L-DOPA-specific sensing electrode based on local acidification of hollow confined structure, preparation method and application

Abstract

The present invention discloses a levodopa-specific sensing electrode based on local acidification of a hollow confined structure, a preparation method and an application thereof, wherein the sensing electrode comprises a basic conductive layer and a confined microcavity layer. The basic conductive layer is used to transfer the electrons generated during the cyclization reaction of levodopa to an electrochemical measurement circuit; the confined microcavity layer is formed by modifying a confined microcavity on the basic conductive layer, and the local acidification enzyme and levodopa catalytic particles are fixed on the inner surface of the confined microcavity by utilizing the difference in the diameter of the macromolecular protein, the local acidification enzyme and the pores of the confined microcavity. During measurement, the local acidification enzyme forms a local acidification environment with a pH value less than 5 in the confined microcavity, and the levodopa catalytic particles are used to catalyze and accelerate the reaction rate of levodopa on the electrode. The sensing electrode can be used for the determination of the levodopa concentration in body fluids, which solves the problem that the existing electrochemical sensors are difficult to distinguish between levodopa and dopamine in body fluids, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical biosensors, and in particular relates to a L-dopa-specific sensing electrode based on local acidification of a hollow confined structure, a preparation method and an application thereof. Background Art

[0002] Levodopa is currently the most effective drug for the treatment of Parkinson's disease. After passing through the blood-brain barrier, it can supplement the dopamine deficiency in the striatum and exert its therapeutic effect. Levodopa is a precursor of dopamine. Dopamine itself cannot penetrate the blood-brain barrier. Therefore, in the treatment of Parkinson's disease, it is necessary to take Levodopa to pass through the blood-brain barrier and convert it into dopamine in the brain, thereby helping to supplement dopamine levels and relieve symptoms.

[0003] The safe dosage range of levodopa is narrow and has many side effects. Free levodopa will be further oxidized or dismutated into toxic metabolites such as dopaquinone, causing adverse reactions including sleep and mental disorders, hyperkinesia, symptom fluctuations and psychiatric symptoms. Since Parkinson's patients need to take medication for a long time, excessive peripheral accumulation of levodopa is very likely to cause the above side effects. Clinically, complication control and dosage adjustment of levodopa have been throughout the entire process of drug use. Therefore, in order to improve the therapeutic effect of the drug and reduce the symptoms of side effects, it is necessary to measure the pharmacokinetic process of levodopa in different individuals and conduct individual analysis of the patient's drug treatment response to facilitate precision medication.

[0004] Currently, the determination of levodopa mainly adopts ultraviolet spectrophotometry, high performance liquid chromatography and gas chromatography-mass spectrometry. In clinical practice, the adjustment of levodopa dosage is basically based on the doctor's experience, and the dosage is formulated according to parameters such as patient weight, and the dosage is adjusted according to the feedback of side effects in the later stage. Therefore, side effects have basically appeared when the medication is adjusted. Therefore, a sensor for real-time determination of levodopa concentration in body fluids is needed for the determination of drug metabolic dynamics, so as to formulate personalized medication plans and adjust drug dosages in time.

[0005] At present, electrochemical sensors are mostly used for real-time determination of levodopa. Levodopa will undergo a cyclization reaction on the electrode, exchanging two electrons and two protons and being oxidized to dopaquinone. When the electrode accepts electrons, a corresponding current signal is generated, and the concentration of levodopa is determined by measuring the magnitude of the current signal. However, it has the problem of poor selectivity. The main reason is that levodopa and dopamine coexist in human body fluids (blood, sweat, saliva, etc.). The two have the same electrochemical oxidation potential, which makes it difficult to distinguish between the two substances when measuring with electrochemical sensors, and it is impossible to determine the actual drug metabolism of levodopa, resulting in limited clinical applications.

[0006] The study found that in an acidic environment, the cyclization reaction of dopamine is inhibited, while levodopa can continue to undergo the cyclization reaction. Therefore, in an environment with a pH value less than 5, this difference can be used to control the cyclization reaction rates of levodopa and dopamine and improve the selectivity of electrochemical detection. Summary of the invention

[0007] In view of the problem that existing sensors have poor selectivity in detecting levodopa, the purpose of the present invention is to provide a levodopa-specific sensing electrode based on local acidification of a hollow confined structure, a preparation method and application. The present invention uses local acidification enzymes such as dehydrogenase or oxidase to co-fix with levodopa catalytic particle materials in a confined microcavity, uses dehydrogenase or oxidase to catalyze sugar metabolites (glucose or lactic acid), forms a local acidification environment, inhibits dopamine cyclization reaction, and forms highly selective test conditions in the confined space of the microcavity.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a levodopa-specific sensing electrode based on local acidification of a hollow confined structure, wherein the sensing electrode uses an electrochemical method to measure the levodopa concentration, and at least comprises:

[0010] A basic conductive layer, used for transferring the electrons generated during the cyclization reaction of L-DOPA to an electrochemical measurement circuit;

[0011] A confined microcavity layer is formed by modifying the confined microcavity on the base conductive layer, and a local acidification enzyme and a levodopa catalytic particle are fixed on the inner surface of the confined microcavity. During measurement, the local acidification enzyme forms a local acidification environment with a pH value less than 5 in the confined microcavity, and the levodopa catalytic particle is used to catalyze and accelerate the rate of cyclization reaction of levodopa on the electrode.

[0012] Preferably, the local acidification enzyme is fixed to the inner surface of the confined microcavity via chemical bonds.

[0013] Preferably, the local acidification enzyme includes an oxidase and / or a dehydrogenase, which is used to catalyze the corresponding catalytic substrate during measurement to reduce the pH value of the surrounding liquid environment.

[0014] Preferably, the conductive material of the base conductive layer includes gold, platinum or carbon.

[0015] Preferably, the confined microcavity is a porous hollow structure with a particle size of 100nm-10µm; the material of the confined microcavity includes mesoporous silicon, metal organic framework or hollow gold particles.

[0016] Preferably, the material of the levodopa catalytic particles includes silver, iridium oxide, titanium oxide or nickel oxide, and is fixed to the inner surface of the confined microcavity by chemical deposition.

[0017] Preferably, the local acidification enzyme includes one or a combination of glucose oxidase, lactate oxidase, glucose dehydrogenase, and lactate dehydrogenase, and the catalytic substrate includes glucose and / or lactic acid.

[0018] The second aspect of the present invention provides a method for preparing a levodopa-specific sensing electrode based on local acidification of a hollow confined structure, which comprises at least the following steps:

[0019] S1. preparing a confined microcavity with a porous hollow structure by a template sacrificial method, wherein the surface of the confined microcavity contains chemical bond sites;

[0020] S2, depositing L-DOPA catalytic particles on the surface of the confined microcavity obtained in step S1 by chemical deposition;

[0021] S3, placing the confined microcavity obtained in step S2 in a macromolecular protein solution, wherein the protein scale of the macromolecular protein is larger than the hole diameter of the confined microcavity, so that the macromolecular protein occupies all the chemical bond sites outside the confined microcavity and covers the L-DOPA catalytic particles outside the confined microcavity, while retaining the chemical bond sites and L-DOPA catalytic particles on the inner surface of the confined microcavity;

[0022] S4, centrifugally separating the confined microcavity obtained in step S3 from the unbound macromolecular protein, and then placing it in a local acidification enzyme solution, wherein the protein size of the local acidification enzyme is smaller than the hole diameter of the confined microcavity, so as to enter the interior thereof; the local acidification enzyme is fixed on the inner surface of the confined microcavity by forming chemical bonds with the chemical bond points on the inner surface of the confined microcavity;

[0023] S5, modifying the confined microcavity obtained in step S4 on the base conductive layer to prepare the sensing electrode.

[0024] The third aspect of the present invention proposes a use of the above-mentioned sensing electrode in the preparation of an electrochemical biosensor for specifically detecting the concentration of L-DOPA in body fluids.

[0025] Preferably, the electrochemical measurement method used by the electrochemical biosensor includes cyclic voltammetry or differential pulse voltammetry, and the body fluid includes blood, sweat or saliva.

[0026] Compared with the prior art, the present invention has the following gain effects:

[0027] The present invention is based on the principle that the cyclization reaction of dopamine is inhibited in an acidic environment, while levodopa can continue to undergo cyclization reaction. By fixing the local acidification enzyme in the confined microcavity, the local acidification enzyme such as dehydrogenase or oxidase is used to catalyze the sugar metabolites (glucose or lactic acid) in the body fluid to form a local acidification environment with a pH less than 5, thereby inhibiting the cyclization reaction of dopamine and forming a highly selective test condition for levodopa in the confined space of the microcavity, thereby ensuring the accuracy of the detection of levodopa in the body fluid. At the same time, the present invention further accelerates the rate of cyclization reaction of levodopa on the electrode by fixing the levodopa catalytic particles in the confined microcavity, thereby improving the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the catalytic reaction equation under different pH conditions;

[0029] Figure 2 This is a schematic structural diagram of a L-DOPA-specific sensing electrode based on local acidification of a hollow confined structure according to an embodiment of the present invention;

[0030] Figure 3 A microscopic schematic diagram of a confined microcavity in which levodopa catalytic particles and local acidifying enzymes are fixed according to an embodiment of the present invention;

[0031] Figure 4 Schematic diagram of detection of levodopa according to an embodiment of the present invention, wherein a is a differential pulse voltammetry test diagram of simulated sweat containing different concentrations of levodopa (0 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM), and b is a diagram showing the corresponding relationship between levodopa concentration and current peak value;

[0032] Figure 5 This is a comparison chart of the sweat levodopa concentration value actually measured by the sensor of an embodiment of the present invention and the HPLC calibration value.

[0033] In the figure: 1. confined microcavity layer; 11. confined microcavity; 12. L-DOPA catalytic particles; 13. local acidification enzyme; 2. basic conductive layer. DETAILED DESCRIPTION

[0034] In order to make the purpose and technical solution of the present invention clearer and more complete, the present invention is further described in detail below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art can make modifications or equivalent substitutions based on the understanding of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, which all belong to the scope of protection of the present invention.

[0035] Unless otherwise specified, the reagents and materials involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

[0036] Example 1

[0037] It should be noted that Figure 1 The equations for the catalytic reaction of L-DOPA and dopamine under different pH conditions are shown. As shown in the figure, when pH>5, both L-DOPA and dopamine can undergo cyclization reaction to generate dopaquinone; when pH<5, the cyclization reaction of dopamine is inhibited, while L-DOPA can continue to undergo cyclization reaction. Therefore, in an environment with a pH less than 5, this difference can be used to control the cyclization reaction rate of L-DOPA and dopamine and improve the selectivity of electrochemical detection.

[0038] Based on the above principle, Figure 2-Figure 3 As shown, this embodiment proposes a levodopa-specific sensing electrode based on local acidification of a hollow confined structure, which at least includes a basic conductive layer 2 and a confined microcavity layer 1. Among them, the basic conductive layer 2 is used to transfer the electrons generated during the cyclization reaction of levodopa to the electrochemical measurement circuit, and the material constituting the basic conductive layer 2 can be any one of the conductive materials with stable chemical properties such as gold, platinum, and carbon. The confined microcavity layer 1 is formed by modifying a large number of confined microcavities 11 on the basic conductive layer 2. The inner surface of the confined microcavity 11 is fixed with a local acidification enzyme 13, which can quickly form a local acidified microenvironment with a pH less than 5 in the confined microcavity 11 after catalyzing the substrate during the measurement process, thereby inhibiting the cyclization reaction of dopamine, while the cyclization reaction of levodopa is not affected. Since the body fluid contains glucose, lactic acid and other sugar metabolites, preferably, the local acidification enzyme 13 includes one or more combinations of oxidases and dehydrogenases such as glucose oxidase, lactate oxidase, glucose dehydrogenase, lactate dehydrogenase, etc., which are used to catalyze the corresponding substrates such as glucose or lactic acid in the body fluid. For example, glucose oxidase and glucose dehydrogenase can catalyze glucose to generate additional hydrogen ions and reduce the pH value in the surrounding liquid environment; lactate oxidase and lactate dehydrogenase can catalyze lactic acid to generate additional hydrogen ions, which can also reduce the pH value in the surrounding liquid environment.

[0039] Furthermore, if Figure 3As shown, in this embodiment, levodopa catalytic particles 12 are also fixed on the inner surface of the confined microcavity 11, which are mainly used to catalyze and accelerate the rate of cyclization reaction of levodopa on the electrode during measurement. Preferably, the material of the levodopa catalytic particles 12 includes silver, iridium oxide, titanium oxide, nickel oxide, etc., and is fixed on the inner surface of the confined microcavity 11 by chemical deposition and other methods. It is worth noting that the main feature of the confined microcavity 11 is that it has a porous hollow structure with a particle size of 100nm-10µm, and the types include mesoporous silicon, metal organic framework, hollow gold particles and other materials. It should be noted that, including but not limited to preparing the confined microcavity 11 by a template sacrificial method, in some embodiments, it is also necessary to chemically modify the inner surface of the confined microcavity 11 to form a chemical bond site, such as modifying the carboxyl group, which is used to improve the fixation efficiency of the local acidification enzyme 13 through chemical bonds.

[0040] Furthermore, this embodiment also proposes a method for preparing a L-DOPA-specific sensing electrode based on local acidification of a hollow confined structure, which at least comprises the following steps:

[0041] S1. A confined microcavity 11 with a porous hollow structure is prepared by a template sacrificial method, wherein the surface of the confined microcavity 11 contains chemical bond sites.

[0042] It should be noted that the surface of the confined microcavity 11 prepared from some materials needs to be chemically modified to form chemical bond sites to fix the local acidifying enzyme 13, while the confined microcavity 11 prepared from other materials itself has chemical bond sites, so no chemical group modification is required. For example, when the selected confined microcavity 11 material is a hollow organic metal framework, the carboxyl group can be modified on the surface to form a chemical bond with the amino group on the dehydrogenase; and when the selected confined microcavity 11 material is a hollow gold nanocage, since it itself has a gold-sulfur bond site, the sulfhydryl group on the oxidase can directly form a chemical bond-gold-sulfur bond with the gold element on the hollow gold nanocage material, so there is no need to modify the chemical group.

[0043] It should be understood that the surface of the confined microcavity 11 includes the inner surface and the outer surface of the confined microcavity 11, and both the inner and outer surfaces of the confined microcavity 11 contain chemical bond sites. If chemical group modification is required to form chemical bond sites, both the inner and outer surfaces of the confined microcavity 11 will be chemically modified. In subsequent steps, the chemical bond sites located on the outside are occupied by macromolecular proteins, and ultimately only the chemical bond sites on the inner surface of the confined microcavity 11 are retained.

[0044] S2. Depositing the L-DOPA catalytic particles 12 on the surface of the confined microcavity 11 obtained in step S1 by chemical deposition.

[0045] It should be noted that, during the chemical deposition process, the L-DOPA catalytic particles 12 are deposited on both the inner and outer surfaces of the confined microcavity 11. In subsequent steps, the L-DOPA catalytic particles 12 located on the outer side are covered by macromolecular proteins, and ultimately only the L-DOPA catalytic particles 12 on the inner surface of the confined microcavity 11 are retained.

[0046] S3. placing the confined microcavity 11 obtained in step S2 in a macromolecular protein solution, wherein the protein scale of the macromolecular protein is larger than the hole diameter of the confined microcavity 11, so that the macromolecular protein occupies all the chemical bond sites outside the confined microcavity 11 and covers the L-DOPA catalytic particles 12 outside the confined microcavity 11, but cannot cover the chemical bond sites and L-DOPA catalytic particles 12 on the inner surface of the confined microcavity 11; wherein the macromolecular protein includes macromolecular collagen (molecular weight>100kDa) and connexin (molecular weight>200 kDa).

[0047] S4, centrifugally separating the confined microcavity 11 obtained in step S3 from the unbound macromolecular proteins, and then placing them in a local acidification enzyme 13 solution, wherein the protein scale of the local acidification enzyme 13 is smaller than the pore diameter of the confined microcavity 11, thereby entering the interior thereof; the local acidification enzyme 13 forms chemical bonds with the chemical bond points on the inner surface of the confined microcavity 11, so that the local acidification enzyme 13 is fixed on the inner surface of the confined microcavity 11.

[0048] S5, modifying the confined microcavity 11 obtained in step S4 on the base conductive layer 2 to prepare a sensing electrode.

[0049] Example 2

[0050] In the embodiment of the levodopa-specific sensing electrode based on local acidification of the hollow confined structure, the base conductive layer 2 uses gold as the conductive material, and the confined microcavity 11 uses a hollow organic metal framework material, the average particle size of the hollow organic metal framework is 1.5 μm, and the surface is modified with carboxyl groups. The material of the levodopa catalytic particles 12 is titanium oxide, which is deposited on the surface of the hollow organic metal framework by chemical deposition.

[0051] The hollow organic metal framework is placed in a solution of macromolecular collagen (molecular weight>100 kDa). The macromolecular collagen occupies all the carboxyl sites on the outside of the organic metal framework and covers the titanium oxide on the outside of the organic metal framework. Since the molecular weight of collagen exceeds 100 kDa, which is larger than the pore diameter of the hollow organic metal framework, it cannot occupy the carboxyl sites inside the hollow organic metal framework. The hollow organic metal framework and the unbound collagen are centrifuged and then placed in a glucose dehydrogenase solution. The glucose dehydrogenase protein is smaller than the pore diameter of the hollow organic metal framework and can enter its interior. The amino group on the glucose dehydrogenase forms a chemical bond with the carboxyl group on the hollow organic metal framework material, so that the glucose dehydrogenase is fixed on the surface inside the organic metal framework material.

[0052] During the measurement process, the prepared sensing electrode is used as a working electrode and immersed together with a reference electrode in a simulated human sweat environment to be tested. The glucose dehydrogenase in the hollow organic metal framework catalyzes the glucose in the simulated sweat to form free hydrogen ions, thereby reducing the local pH value in the hollow environment and inhibiting the electrochemical oxidation reaction of dopamine. Figure 4 As shown in a, the oxidation reaction of L-dopa at different concentrations (0 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM) in simulated sweat on titanium oxide was measured by differential pulse voltammetry, and an obvious L-dopa oxidation peak appeared at 0.12 V. The L-dopa concentration can be calculated by calculating the oxidation peak, as shown in Figure 4 As shown in b, the peak current of levodopa oxidation increases with its concentration, showing a good linear relationship. The simulated sweat contains 40nM dopamine, as shown in Figure 4 As shown in a in FIG. 1 , a dopamine oxidation peak at 0.29 V was observed in this measurement system, and the peak value was relatively low, indicating that the oxidation reaction of dopamine was inhibited in the local acidic environment.

[0053] Example 3

[0054] In the embodiment of the L-DOPA-specific sensing electrode based on the local acidification of the hollow confined structure, the base conductive layer 2 is a glassy carbon electrode, the confined microcavity 11 is a hollow gold nanocage material, and the average particle size of the hollow gold nanocage is 800nm. The material of the L-DOPA catalytic particles 12 is nickel oxide, which is deposited on the surface of the hollow gold nanocage by chemical deposition.

[0055] The hollow gold nanocage is placed in a connexin (molecular weight>200 kDa) solution. The connexin occupies all the gold-sulfur bond sites on the outside of the hollow gold nanocage and covers the nickel oxide on the outside of the hollow gold nanocage. Since the molecular weight of the connexin exceeds 200 kDa, which is larger than the hole diameter of the hollow gold nanocage, it cannot occupy the gold-sulfur bond sites inside the hollow gold nanocage. The hollow gold nanocage and the unbound connexin are centrifuged and then placed in a glucose oxidase solution. The glucose oxidase protein is smaller than the hole diameter of the hollow gold nanocage and can enter its interior. The thiol group on the glucose oxidase forms a chemical gold-sulfur bond with the gold element on the hollow gold nanocage material, so that the glucose oxidase is fixed on the surface inside the hollow gold nanocage material.

[0056] During the measurement process, the prepared sensing electrode is used as a working electrode and immersed in the human sweat environment to be tested together with the reference electrode. The glucose oxidase in the hollow gold nanocage will catalyze the glucose in the simulated sweat to form gluconic acid, reduce the local pH value in the hollow environment, and inhibit the electrochemical oxidation reaction of dopamine. The oxidation reaction of levodopa in the simulated sweat on nickel oxide is measured by differential pulse voltammetry, and the levodopa concentration can be calculated by calculating the oxidation peak. At the same time, the simulated sweat is subjected to high performance liquid chromatography analysis to obtain the actual concentration of levodopa therein. Figure 5 As shown, the levodopa concentration measured by the sensing electrode of this embodiment is close to the concentration measured by the standard method, and the average deviation is less than 10%.

Claims

1. A L-DOPA-specific sensing electrode based on local acidification of a hollow confined structure, characterized in that: The sensing electrode uses an electrochemical method to measure the concentration of levodopa, which at least includes: The base conductive layer (2) is used to transfer the electrons generated during the cyclization reaction of L-DOPA to the electrochemical measurement circuit; A confined microcavity layer (1) is formed by modifying a confined microcavity (11) on the base conductive layer (2), wherein a local acidification enzyme (13) and a levodopa catalytic particle (12) are fixed on the inner surface of the confined microcavity (11); during measurement, the local acidification enzyme (13) forms a local acidified environment with a pH value less than 5 in the confined microcavity (11), and the levodopa catalytic particle (12) is used to catalyze and accelerate the rate of cyclization reaction of levodopa on the electrode.

2. The L-DOPA-specific sensing electrode based on local acidification of a hollow confined structure according to claim 1, characterized in that: The local acidifying enzyme (13) is fixed to the inner surface of the confined microcavity (11) via chemical bonds.

3. The L-DOPA-specific sensing electrode based on local acidification of a hollow confined structure according to claim 1, characterized in that: The local acidification enzyme (13) includes oxidase and / or dehydrogenase, which is used to catalyze the corresponding catalytic substrate during measurement to reduce the pH value of the surrounding liquid environment.

4. The L-DOPA-specific sensing electrode based on local acidification of a hollow confined structure according to claim 1, characterized in that: The conductive material of the base conductive layer (2) includes gold, platinum or carbon.

5. The L-DOPA-specific sensing electrode based on local acidification of a hollow confined structure according to claim 1, characterized in that: The confined microcavity (11) is a porous hollow structure with a particle size of 100 nm-10 µm; the material of the confined microcavity (11) includes mesoporous silicon, metal organic framework or hollow gold particles.

6. The L-DOPA-specific sensing electrode based on local acidification of a hollow confined structure according to claim 1, characterized in that: The material of the levodopa catalytic particles (12) includes silver, iridium oxide, titanium oxide or nickel oxide, and is fixed to the inner surface of the confined microcavity (11) by chemical deposition.

7. The L-DOPA-specific sensing electrode based on local acidification of a hollow confined structure according to claim 3, characterized in that: The local acidification enzyme (13) includes one or a combination of glucose oxidase, lactate oxidase, glucose dehydrogenase, and lactate dehydrogenase, and the catalytic substrate includes glucose and / or lactic acid.

8. The method for preparing the L-DOPA-specific sensing electrode based on local acidification of the hollow confined structure according to any one of claims 1 to 7, characterized in that: At least the following steps are included: S1. preparing a confined microcavity (11) with a porous hollow structure by a template sacrificial method, wherein the surface of the confined microcavity (11) comprises chemical bond sites; S2, depositing the L-DOPA catalytic particles (12) on the surface of the confined microcavity (11) obtained in step S1 by chemical deposition; S3, placing the confined microcavity (11) obtained in step S2 in a macromolecular protein solution, wherein the protein scale of the macromolecular protein is larger than the hole diameter of the confined microcavity (11), so that the macromolecular protein occupies all the chemical bond sites outside the confined microcavity (11) and covers the L-DOPA catalytic particles (12) outside the confined microcavity (11), while retaining the chemical bond sites and L-DOPA catalytic particles (12) on the inner surface of the confined microcavity (11); S4, centrifugally separating the confined microcavity (11) obtained in step S3 from the unbound macromolecular proteins, and then placing them in a solution of a local acidifying enzyme (13), wherein the protein size of the local acidifying enzyme (13) is smaller than the hole diameter of the confined microcavity (11), thereby entering the interior thereof; the local acidifying enzyme (13) is fixed on the inner surface of the confined microcavity (11) by forming chemical bonds with chemical bond points on the inner surface of the confined microcavity (11); S5, modifying the confined microcavity (11) obtained in step S4 on the base conductive layer (2) to prepare the sensing electrode.

9. Use of the sensing electrode according to any one of claims 1 to 7 in the preparation of an electrochemical biosensor for specifically detecting the concentration of levodopa in body fluids.

10. Use of the sensing electrode according to claim 9 in preparing an electrochemical biosensor for specifically detecting the concentration of levodopa in body fluids, characterized in that: The electrochemical measurement method used by the electrochemical biosensor includes cyclic voltammetry or differential pulse voltammetry, and the body fluid includes blood, sweat or saliva.

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