Uric acid biosensor electrode substrate and preparation method and application thereof

Abstract

The invention discloses a uric acid biosensor electrode substrate and a preparation method and application thereof, and relates to the technical field of biosensors. The uric acid biosensor electrode substrate is prepared by modifying the surface of an electrode with a poly (3-methylthiophene) membrane and assembling a gold nanocage on the surface of the poly (3-methylthiophene) membrane. The electrode substrate is modified by using poly-3-methylthiophene, the assembly efficiency of the gold nanocage on the surface of the electrode is effectively improved, the poly3-methylthiophene and the gold nanocage are synergistically catalyzed, the redox peaks of interferents can be effectively separated, the uric acid detection effect is ensured, and the accuracy and feasibility of the detection technology are improved. The uric acid biosensor provided by the invention has the advantages of high accuracy, good selectivity, high sensitivity and the like, can realize quantitative detection of uric acid, and has important application value in the fields of clinical diagnosis and drug research of related diseases such as purine metabolic disorder and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and particularly to a uric acid biosensor electrode substrate, a preparation method thereof, and an application thereof. Background Art

[0002] Uric acid (UA), as the final product of purine metabolism in organisms, its content disorder can cause the occurrence of diseases such as hyperuricemia, gout, leukemia, etc. Therefore, UA is an effective biomarker for diagnosing purine metabolism disorders and related diseases.

[0003] Currently, the methods for detecting UA mainly include electrochemical sensing method, fluorescence spectroscopy method, sol-gel method, capillary electrophoresis method, colorimetry method, high performance liquid chromatography method, etc. Among them, electrochemical technology has the advantages of simple operation, rapid detection, high sensitivity, portable instrument, etc. It can perform quantitative detection by detecting the redox signal of UA. However, the composition of human body fluids is complex, and UA usually coexists with substances such as ascorbic acid (AA) and dopamine (DA), and the redox potentials of these substances are relatively close, making it a great challenge to directly detect UA in biological samples.

[0004] Due to its good electrical conductivity, gold nanomaterials are widely used in the field of electrochemical sensing. Existing research mostly uses electrodeposition technology to prepare electrodes modified with nano-gold. This method is easy to operate, but there is a problem of non-uniform size of nano-gold particles, and the electrodeposited nano-gold particles are usually solid structures, which limits the electrocatalytic performance and detection efficiency of nano-gold electrodes. And hollow-structured gold nanomaterials (such as gold nanocages, GNC) have a larger specific surface area and more reaction sites. Research shows that the morphology and size of nano-gold can be regulated by changing the local microenvironment and electrode substrate. Summary of the Invention

[0005] The purpose of the present invention is to provide a uric acid biosensor electrode substrate, a preparation method thereof, and an application thereof to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] One of the technical solutions of the present invention: Provide a biosensor electrode substrate, which is prepared by modifying a poly(3-methylthiophene) film on the electrode surface and assembling gold nanocages on the surface of the poly(3-methylthiophene) film.

[0008] As a further preference of the present invention, the electrode is a glassy carbon electrode.

[0009] As a further preference of the present invention, 3-methylthiophene is used as a monomer, and the poly(3-methylthiophene) film is modified on the electrode surface by electro-polymerization.

[0010] As a further preference of the present invention, in the process of electro-polymerization, the electrode is placed in an acetonitrile solution containing 3-methylthiophene and NaClO4, and cyclic voltammetry scanning is performed (preferably 0.0 - 1.7 V, and not 0 V), and then potentiostatic growth is carried out (preferably 0.7 V), so that 3-methylthiophene (3MT) is polymerized into a film. Further, it also includes the step of scanning the electro-polymerized electrode material in a buffer solution to obtain a stable signal.

[0011] As a further preference of the present invention, the glassy carbon electrode is pretreated, and the pretreatment process includes: polishing the glassy carbon electrode into a mirror surface with an alumina suspension, and ultrasonically cleaning it with absolute ethanol and deionized water, and drying it with nitrogen for standby.

[0012] As a further preference of the present invention, a gold nanocage solution is placed on the surface of the poly(3-methylthiophene) film for gold nanocage assembly.

[0013] As a further preference of the present invention, the gold nanocages are prepared by a replacement reaction using silver nanoparticles as sacrificial templates and polyvinylpyrrolidone as a stabilizer.

[0014] Even further, the preparation method of the gold nanocages includes the following steps:

[0015] Under a protective atmosphere, ethylene glycol and a Na2S solution are subjected to condensation reflux reaction (preferably 10 min), and then polyvinylpyrrolidone is added for a mixing reaction (preferably 10 min), and then an AgNO3 solution is added and reacted until the system shows a greenish-brown color, and the product silver nanoparticles are collected;

[0016] The silver nanoparticles and a polyvinylpyrrolidone solution are subjected to a mixed heating reaction (preferably 90 °C), and then chloroauric acid HAuCl4 is added, and AuCl4 - ions are gradually deposited on the surface of the silver nanoparticles as elemental gold, and at the same time, the elemental silver inside the nanoparticles is replaced by silver ions and enters the solution. After the reaction until the color of the system is stable, it is washed with a saturated saline solution and ultrapure water to obtain gold nanocages.

[0017] The conductive polymer thin film modified electrode can introduce a high density of functional groups at the sensing interface, can provide a three-dimensional space reaction field and stable functional groups, and has excellent electrocatalytic performance and unique applications. In the present invention, a stable conductive polymer poly(3-methylthiophene) (P3MT) is electro-polymerized on the electrode surface through the electro-oxidation of 3-methylthiophene monomers. The heterocyclic structure of P3MT contains sulfur atoms, which can form strong Au-S chemical bonds with gold nanoparticles. P3MT can provide a good substrate and microenvironment for the assembly of hollow-structured gold nanocages on the electrode surface.

[0018] The second technical solution of the present invention: Provide a method for preparing the above-mentioned biosensor electrode substrate, including the following steps:

[0019] Using 3-methylthiophene as a monomer, electrochemically polymerize to modify a poly-3-methylthiophene film on the electrode surface, and then assemble gold nanocages onto the surface of the poly-3-methylthiophene film to prepare the biosensor electrode substrate.

[0020] The third technical solution of the present invention: Provide the application of the above-mentioned biosensor electrode substrate as a biosensor electrode substrate for uric acid detection.

[0021] The fourth technical solution of the present invention: A method for detecting uric acid based on the above-mentioned biosensor electrode substrate, including the following steps:

[0022] Add the sample to be tested into a phosphate buffer solution and mix it to serve as the electrolyte;

[0023] Use an Ag / AgCl electrode as the reference electrode, a platinum wire as the auxiliary electrode, and the above-mentioned biosensor electrode substrate as the working electrode to form a three-electrode system;

[0024] Place the three-electrode system in the electrolyte to detect uric acid in the sample to be tested.

[0025] More preferably, the concentration of the phosphate buffer is 0.1 M and the pH is 7.0; the mixing volume ratio of the phosphate buffer solution to the sample to be tested is 20:1.

[0026] More preferably, place the three-electrode system in the electrolyte, use an electrochemical workstation to record the differential pulse voltammetry (DPV) signal, and calculate the uric acid content according to the linear standard curve; even more preferably, the voltage range of DPV is -0.2 to 0.6 V, the pulse amplitude is 50 mV, the pulse width is 50 ms, the sampling width is 20 ms, and the pulse period is 200 ms.

[0027] The fifth technical solution of the present invention: Provide a uric acid detection biosensor, including the above-mentioned biosensor electrode substrate.

[0028] The sixth technical solution of the present invention: Provide the application of the above-mentioned uric acid detection biosensor in detecting uric acid.

[0029] The present invention discloses the following technical effects:

[0030] The present invention uses poly-3-methylthiophene to modify the electrode substrate, effectively improving the assembly efficiency of gold nanocages on the electrode surface. The poly-3-methylthiophene and gold nanocages synergistically catalyze, can effectively separate the oxidation-reduction peaks of interfering substances, ensure the uric acid detection effect, and improve the accuracy and feasibility of the detection technology.

[0031] The uric acid biosensor of the present invention has advantages such as high accuracy, good selectivity, and high sensitivity, can achieve quantitative detection of uric acid, and has important application value in the clinical diagnosis of related diseases such as purine metabolism disorders and the field of drug research. Description of the Drawings

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0033] Figure 1 SEM imaging and CV characterization diagrams of GNC / P3MT / GCE prepared for Example 1; among them, (A) is the SEM diagram of GNC / P3MT / GCE; (B) is the CV response diagram of different electrodes in a mixed solution of 5 mM K3[Fe(CN)6] and 0.1 M KCl, and curves a to d correspond to GCE, GNC / GCE, P3MT / GCE, and GNC / P3MT / GCE in sequence;

[0034] Figure 2 Response diagrams of different electrodes to UA and AA in Example 2; among them, (A) and (B) are the CV response diagrams of different electrodes to PBS buffer solutions containing 0.01 mM UA and 0.04 mM AA alone, and curves a to d correspond to GCE, GNC / GCE, P3MT / GCE, and GNC / P3MT / GCE in sequence; (C) and (D) are the CV response and DPV response diagrams of different electrodes to PBS buffer solutions containing 0.01 mM UA and 0.04 mM AA at the same time, and curves a and b correspond to GCE and GNC / P3MT / GCE respectively;

[0035] Figure 3 Response diagrams of GNC / P3MT / GCE prepared for Example 1 at different scan rates and pH values; among them, (A) is the CV response diagram of GNC / P3MT / GCE to 0.01 mM UA at different scan rates, and curves a to i correspond to 25, 50, 75, 100, 150, 200, 250, 300, and 350 mV s -1 ; (B) is the linear relationship diagram between the peak current and the scan rate; (C) is the current response diagram of GNC / P3MT / GCE to 0.01 mM UA at different pH values; (D) is the relationship diagram between the peak potential and pH;

[0036] Figure 4Detection results of different concentrations of UA samples by GNC / P3MT / GCE; among them, (A) is the DPV response curve of GNC / P3MT / GCE to different concentrations of UA, and the curves a to k are UA at 0, 1, 2, 5, 7, 9, 12, 15, 18, 22, and 26 μM in sequence, and the concentration of AA is 0.02 mM. (B) is the linear relationship diagram between the peak current value and the UA concentration;

[0037] Figure 5 Detection results of blood samples from healthy volunteers (numbered a to c) and blood samples from gout patients (numbered d to f). Detailed implementation manners

[0038] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation manners of the present invention.

[0039] It should be understood that the terms described in the present invention are only used to describe specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0040] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0041] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0042] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0043] Example 1

[0044] Preparation of the sensing substrate:

[0045] (1) Preparation of gold nanocages GNC:

[0046] In a nitrogen atmosphere, 30 mL of ethylene glycol was heated to 150 °C, and a newly prepared Na2S solution (3 mM) was slowly added and refluxed for 10 minutes; then 7.5 mL of polyvinylpyrrolidone PVP (20 mg / mL) was added and reacted for 10 minutes, followed by the addition of 2.5 mL of AgNO3 solution (48 mg / mL) and reacted until it turned green-brown. After washing the resulting silver nanoparticles, they were stored in water. Take 5 mL of silver nanoparticles (10 mg / mL) and mix and stir with 45 mL of PVP solution (5 mg / mL) and heat to 90 °C. Dropwise add chloroauric acid solution (0.5 mM) until the color of the mixture is stable. Finally, wash with saturated saline solution and ultrapure water in turn, and disperse in ultrapure water to obtain a GNC dispersion.

[0047] (2) Construction of the sensing interface GNC / P3MT / GCE:

[0048] a. First, the glassy carbon electrode (GCE) was polished into a mirror surface with 1.0 μm, 0.3 μm, and 0.05 μm alumina suspensions in turn, and ultrasonically cleaned with absolute ethanol and deionized water for 5 min each, and dried with nitrogen for standby;

[0049] b. The GCE was placed in an anhydrous acetonitrile solution containing 3-methylthiophene (3MT, 0.1 M) and NaClO4 (0.1 M), and cyclic voltammetry (CV) scanning was performed at a voltage of 0.0 to 1.7 V (scanning rate 20 mV s -1 , 3 cycles), then potentiostatic growth was carried out at 0.7 V for 10 s to polymerize 3MT into a film, and the electrode was placed in a phosphate buffer solution (PBS, pH 7.0) and scanned 10 cycles in the range of 0.0 to 0.7 V (scanning rate 50 mV s -1 ), and then scanned repeatedly between -0.2 and +0.5 V at a scanning rate of 100 mV s -1 until a stable signal was obtained to get P3MT / GCE;

[0050] c. The GNC dispersion (2 mM) was drop-coated onto the surface of P3MT / GCE and dried at room temperature for 12 hours to obtain the sensing substrate GNC / P3MT / GCE.

[0051] Comparative Example 1

[0052] Preparation of GNC / GCE substrate:

[0053] a. The GCE was polished into a mirror surface with 1.0 μm, 0.3 μm, and 0.05 μm alumina suspensions in turn, and ultrasonically cleaned with absolute ethanol and deionized water for 5 min each, and dried with nitrogen for standby;

[0054] b. Prepare gold nanocages by the method in Example 1, dropwise coat its dispersion (2 mM) onto the surface of GCE, and dry it at room temperature for 12 hours to obtain the sensing substrate GNC / GCE.

[0055] The present invention uses poly(3-methylthiophene) to improve the microenvironment of the biosensor electrode. The hollow structure of the gold nanocages provides more reaction sites, and the two act synergistically to form the sensing substrate of the biosensor.

[0056] Characterize the sensing substrate GNC / P3MT / GCE prepared in Example 1 and the GNC / GCE substrate prepared in Comparative Example 1.

[0057] The present invention uses scanning electron microscopy (SEM) to characterize the morphology of GNC / P3MT / GCE. As can be seen from (A) in Figure 1 , the GNC presents a hollow spherical structure with an average particle size of about 50 nm, and is uniformly distributed on the electrode surface. CV tests were carried out in a 5 mM K3[Fe(CN)6] solution to characterize the construction of the sensing interface. As shown in (B) in Figure 1 , compared with the bare GCE (curve a), the peak current value obtained by GNC / GCE (curve b) increases, and the peak potential shifts negatively. This is because GNC has good conductivity and electrocatalytic performance, which can reduce the overpotential and accelerate electron transfer. Similarly, the peak current obtained by P3MT / GCE (curve c) is larger than that of the bare GCE, and the peak potential remains basically unchanged. This is due to the excellent conductivity of P3MT itself, which can effectively enhance the electron transfer of the sensing interface. In addition, after further modifying GNC on the surface of P3MT / GCE, the peak current value reaches the maximum (curve d). This phenomenon can be attributed to the synergistic effect of P3MT and GNC, and the hollow structure of GNC increases the specific surface area of the sensing interface.

[0058] The present invention uses poly(3-methylthiophene) and gold nanocages to modify the electrode, effectively separating the electrochemical oxidation signals of UA and AA and improving the detection efficiency of UA. Therefore, monitor and verify the electrochemical signals of UA and AA, the effective discrimination of the oxidation peaks of UA and AA, the detection conditions of UA, and the detection mechanism:

[0059] (1) Characterization of the electrochemical signals of UA and AA: In a PBS buffer solution with a pH of 7.0, test the CV responses of different electrodes to UA and AA:

[0060] Figure 2(A) shows the test results of different electrodes for UA (0.01 mM). The bare GCE exhibits a small and broad oxidation peak (curve a), indicating that the oxidation process of UA on the bare GCE is completely irreversible, and the bare GCE is not suitable for UA detection. When GNC (curve b) and P3MT (curve c) are modified onto the electrode surface, it can be seen that the peak current increases significantly, and the current signals increase by 2 times and 7 times respectively, indicating that both GNC and P3MT modifications on the electrode can accelerate the interfacial electron transfer. Importantly, after GNC and P3MT are simultaneously modified onto the bare GCE (curve d), the peak current increases by 10 times, and there is a well-defined reversible redox peak, indicating that GNC / P3MT / GCE has a strong catalytic effect on the oxidation of UA. Figure 2 (B) shows the test results of different electrodes for AA (0.04 mM AA). For the electrochemical oxidation of AA, the peak current of GNC / P3MT / GCE (curve d) increases by 7 times compared to the bare GCE (curve a), and the peak potential value shifts negatively by 17 mV. While for GNC / GCE (curve b) and P3MT / GCE (curve c), the increases are about 2 times and 5 times respectively, also indicating that GNC / P3MT / GCE has the best catalytic effect on the oxidation of AA. Therefore, it can be concluded that GNC / P3MT / GCE exhibits excellent electrocatalytic activity towards the oxidation of UA and AA.

[0061] Effective discrimination of the oxidation peaks of UA and AA. In body fluids such as cell fluid and serum, UA usually coexists with AA, and their oxidation potentials are relatively close, resulting in difficulties in detecting overlapping signals in the actual sample detection. The sensing substrate GNC / P3MT / GCE prepared in this invention can effectively separate the oxidation peak signals of UA and AA. As Figure 2 shown in (C), when the bare GCE detects a mixed solution of UA and AA (UA concentration is 0.01 mM, AA concentration is 0.04 mM), two relatively broad and partially overlapping oxidation peaks (curve a) are obtained, and the individual concentrations of UA and AA cannot be determined; while for the CV curve obtained by GNC / P3MT / GCE (curve b), two independent and narrow oxidation peaks are shown, with peak potentials of 323 mV and 143 mV respectively, and the difference between the two peak potentials is large enough, indicating that GNC / P3MT / GCE can effectively distinguish the electrochemical signals of UA and AA and can perform the simultaneous determination of UA and AA. In addition, the experimental results detected by differential pulse voltammetry (DPV) are consistent with those of CV, as Figure 2 shown in (D). When measured by the bare GCE, the oxidation peaks of UA and AA overlap, and their respective concentrations cannot be determined from the mixture. While when GNC / P3MT / GCE is used as the detection electrode, the oxidation peaks of UA and AA appear at 290 mV and 111 mV respectively, and the difference between their peak potentials is 179 mV, enabling the simultaneous detection of UA and AA.

[0062] (2) UA detection conditions and detection mechanism:

[0063] In this invention, the responses of GNC / P3MT / GCE to UA at different scanning rates were investigated. The CV peak current increased linearly with the increase of the scanning rate, as shown in (A)-(B) below. The linear equation was I = 6.211 + 0.0630v (R Figure 3 = 0.9989), indicating that the adsorption of UA on the surface of GNC / P3MT / GCE was controlled. 2 Figure (C) below shows the relationship curve between the response current of UA and pH. As shown in the figure, UA had a large signal response in a weakly acidic medium. When pH was equal to 4, the current signal of UA was the largest, and the optimal pH value of the buffer solution was 4. However, considering that the human body fluid was in a neutral environment, PBS buffer solution with pH 7 was still selected as the supporting electrolyte for the subsequent experiments. Figure 3 Figure (D) below analyzed the change of the peak potential of UA at different pH values. It was obtained that the peak potential of UA shifted negatively with the increase of the pH value, indicating that the electrochemical process of UA was related to the proton transfer process. At the same time, the peak potential was linearly correlated with the pH value, and the linear equation was E = 0.7212 - 0.0610pH (R Figure 3 = 0.9994), and the slope was 61 mV / pH, which was close to the theoretical value of 59 mV / pH, indicating that the electrochemical reaction process of UA was a process of equal protons and equal electrons. 2

[0064] In order to simulate the interference of substances such as AA, DA, or glucose that UA in the actual sample might be affected by, this invention took AA interference as an example and detected UA under the condition that the concentration of AA remained unchanged. In the presence of 0.02 mM AA, the DPV responses of GNC / P3MT / GCE to 0, 1, 2, 5, 7, 9, 12, 15, 18, 22, and 26 μM UA were investigated ( Figure 4 ), and the DPV peak current increased linearly with the increase of the UA concentration. The linear equation was I UA = 0.5960 + 2.067c (R 2 = 0.9993). According to the 3-fold relative standard deviation rule, the detection limit was calculated to be 0.3 μM. When the concentration of UA continued to increase, the peak current and peak potential of AA during the measurement basically remained unchanged, indicating that this GNC / P3MT / GCE could be used for the quantitative detection of UA in the presence of AA, and GNC / P3MT / GCE had good stability and anti-interference ability.

[0065] (3) Verification of the detection performance of the biosensor:

[0066] ​The detection performance of the sensing substrate of Example 1 was verified using blood samples from healthy volunteers (numbered a - c) and blood samples from gout patients (numbered d - f). The specific process is as follows:

[0067] The sample to be tested was diluted 50 - fold with phosphate buffer without incubation. The sensing substrate GNC / P3MT / GCE was used as the working electrode, which, together with an Ag / AgCl reference electrode and a platinum wire auxiliary electrode, formed a three - electrode system. The DPV signal of the three - electrode system was detected using a CH Instrument CHI 660E electrochemical workstation.

[0068] Calculations were performed using the above linear regression equation (I UA = 0.5960 + 2.067c) to obtain the concentration of UA in the blood sample. The results are as Figure 5 shown. The UA content in the blood samples of healthy individuals (a, b, and c) was low, with an average concentration of 288.7 μM. The UA concentration in the blood samples of gout patients (d, e, and f) was significantly higher than that of healthy individuals, with an average concentration of 682.1 μM. In addition, a commercial kit used for clinical diagnosis in the hospital was used to test the same blood samples, and the test results were consistent with those of this sensor, with a concentration error of less than 4.5%. At the same time, 12 groups of standard addition recovery experiments were carried out using blood samples from healthy individuals in this invention. The obtained standard addition recovery rates were between 94.5% and 106.9%, and the relative standard deviations were between 2.3% and 4.7%, indicating that the biosensor designed in this invention has good detection ability and application even in complex blood samples.

[0069] This invention prepared a substrate material for a uric acid biosensor. Based on the enhanced catalytic efficiency of the polymer P3MT for gold nanocages, the clinical detection of UA in the blood of gout patients was realized. In this invention, a poly(3 - methylthiophene) film was modified on the surface of a glassy carbon electrode by electro - polymerization, and then gold nanocages were further assembled onto the surface of poly(3 - methylthiophene). Poly(3 - methylthiophene) itself has catalytic ability, good conductivity, and can provide a good substrate and micro - environment for the assembly of gold nanocages on the electrode surface, which is conducive to the modification of gold nanocages at the electrode interface. More, more uniform, and more stable gold nanocages can be loaded on the surface of poly(3 - methylthiophene), thereby enhancing the catalytic efficiency of gold nanocages. Therefore, when detecting complex biological samples, this sensing substrate can effectively distinguish the electrochemical oxidation signals of UA and AA, and then eliminate the signal interference of AA, realizing the sensitive determination of UA in actual samples.

[0070] In the present invention, P3MT is used for electrode modification to improve the microenvironment of the glassy carbon electrode; the hollow structure of GNC is utilized to increase the specific surface area of the electrode; based on the synergistic catalytic separation of the electrochemical signals of interfering substances between P3MT and GNC, the accuracy and feasibility of the biosensor are improved. Therefore, the biosensor electrode material prepared by the present invention has the advantages of high accuracy, high sensitivity, good selectivity, etc., and can be applied to the accurate detection of the UA content in the blood of gout patients, and has important application value in the clinical diagnosis and drug research fields of related diseases such as purine metabolism disorders.

[0071] The embodiments described above are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A biosensor electrode substrate, characterized in that, It is prepared by modifying the surface of the electrode with a poly(3-methylthiophene) film and assembling gold nanocages onto the surface of the poly(3-methylthiophene) film.

2. The biosensor electrode substrate according to claim 1, wherein The electrode is a glassy carbon electrode.

3. The biosensor electrode substrate according to claim 1, characterized in that, Using 3-methylthiophene as a monomer, a poly(3-methylthiophene) film is modified on the surface of the electrode by electro-polymerization.

4. The biosensor electrode substrate according to claim 1, wherein The gold nanocages are prepared by a substitution reaction using silver nanoparticles as sacrificial templates and polyvinylpyrrolidone as a stabilizer.

5. The preparation method of the biosensor electrode substrate according to any one of claims 1-4, characterized in that, It includes the following steps: Using 3-methylthiophene as a monomer, a poly(3-methylthiophene) film is modified on the surface of the electrode by electro-polymerization, and then gold nanocages are assembled onto the surface of the poly(3-methylthiophene) film to prepare the biosensor electrode substrate.

6. The application of the biosensor electrode substrate according to any one of claims 1-4 as a biosensor electrode substrate for uric acid detection.

7. A method for uric acid detection based on the biosensor electrode substrate according to any one of claims 1-4, characterized in that, It includes the following steps: The sample to be tested is added to phosphate buffer and mixed to serve as the electrolyte; Using an Ag / AgCl electrode as the reference electrode, a platinum wire as the auxiliary electrode, and the biosensor electrode substrate as the working electrode to form a three-electrode system; The three-electrode system is placed in the electrolyte to detect uric acid in the sample to be tested.

8. A uric acid detection biosensor, characterized in that, It includes the biosensor electrode substrate according to any one of claims 1-4.

9. The application of the uric acid detection biosensor according to claim 8 in detecting uric acid.

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