Furthermore, the electrochemical sensor for continuously monitoring uric acid can be used to monitor the uric acid concentration in interstitial fluid in real time.

[0037] (3) Beneficial effects Compared with the prior art, the present invention has the following advantages: (1) The present invention uses a cross-linkable conductive metal complex as a sensitive layer. The metals Os, Ir, and Ru used can catalyze the oxidation of uric acid, reduce the potential required for uric acid oxidation, and improve the detection sensitivity. The selected polymers include poly-4-vinylpyridine, poly-2-vinylimidazole, etc., which provide material cross-linking sites. The material and the cross-linking agent are fixed to the working electrode surface of the base electrode, and a stable sensitive film layer is formed after cross-linking. Therefore, the sensitive film layer prepared by the present invention is conducive to detecting the uric acid concentration when the uric acid concentration is low, and can be reused, that is, the uric acid concentration can be continuously measured.

[0038] (2) The sensor of the present invention has a special anti-interference layer that can effectively eliminate the interference of ascorbic acid. The anti-interference layer is a cross-linker-modified ascorbic acid oxidase layer. Through specific chemical modification and cross-linking technology, ascorbic acid oxidase can specifically recognize and oxidize ascorbic acid, thereby avoiding the interference of ascorbic acid when detecting uric acid, and improving the accuracy and reliability of the test results.

[0039] (3) The detection range of the sensor of the present invention is significantly broadened due to the addition of the diffusion-limiting layer. The diffusion-limiting layer regulates the diffusion rate of uric acid, so that uric acid can be accurately detected even at a lower overpotential, thereby expanding the detection range to 100μM-2000μM, which is suitable for a variety of detection scenarios. At the same time, the diffusion-limiting layer has good biocompatibility, which can reduce the risk of immune and inflammatory reactions, improve patient comfort and safety, and make it more compatible with biological tissues or body fluids, ensuring that the sensor remains stable and reliable in the body or when in contact with biological tissues.

[0040] (4) The preparation method of the electrochemical sensor of the present invention is simpler than that of the enzyme electrode and has better stability. The preparation process includes coating a sensitive layer on the surface of the base electrode and then coating an anti-interference layer and a diffusion restriction layer layer by layer. This method not only simplifies the preparation steps, but also avoids the instability of the enzyme in the enzyme electrode and the complex modification process, making the sensor more stable and reliable during preparation and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0042] Figure 1Schematic diagram of the chemical structure of the sensitive layer material in an electrochemical sensor for continuous monitoring of uric acid prepared in Example 1 of the present invention.

[0043] Figure 2 This is a graph showing the current response results of the electrochemical sensor prepared in Example 1 of the present invention to different uric acid concentrations in phosphate buffer.

[0044] Figure 3 This is a comparison chart of the current response results of the electrochemical sensors prepared in Example 1 of the present invention and Comparative Example 1 to different uric acid concentrations in phosphate buffer.

[0045] Figure 4 This is a graph showing the current response results of the electrochemical sensor prepared in Comparative Example 2 of the present invention to different uric acid concentrations in phosphate buffer. DETAILED DESCRIPTION

[0046] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0047] The following describes the embodiments of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of this application.

[0048] It should be noted that various aspects of the embodiments within the scope of the appended claims are described below. It should be apparent that the aspects described herein can be embodied in a wide variety of forms, and any specific structure and / or function described herein is merely illustrative. Based on this application, it should be understood by those skilled in the art that an aspect described herein can be implemented independently of any other aspect, and two or more of these aspects can be combined in various ways. For example, any number and aspect described herein can be used to implement an apparatus and / or practice a method. In addition, other structures and / or functionalities other than one or more of the aspects described herein can be used to implement this apparatus and / or practice this method.

[0049] Additionally, in the following description, specific details are provided to provide a thorough understanding of the examples, however, one skilled in the art will appreciate that the examples can be practiced without these specific details.

[0050] Unless otherwise specified, the raw materials or devices used in the following examples are commercially available raw materials or conventional experimental devices.

[0051] The following describes the technical solutions provided by various embodiments of the present application in conjunction with the accompanying drawings. Example 1

[0052] This embodiment provides an electrochemical sensor for continuously monitoring uric acid, wherein the sensor includes a base electrode, a sensitive layer, an anti-interference layer, and a diffusion-limiting layer.

[0053] The base electrode, comprising a reference electrode, a working electrode, and a counter electrode, was prepared by screen-printing Ag / AgCl ink onto polyethylene glycol (PET) in separate steps. The reference electrode was prepared by screen-printing a conductive carbon paste and then heat-curing it at 80°C for 30 minutes. The working and counter electrodes were both prepared by screen-printing a conductive carbon paste and then heat-curing it at 80°C for 30 minutes. To form a three-electrode system, an insulating paste was screen-printed between the electrodes and cured using UV light.

[0054] The sensitive layer covers the surface of the base electrode and contains a conductive metal complex that catalyzes the oxidation of uric acid, which is prepared from metal Os and a polymer ligand poly-2-vinyl imidazole. The synthesis method of the conductive metal complex is as follows: the structure of the prepared sensitive layer material is as follows Figure 1 As shown: (1) Synthesis of poly(vinylimidazole) (PVI): 10.0 g of vinylimidazole was dissolved in 100 mL of N,N-dimethylformamide, followed by deoxygenation for 30 min. 50 mg of azobisisobutyronitrile was added and the mixture was polymerized at 75 °C for 24 h. After the reaction, the product was precipitated with ether to obtain poly(vinylimidazole).

[0055] (2) Synthesis of Os complex PVI-Os(bpy)2Cl2: 1.0 g of ammonium chloroosmate and 0.72 g of 2,2-bipyridine were mixed in 50 mL of ethylene glycol, deoxygenated, and then reacted at 140°C under anhydrous and oxygen-free conditions for 1 h. After the reactants were cooled, an equal volume of sodium dithionite aqueous solution was added to quench the reaction. Finally, Os(bpy)2Cl2 was obtained by filtration and washing with ether and pure water.

[0056] (3) Synthesis of PVI-Os(bpy2)Cl: 0.30 g of PVI and 0.30 g of Os(bpy)2Cl2 were dissolved in 50 mL of ethylene glycol and reacted at 140 °C for 24 h under anhydrous and oxygen-free conditions. The conductive metal complex was obtained by ether precipitation and washing, and then dialysis with pure water.

[0057] The anti-interference layer covers the surface of the sensitive layer and is an ascorbic acid oxidase layer modified by an epoxy cross-linking agent, and is used to eliminate the interference of ascorbic acid.

[0058] The diffusion limiting layer covers the surface of the anti-interference layer and is used to control the diffusion rate of uric acid.

[0059] The preparation method of the electrochemical sensor is as follows: (1) Preparation of sensitive layer: The conductive metal complex PVI-Os(bpy)2Cl2 was prepared into a 50 mg / ml aqueous solution, 10 wt% polyethylene glycol diglycidyl ether was added to the solution, the solution was mixed and then evenly modified on the surface of the substrate electrode by dot coating, a total of 6 μL, and cross-linked and cured at 37°C and 40% humidity for 24 hours to obtain a substrate electrode modified with the sensitive layer; (2) Preparation of anti-interference layer: ascorbic acid oxidase (AAOx) solution dissolved in 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer solution was prepared at a concentration of 20 mg / ml. 10 wt% bovine serum albumin and 10 wt% polyethylene glycol diglycidyl ether were added to the solution relative to the mass percentage of AAOx. The enzyme solution was mixed and fixed on the surface of the sensitive membrane layer by dip coating. The dip coating was repeated twice, with an interval of 30 minutes between each times. The anti-interference layer was cross-linked and cured at 37°C and 40% humidity for 24 hours to obtain the anti-interference layer. (3) Preparation of a diffusion limiting layer: a mixture of 75% ethanol and 25% water was used as a solvent, poly (4-vinyl pyridine) was added, and the mixture was stirred and dissolved to obtain a solution with a concentration of 100 mg / mL. 5 wt% of trimethylolpropane triglycidyl ether was added to the solution and mixed to obtain a membrane solution for preparing a diffusion limiting layer. The membrane solution was evenly coated on the surface of the anti-interference layer by dip coating for a total of 6 times, with an interval of 30 minutes each time. The membrane solution was cross-linked and cured at 37°C and 40% humidity for 24 hours to obtain the electrochemical sensor.

[0060] The electrochemical sensor prepared in Example 1 was used to measure uric acid in tissue fluid. The results were as follows: Figure 2 As shown by Figure 2 It can be seen that the detection range of the electrode of the present invention is extended to 100 μM – 2000 μM, which is much larger than the fingertip blood detection product, and the response current of each concentration of uric acid varies in size. Therefore, the electrochemical sensor prepared by the present invention can be used to accurately and continuously measure uric acid concentration in a liquid environment. Example 2

[0061] This embodiment provides an electrochemical sensor for continuously monitoring uric acid, wherein the sensor includes a base electrode, a sensitive layer, an anti-interference layer, and a diffusion-limiting layer.

[0062] The base electrode, consisting of a reference electrode, a working electrode, and a counter electrode, was prepared by screen-printing Ag / AgCl ink onto polyethylene glycol (PET) in separate steps. The reference electrode was prepared by screen-printing Ag / AgCl ink and then heat-curing it at 80°C for 30 minutes. The working and counter electrodes were both prepared by screen-printing carbon nanotubes and then heat-curing them at 80°C for 30 minutes. To form a three-electrode system, an insulating paste was screen-printed between the electrodes and cured using UV light.

[0063] The sensitive layer covers the surface of the base electrode and contains a conductive metal complex that catalyzes the oxidation of uric acid, which is prepared from metal Ru and a polymer ligand poly-4-vinylpyridine. The synthesis method of the conductive metal complex is as follows: (1) Synthesis of Ru complex Ru(bpy)2Cl2: 1.0 g of ruthenium trichloride and 0.72 g of 2,2-bipyridine were mixed in 50 mL of ethylene glycol, deoxygenated, and then reacted at 140°C under anhydrous and oxygen-free conditions for 1 h. After the reactants were cooled, an equal volume of sodium dithionite aqueous solution was added to quench the reaction. Finally, Ru(bpy)2Cl2 was obtained by filtration and washing with ether and pure water.

[0064] (2) Synthesis of PVP-Ru(bpy)2Cl2: 0.30 g of poly (4-vinyl pyridine) and 0.30 g of Ru(bpy)2Cl2 were dissolved in 50 mL of N,N-dimethylformamide and reacted at 140°C for 24 h under anhydrous and oxygen-free conditions. The conductive metal complex was obtained by precipitation and washing with ether and dialysis with pure water.

[0065] The anti-interference layer covers the surface of the sensitive layer and is a glutaraldehyde cross-linked ascorbic acid oxidase layer, which is used to eliminate the interference of ascorbic acid.

[0066] The diffusion limiting layer covers the surface of the anti-interference layer and is used to control the diffusion rate of uric acid.

[0067] The preparation method of the electrochemical sensor is as follows: (1) Preparation of sensitive layer: The conductive metal complex PVP-Ru(bpy)2Cl2 was prepared into a 50 mg / ml aqueous solution, 10 wt% polyethylene glycol diglycidyl ether was added to the solution, the solution was mixed and then evenly modified on the surface of the substrate electrode by dot coating, a total of 4 μL, and cross-linked and cured at 37°C and 40% humidity for 24 hours to obtain a substrate electrode modified with the sensitive layer; (2) Preparation of anti-interference layer: ascorbic acid oxidase (AAOx) solution dissolved in 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer solution was used at a concentration of 20 mg / ml. 10 wt% bovine serum albumin and 10 wt% glutaraldehyde were added to the solution relative to the mass percentage of AAOx. The enzyme solution was mixed and fixed on the surface of the sensitive membrane layer by dip coating. The dip coating was repeated twice, with an interval of 30 minutes between each times. The anti-interference layer was cross-linked and cured at 37°C and 40% humidity for 24 hours to obtain the anti-interference layer. (3) Preparation of the diffusion limiting layer: Tetrahydrofuran was used as a solvent, and Lubrizol Tecophilic™ SP-80A was added and stirred to dissolve to obtain a solution with a concentration of 100 mg / mL. The solution was evenly coated on the surface of the anti-interference layer by dip coating for 5 times, with an interval of 30 minutes each time. The solution was cross-linked and cured at 37°C and 40% humidity for 24 hours to obtain the electrochemical sensor.

[0068] Comparative Example 1 Comparative Example 1 provides an electrochemical sensor, which includes a base electrode, an anti-interference layer and a diffusion limiting layer. Except that no sensitive layer material is provided in this comparative example, the rest is the same as that of Example 1.

[0069] The electrochemical sensor prepared in Comparative Example 1 was used to measure uric acid in tissue fluid. The results of the comparison between Comparative Example 1 and Example 1 are shown in FIG. Figure 3 As shown, the sensor without a sensitive layer (Comparative Example 1) exhibited a moderate response to uric acid, primarily due to the catalytic activity of the conductive graphite in the sensor electrode substrate and the oxidation of uric acid itself. However, the sensor with a sensitive layer (Example 1) exhibited a higher response and a wider linear range. This comparison validated the catalytic activity of the sensitive layer towards uric acid.

[0070] Comparative Example 2 Comparative Example 2 provides an electrochemical sensor comprising a base electrode, a sensitive layer, an anti-interference layer, and a diffusion-limiting layer. This comparative example is identical to Example 1, except that the diffusion-limiting layer is a conventional diffusion-limiting layer. The conventional diffusion-limiting layer used in Comparative Example 2 is a purchased polyurethane (Selectophore™, Sigma-Aldrich). This polyurethane is dissolved in tetrahydrofuran and then dip-coated on the surface of the anti-interference layer.

[0071] The electrochemical sensor prepared in Comparative Example 1 was used to measure uric acid in tissue fluid. The results were as follows: Figure 4As shown in Table 1, in Comparative Example 1, the sensor without the added sensitive layer exhibited poor uric acid response, with sensitivity and linearity failing to meet the required standards. In contrast, compared with Comparative Example 2, which used commercial polyurethane, the diffusion-limiting layer solution of the present invention extends the linear range to 2000 μM, far exceeding the detection range of conventional methods and fully meeting the required standards. Therefore, the electrochemical sensor prepared by the present invention can be used for precise and continuous measurement of uric acid concentration in liquid environments.

[0072] Table 1 Response sensitivity and linear range of sensors in Example 1 and Comparative Example 2 to uric acid

[0073] The same or similar parts between the various embodiments in this specification can be referred to each other, and each embodiment focuses on the differences from other embodiments.

[0074] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present application should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. An electrochemical sensor for continuous monitoring of uric acid, characterized in that: The sensor includes a base electrode, a sensitive layer, an anti-interference layer and a diffusion limiting layer; The sensitive layer covers the surface of the base electrode and contains a conductive metal complex that catalyzes the oxidation of uric acid; The anti-interference layer covers the surface of the sensitive layer and is used to eliminate the interference of ascorbic acid; The diffusion limiting layer covers the surface of the anti-interference layer and is used to control the diffusion rate of uric acid.

2. An electrochemical sensor for continuous monitoring of uric acid according to claim 1, characterized in that: The conductive metal complex is prepared from a metal M and a polymer ligand, wherein the metal M includes any one of Os, Ir, and Ru; and the polymer ligand includes a monomer and a copolymer.

3. An electrochemical sensor for continuous monitoring of uric acid according to claim 2, characterized in that: The monomer includes any one of poly-4-vinylpyridine and poly-2-vinylimidazole; The copolymer includes any one of poly(vinylimidazole-co-methacrylic acid), poly(vinylimidazole-co-hydroxyethyl methacrylate), and poly(vinylimidazole-co-hydroxypropyl methacrylate).

4. The electrochemical sensor for continuous monitoring of uric acid according to claim 2, characterized in that: The preparation method of the conductive metal complex comprises the following steps: S1: subjecting the polymer monomer to a free radical polymerization reaction in the presence of an initiator to obtain a polymer ligand; S2: heating a soluble metal salt containing metal M and a nitrogen-containing bidentate ligand in a solvent to react to obtain a metal complex; S3: mixing the polymer ligand obtained in step S1 and the metal complex obtained in step S2 in a polar solvent, and heating the mixture to react to form the conductive metal complex.

5. The electrochemical sensor for continuous monitoring of uric acid according to claim 1, characterized in that: The anti-interference layer is an ascorbic acid oxidase layer modified by a cross-linking agent; the cross-linking agent includes any one of an epoxy cross-linking agent and an aldehyde cross-linking agent.

6. The electrochemical sensor for continuous monitoring of uric acid according to claim 1, characterized in that: The diffusion limiting layer is a hydrophilic polymer membrane layer.

7. The electrochemical sensor for continuous monitoring of uric acid according to claim 6, characterized in that: When the diffusion limiting layer is a cross-linked hydrophilic polymer membrane layer, the membrane solution for preparing the diffusion limiting layer uses a mixture of 75%-100% ethanol and 0-25% water as a solvent, and the components include 5%-10% hydrophilic polyurethane or polyvinyl pyridone and 0.5%-3% epoxy cross-linking agent.

8. The electrochemical sensor for continuous monitoring of uric acid according to claim 6, characterized in that: When the diffusion limiting layer is a non-crosslinked hydrophilic polymer membrane layer, the membrane solution for preparing the diffusion limiting layer uses tetrahydrofuran as a solvent, and the components include 5%-15% polyurethane.

9. A method for preparing an electrochemical sensor for continuous monitoring of uric acid as claimed in claim 1, characterized in that: The following steps are involved: A mixed solution of a conductive metal complex and a cross-linking agent is coated on the surface of the working electrode region of the base electrode to form a sensitive layer through a cross-linking reaction; The anti-interference layer and the diffusion limiting layer are coated layer by layer on the surface of the sensitive layer, and each layer is heat-treated and cured at 20-50° C. after coating to obtain the electrochemical sensor.

10. Use of the electrochemical sensor for continuously monitoring uric acid as claimed in claim 1 for continuously monitoring uric acid in a liquid environment.

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