Hollow gold-carbon nanospheres / dog liver aminease double modified platinum microelectrode, its preparation and application

Abstract

The application discloses a hollow gold-carbon nanosphere / kynureninase double-modified platinum microelectrode and a preparation method thereof, and relates to the technical field of microelectrode analysis systems for kynureninase detection. (1) silica spheres are dispersed in a dopamine solution to prepare a silica / polydopamine composite product, potassium chloroaurate is reacted with the composite product to obtain a gold particle modified composite product, the silica core is removed after high-temperature carbonization, and a gold modified hollow carbon nanosphere is obtained; (2) the gold modified hollow carbon nanosphere suspension is fully ultrasonically dispersed, and is coated on the surface of a solid microelectrode to obtain a microelectrode coated with a nanometer material layer after drying at room temperature; (3) a mixed solution containing kynureninase and bovine serum albumin is prepared, is dropped on the microelectrode coated with the nanometer material layer, and after incubation at room temperature, a conductive adhesive is added to obtain the hollow gold-carbon nanosphere / kynureninase double-modified platinum microelectrode. The hollow gold-carbon nanosphere / kynureninase double-modified platinum microelectrode is used in a microelectrode analysis system for kynureninase detection.

Description

Technical Field

[0001] This invention relates to a platinum microelectrode dually modified with hollow gold carbon nanospheres and kynurenase, its preparation method, and its application. Background Technology

[0002] Over 95% of free tryptophan in the human body is degraded as a substrate of the kynurenine pathway (KP), producing metabolites with different biological activities in immune responses and neurotransmission. Kynurenine (Kyn) is the most important and widely studied product among these. Kyn metabolic imbalance is widely associated with human diseases, including malignant tumors, inflammatory bowel disease, pancreatitis, diabetes, and microbial infections. During tumorigenesis, tryptophan is catalyzed by indoleamine-2,3-dioxygenase and tryptophan 2,3-dioxygenase, which are highly expressed in tumors, to produce large amounts of Kyn. Kyn promotes tumor development through mechanisms such as mediating immune escape, stimulating tumor growth, and inducing drug resistance. Therefore, overactivation of the KP pathway is often highly correlated with poor tumor prognosis and can serve as an independent prognostic factor for multiple cancer types. Developing an efficient, convenient, and accurate Kyn detection platform is of great significance for guiding the management and treatment of clinical oncology patients, elucidating the relationship between the KP pathway and tumor development, and accelerating the development of inhibitors targeting this metabolic pathway.

[0003] Currently, the main mature detection methods for kynurenine are high-performance liquid chromatography (HPLC) and high-performance liquid chromatography-mass spectrometry (HPLC-MS). Chinese invention patent specification CN115236248A discloses a method for simultaneously determining the content of tryptophan and kynurenine. This method uses an HPLC-UV instrument, with rat cerebrospinal fluid as the sample. The test conditions are as follows: chromatographic column: Kromasil 100-5-C18; mobile phase: ammonium formate solution in aqueous phase, methanol in organic phase; kynurenine is detected using ultraviolet light at a wavelength of 225 nm, and tryptophan is detected using ultraviolet light at a wavelength of 279 nm. Chinese invention patent specification CN116879460A discloses a method for simultaneously detecting multiple amino acids (including kynurenine) and hormones in blood or cerebrospinal fluid using liquid chromatography-tandem mass spectrometry. This method pretreatment of the sample involves a combination of protein precipitation and solid-phase extraction to obtain an injection sample for detecting amino acids and hormones. The injection sample is then detected using high-performance liquid chromatography-mass spectrometry. The sample detection results are substituted into a standard curve equation to obtain the content of multiple amino acids and hormones in the sample to be tested.

[0004] With the advancement of materials science, organic materials capable of specifically binding to biomolecules are increasingly widely used in the field of biodetection. Chinese Invention Patent Specification CN113880868A discloses a binuclear rare-earth complex crystalline material constructed from rare-earth (III) elements, the ligand 2,5-difluorobenzoic acid, and the auxiliary ligand 1,10-phenanthroline. This material can be used as a fluorescent probe for detecting the tumor marker L-kynurenine in human serum and urine. Dissolving this material in an organic solvent and then reacting it with the tumor marker L-kynurenine in serum or urine allows for quantitative detection of L-kynurenine through fluorescence enhancement at 532 nm.

[0005] HPLC is the most commonly used method for kynurenine detection, applicable to various biological fluids, and boasts advantages such as high resolution, high selectivity, and high sensitivity. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) combines liquid chromatography and mass spectrometry, offering even higher sensitivity and selectivity, making it particularly suitable for analyzing complex samples. However, both techniques suffer from drawbacks such as long analysis times, expensive equipment, and high operating costs, and require complex sample pretreatment procedures (e.g., protein precipitation, solid-phase extraction). Furthermore, the advanced instruments used in these methods must be operated by qualified trained technicians, resulting in time-consuming and highly specialized operations, thus limiting their practical application. Summary of the Invention

[0006] The purpose of this invention is to provide a platinum microelectrode modified with hollow gold carbon nanospheres / kynurenase, its preparation method and application, in order to solve the problems of slow analysis speed and cumbersome sample pretreatment steps in the detection of kynurenine in biological fluids by high performance liquid chromatography and liquid chromatography-mass spectrometry tandem techniques.

[0007] The technical solution provided by this invention is: a method for preparing a platinum microelectrode dual-modified with hollow gold carbon nanospheres and kynurenase, comprising the following steps: (1) Dissolve water and ammonium hydroxide in ethanol, then add tetraethyl orthosilicate, centrifuge to collect silica sphere template after reaction, disperse silica spheres in dopamine solution for reaction, centrifuge and freeze dry the solid product to obtain silicon / polydopamine composite product, potassium chloroaurate reacts with composite product to obtain gold particle modified composite product, carbonize at high temperature under argon, remove silicon core with hydrofluoric acid to obtain gold modified hollow carbon nanosphere powder; (2) The gold-modified hollow carbon nanosphere powder obtained in step (1) is fully ultrasonically dispersed in deionized water. The gold-modified hollow carbon nanosphere suspension is coated on the surface of the solid microelectrode by drop coating, spin coating, spray coating or dip coating method to make the solid microelectrode covered with a nanomaterial coating. The microelectrode coated with nanomaterial layer is obtained by drying at room temperature. (3) Prepare a mixed solution containing kynurenase and bovine serum albumin, drop it onto the microelectrode coated with nanomaterial layer obtained in step (2), incubate at room temperature and wash away the unbound free enzyme, then add conductive adhesive to improve the physical stability of the dual-modified structure, and obtain a platinum microelectrode with hollow gold carbon nanospheres / kynurenase dual modification.

[0008] The solid microelectrode is a platinum microelectrode.

[0009] The silica sphere template was prepared by adding 1.5 ml of deionized water and 5 ml of concentrated ammonia solution with a weight percentage of 25-28% to 100 ml of ethanol, stirring magnetically until homogeneous, and slowly adding 1.5 ml of tetraethyl orthosilicate dropwise (20 drops / minute). The mixture was then hydrolyzed magnetically at room temperature for 4 hours, followed by centrifugation at 12,000 rpm for 15 minutes at 4 degrees Celsius. The resulting white precipitate was washed three times with deionized water to remove unreacted tetraethyl orthosilicate, thus obtaining the silica sphere template.

[0010] The preparation of the silicon / polydopamine composite product is as follows: 200 mg of silica spherical template is weighed and added to 60 mL of deionized water. After ultrasonic dispersion, 72 mg of tris(hydroxymethyl)aminomethane is added and magnetically stirred to mix. Then, 180 mg of dopamine hydrochloride is added and the mixture is stirred at room temperature for 48 hours. The solid product is collected by filtration, washed three times with deionized water, and then freeze-dried to obtain the silicon / polydopamine composite product.

[0011] The preparation of the gold particle-modified composite product is as follows: 200 mg of silicon / polydopamine composite product is weighed and ultrasonically dispersed in 50 mL of deionized water. Then, 2 mL of 50 mg / mL potassium chloroaurate solution is slowly added (10 drops / min), and the mixture is stirred in an ice bath for 30 minutes. The solid product is collected by vacuum filtration, washed three times with deionized water, and then freeze-dried to obtain the gold particle-modified composite product.

[0012] The gold-modified hollow carbon nanospheres were prepared by calcining the composite product modified with gold particles at 500°C for 3 hours under argon protection, followed by the addition of 4% hydrofluoric acid by weight and thorough stirring to digest the silicon cores. After digestion for 4 hours, 30 mL of 20% sodium bicarbonate solution was added to neutralize the hydrofluoric acid, and the pH of the mixture was adjusted to alkaline to terminate the reaction. The solid product was collected by filtration, washed, and freeze-dried to obtain gold-modified hollow gold carbon nanospheres.

[0013] The microelectrode coated with the nanomaterial layer was prepared by ultrasonically dispersing a 5 mg / mL hollow gold carbon nanosphere suspension and then adding 3 μL of the suspension to the tip of the solid microelectrode, followed by natural air drying at room temperature. The platinum microelectrode dual-modified with hollow gold carbon nanospheres and kynurenase was prepared by adding 3 μL of a 200 μg / mL kynurenase solution containing bovine serum albumin to the microelectrode coated with the nanomaterial layer, incubating at room temperature for 4 hours to allow the enzyme protein to bind to the gold nanoparticles through gold-sulfur bonds and physical adsorption, then gently rinsing the electrode with phosphate buffer to remove unbound free enzyme protein, and finally adding 2 μL of 0.5% Nafion solution to maintain the physical stability of the modified material.

[0014] The present invention also provides a platinum microelectrode modified with hollow gold carbon nanospheres / kynurenase by the above preparation method.

[0015] This invention also provides the application of the above-mentioned hollow gold carbon nanosphere / kynurenase dual-modified platinum microelectrode for kynurenine detection in a microelectrode analysis system.

[0016] Preferably, the working electrode of the microelectrode system is one or more of non-metallic microelectrodes (carbon fiber, glass, diamond, etc.) and metallic microelectrodes (platinum, copper, tungsten, etc.); the counter electrode material is one or more of gold, silver, and platinum; and the reference electrode is one or more of silver / silver chloride and calomel electrodes.

[0017] Preferably, the working electrode has a length between 500 mm and 2000 mm and a bottom diameter between 5 μm and 500 μm.

[0018] Preferably, the nanomaterial used for modifying the working electrode is one or more of carbon nanomaterials (graphene, carbon nanotubes, etc.), metal nanomaterials, and nanocomposites.

[0019] Preferably, the nanomaterial is coated onto the surface of the working electrode by one or more of the following methods: drop coating, spin coating, dip coating, and electrodeposition; the electrodeposition method is one or more of the following: cyclic voltammetry, potentiostatic method, and galvanostatic method.

[0020] Preferably, the enzyme loaded is one or more of hydrolases, oxidases, and dehydrogenases, and is loaded onto the nanomaterial coating on the surface of the working electrode through one or more of doping, physical embedding, and covalent bonding.

[0021] Preferably, the conductive adhesive drop-coated on the surface of the microelectrode is one or more of perfluorosulfonic acid polymer, chitosan, and epoxy resin.

[0022] Preferably, the enzyme loading capacity is adjusted by changing one or more of the electrode surface area, nanomaterial composition, and film thickness.

[0023] Preferably, the method for acquiring the response signal changes of the microelectrode system to changes in amino acid metabolite concentration using an electrochemical workstation includes one or more of the following: differential pulse voltammetry, cyclic voltammetry, chronoamperometry, square wave voltammetry, and linear scan voltammetry.

[0024] This invention uses a platinum microelectrode modified with hollow gold carbon nanospheres and kynurenase as the working electrode, a silver / silver chloride electrode as the reference electrode, and a bare platinum electrode as the counter electrode. These electrodes are connected to the corresponding ports of an electrochemical workstation. Cyclic voltammetry, electrochemical impedance spectroscopy, and differential pulse voltammetry are used to analyze the kynurenase sensing performance of the microelectrode system.

[0025] This invention relates to a microworking electrode based on a dual modification of hollow gold carbon nanospheres and an enzyme. The carbon nanospheres have a large specific surface area, excellent conductivity, and abundant electron transfer active sites, which can reduce the overpotential required during material analysis. Their stable skeletal structure facilitates the loading of gold nanoparticles. The gold nanoparticles accelerate charge carrier migration and improve the response of biosensors. Kynureinase is stably modified on the electrode surface via gold-sulfur bonds, specifically catalyzing the hydrolysis of kynureine to generate electroactive downstream products. This multifunctional modification synergistically amplifies the electrochemical reaction signal of the electrode, improving the speed, sensitivity, and specificity of electrochemical detection. Attached Figure Description

[0026] Figure 1 A schematic diagram showing a microelectrode system connected to an electrochemical workstation and operating in a supporting electrolyte solution; Figure 2 Differential pulse voltammetric response and calibration curves of a system constructed using hollow gold carbon nanospheres / kynurenase dual-modified platinum wire as the working electrode, platinum as the counter electrode, and silver / silver chloride as the reference electrode to phosphate buffer containing different concentrations of kynurenine. Figure 3 This is a comparison of the relative signal intensities of the above microelectrode system for kynurenine, a series of electroactive and redox active substances in the body.

[0027] Figure 4 This paper presents a comparative analysis of the results of the microelectrode system of the present invention in determining the concentration of kynurenine in human serum with those obtained by HPLC.

[0028] Figure labels: 1-working electrode, 2-reference electrode, 3-counter electrode, 4-electrochemical workstation. Implementation

[0029] See Figure 1 The present invention provides a method for preparing a microelectrode analysis system for kynurenine detection as follows: (1) Silver wire and platinum wire microneedle electrodes are obtained through melting, pressing into square strips, drawing, shaping and polishing processes. Polishing makes the surface smooth and repeated cleaning removes surface impurities. (2) The silver wire microelectrode described in step (1) is loaded with a layer of silver chloride on the electrode surface by constant potential electrodeposition in hydrochloric acid solution to obtain a silver-silver chloride microelectrode as a reference electrode; (3) Dissolve water and ammonium hydroxide in ethanol, then add tetraethyl orthosilicate, centrifuge to collect silica sphere template after reaction, disperse the silica spheres in dopamine solution for reaction, centrifuge and freeze dry the solid product to obtain silicon / polydopamine composite product, potassium chloroaurate reacts with the composite product to obtain gold particle modified composite product, carbonize at high temperature under argon, remove silicon core with hydrofluoric acid to obtain gold modified hollow carbon nanospheres; (4) The composite nanomaterial suspension obtained in step (3) is fully ultrasonically dispersed, and a nanomaterial coating is applied to the surface of the solid microelectrode by drop coating, spin coating, spray coating or dip coating, and then dried at room temperature; (5) Prepare a mixed solution containing kynurenase and bovine serum albumin, drop it onto the microelectrode coated with nanomaterial layer obtained in step (4), incubate at room temperature, wash away the unbound free enzyme, and then add conductive adhesive to improve the physical stability of the double-modified structure to obtain the working electrode. (6) The double-modified platinum wire obtained in step (5) is the working electrode 1, the silver / silver chloride electrode obtained in step (2) is the reference electrode 2, and the bare platinum electrode is the counter electrode 3. They are connected to the corresponding ports of the electrochemical workstation 4 respectively. Cyclic voltammetry, electrochemical impedance spectroscopy and differential pulse voltammetry are used to analyze the kynurenine sensing performance of the microelectrode system.

[0030] Specific examples are as follows: Synthesis of hollow gold-carbon nanospheres: First, SiO2 sphere template was synthesized: 1.5 mL of deionized water and 5 mL of concentrated ammonia (25-28%) were added to 100 mL of ethanol and magnetically stirred until homogeneous. 1.5 mL of tetraethyl orthosilicate was added dropwise (20 drops / min) and hydrolyzed magnetically at room temperature for 4 hours. After centrifugation at 12000 rpm for 15 minutes, a white precipitate was obtained. This precipitate was washed three times with deionized water to remove unreacted tetraethyl orthosilicate, thus obtaining the SiO2 sphere template. Next, the SiO2@PDA complex was synthesized: 200 mg of SiO2 spheres were weighed and added to 60 mL of deionized water. After thorough ultrasonic dispersion, 72 mg of tris(hydroxymethyl)aminomethane was added and magnetically stirred until homogeneous. Subsequently, 180 mg of dopamine hydrochloride was added, and the reaction was stirred at room temperature for 48 hours. The reaction system gradually changed from pale white to black. The solid product was collected by filtration, washed three times with deionized water, and lyophilized to obtain the SiO2@PDA complex. Then, SiO2@PDA@Au was synthesized: 200 mg of the SiO2@PDA complex was weighed and ultrasonically dispersed in 50 mL of deionized water. Then, 2 mL of 50 mg / mL potassium chloroaurate solution was slowly added (10 drops / min), and the reaction was vigorously stirred in an ice bath for 30 minutes. The solid product was collected by filtration, washed three times with deionized water, and then lyophilized to obtain SiO2@PDA@Au. Finally, the obtained SiO2@PDA@Au was calcined at 500°C for 3 hours under argon protection to carbonize it. The calcined product was transferred to a plastic reaction vessel, and 4% hydrofluoric acid was added. The mixture was stirred thoroughly to digest the silicon core. After 4 hours of digestion, 30 mL of sodium bicarbonate solution (20%) was added to neutralize the hydrofluoric acid, and the pH of the mixture was adjusted to alkaline to terminate the reaction. The solid product was collected by filtration, washed, and lyophilized to finally obtain hollow gold carbon nanospheres (AuHCNs).

[0031] Preparation of platinum and silver / silver chloride microelectrodes: Platinum of 99.999% purity was melted at high temperature to obtain liquid platinum, which was then pressed into square strips, drawn into wires, shaped, and polished to obtain platinum wire microelectrodes with a diameter of 200 micrometers. Similarly, silver of 99.999% purity was melted at high temperature to obtain liquid silver, which was then pressed into square strips, drawn into wires, shaped, and polished to obtain silver wire microelectrodes. The silver wires were then deposited at a constant potential of -0.1 V for 1800 seconds in a 0.1 mol / L hydrochloric acid solution to obtain silver / silver chloride electrodes, which served as reference electrodes.

[0032] Modification of platinum microelectrodes: Small amounts of 0.3 μm and 0.05 μm aluminum oxide polishing powder were sequentially placed on a chamois polishing disc, and a suitable amount of deionized water was added, stirring until a slurry was formed. The platinum electrode was slowly polished, followed by ultrasonic cleaning with deionized water, anhydrous ethanol, and deionized water for 3 minutes each, and then allowed to air dry at room temperature until the platinum electrode surface was smooth and free of impurities. Modification material was then applied by ultrasonically dispersing a 5 mg / mL hollow gold carbon nanosphere suspension and carefully adding 3 μL to the tip of the bare platinum electrode. The electrode was allowed to air dry at room temperature, followed by adding 3 μL of a 200 μg / mL solution of kynurenase containing bovine serum albumin. The electrode was incubated at room temperature for 4 hours to allow the enzyme protein to bind to the gold nanoparticles through gold-sulfur bonds and physical adsorption. The electrode was then gently rinsed with phosphate buffer to remove unbound free enzyme protein. Finally, 2 μL of Nafion solution (0.5%) was added to maintain the physical stability of the modified material, and the electrode was stored at low temperature.

[0033] In the microelectrode based on the dual modification of hollow gold carbon nanospheres and enzymes described in the above embodiments, the carbon nanospheres have a large specific surface area, excellent conductivity, and abundant electron transfer active sites, which can reduce the overpotential required during material analysis. Their stable skeletal structure facilitates the loading of gold nanoparticles. The gold nanoparticles accelerate charge carrier migration and improve the response of biosensors. Kynurenase is stably modified on the electrode surface via gold-sulfur bonds, specifically catalyzing the hydrolysis of kynurenine to generate electroactive downstream products. This multifunctional modification synergistically amplifies the electrochemical reaction signal of the electrode, improving the speed, sensitivity, and specificity of electrochemical detection.

[0034] The hollow gold carbon nanosphere / kynurenase dual-modified platinum microelectrode fabricated in this invention is used to test phosphate buffer containing different concentrations of kynurenine. Figure 2 The differential pulse voltammetric response and linear calibration curves (0.1–80 μM) of the microelectrode system to phosphate buffer containing different concentrations of kynurenine are shown. According to the calibration curves, the sensitivity of the hollow gold carbon nanosphere / kynurenase / platinum wire microelectrode is 0.06594 μA / (μM mm2), and the detection limit is as low as 0.092 μM. Figure 3 The comparison of the relative signal intensities of the microelectrode system for kynurenine and a series of electroactive and redox reactive substances in the body (Trp: tryptophan, GSH: glutathione, H2O2: hydrogen peroxide, Glu: glucose, DA: dopamine, AA: ascorbic acid, UA: uric acid) demonstrates that the generated current signal is not easily interfered with by other substances. Crucially, the microelectrode system requires only 90 seconds for a single differential pulse voltammetric scan, a speed far exceeding that of high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) tandem techniques (which take several hours). Furthermore, biological fluids such as serum can be directly sampled and detected without pretreatment steps such as protein precipitation, solid-phase extraction, or derivatization. Figure 4The data show the accuracy verification of kynurenine content in the serum of 12 patients before radical resection of colorectal cancer using the microelectrode system. The results are basically consistent with those of high performance liquid chromatography analysis, indicating that the microelectrode system can accurately quantify kynurenine in serum.

Claims

1. A method for preparing a platinum microelectrode dual-modified with hollow gold carbon nanospheres and kynurenase, comprising the following steps: (1) Dissolve water and ammonium hydroxide in ethanol, then add tetraethyl orthosilicate, centrifuge to collect silica sphere template after reaction, disperse silica spheres in dopamine solution for reaction, centrifuge and freeze dry the solid product to obtain silicon / polydopamine composite product, potassium chloroaurate reacts with the composite product to obtain gold particle modified composite product, carbonize at high temperature under argon, remove silicon core with hydrofluoric acid to obtain gold modified hollow carbon nanosphere powder; (2) Disperse the gold modified hollow carbon nanosphere powder obtained in step (1) in deionized water by ultrasonication, coat the gold modified hollow carbon nanosphere suspension on the surface of solid microelectrode by drop coating, spin coating, spray coating or dip coating to coat the solid microelectrode with nanomaterial coating, and dry at room temperature to obtain microelectrode coated with nanomaterial layer; (3) Prepare a mixed solution containing kynurenase and bovine serum albumin, drop it onto the microelectrode coated with nanomaterial layer obtained in step (2), incubate at room temperature and wash away the unbound free enzyme, then add conductive adhesive to improve the physical stability of the dual-modified structure, and obtain a platinum microelectrode with hollow gold carbon nanospheres / kynurenase dual modification.

2. The preparation method according to claim 1, characterized in that: The silica sphere template was prepared by adding 1.5 ml of deionized water and 5 ml of concentrated ammonia (25-28% by weight) to 100 ml of ethanol, stirring magnetically until homogeneous, adding 1.5 ml of tetraethyl orthosilicate dropwise, and hydrolyzing magnetically at room temperature for 4 hours. After centrifugation at 12,000 rpm for 15 minutes at 4 degrees Celsius, a white precipitate was obtained. The precipitate was washed three times with deionized water to remove unreacted tetraethyl orthosilicate, which yielded the silica sphere template.

3. The preparation method according to claim 1, characterized in that: The preparation of the silicon / polydopamine composite product is as follows: 200 mg of silica sphere template is weighed and added to 60 mL of deionized water. After ultrasonic dispersion, 72 mg of tris(hydroxymethyl)aminomethane is added and magnetically stirred to mix. Then, 180 mg of dopamine hydrochloride is added and the mixture is stirred at room temperature for 48 hours. The solid product is collected by filtration, washed three times with deionized water, and then freeze-dried to obtain the silicon / polydopamine composite product.

4. The preparation method according to claim 1, characterized in that: The preparation of the gold particle-modified composite product is as follows: 200 mg of silicon / polydopamine composite product is weighed and ultrasonically dispersed in 50 mL of deionized water. Then, 2 mL of 50 mg / mL potassium chloroaurate solution is added, and the mixture is stirred in an ice bath for 30 minutes. The solid product is collected by vacuum filtration, washed three times with deionized water, and then freeze-dried to obtain the gold particle-modified composite product.

5. The preparation method according to claim 1, characterized in that: The gold-modified hollow carbon nanospheres are composite products modified with gold particles. After calcination at 500°C for 3 hours under argon protection, 4% hydrofluoric acid is added and stirred thoroughly to digest the silicon core. After digestion for 4 hours, 30 mL of 20% sodium bicarbonate solution is added to neutralize the hydrofluoric acid, the pH of the mixture is adjusted to alkaline, and the reaction is terminated. The solid product was collected by vacuum filtration, washed, and freeze-dried to obtain gold-modified hollow gold carbon nanosphere powder.

6. The preparation method according to claim 1, characterized in that: The microelectrode coated with the nanomaterial layer was prepared by ultrasonically dispersing a 5 mg / mL hollow gold carbon nanosphere suspension and then adding 3 μL of the suspension to the tip of the solid microelectrode, followed by natural air drying at room temperature. The platinum microelectrode dual-modified with hollow gold carbon nanospheres and kynurenase was prepared by adding 3 μL of a 200 μg / mL kynurenase solution containing bovine serum albumin to the microelectrode coated with the nanomaterial layer, incubating at room temperature for 4 hours to allow the enzyme protein to bind to the gold nanoparticles through gold-sulfur bonds and physical adsorption, then gently rinsing the electrode with phosphate buffer to remove unbound free enzyme protein, and finally adding 2 μL of 0.5% Nafion solution to maintain the physical stability of the modified material.

7. A platinum microelectrode dually modified with hollow gold carbon nanospheres and kynurenase, prepared by the preparation method according to any one of claims 1 to 6.

8. A microelectrode analysis system for the detection of kynurenine, wherein the platinum microelectrode is the platinum microelectrode with hollow gold carbon nanospheres / kynurenase dual modification as described in claim 7.

9. The microelectrode analysis system according to claim 8, characterized in that: A platinum microelectrode modified with hollow gold carbon nanospheres / kynurenase was used as the working electrode, a silver / silver chloride electrode as the reference electrode, and a bare platinum electrode as the counter electrode. These electrodes were connected to the corresponding ports of an electrochemical workstation. Cyclic voltammetry, electrochemical impedance spectroscopy, and differential pulse voltammetry were used to analyze the kynurenine sensing performance of the microelectrode system.

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