Stable enzyme type sensing electrode as well as preparation method and application thereof

Abstract

The invention discloses a stable enzyme type sensing electrode as well as a preparation method and application thereof, and belongs to the technical field of enzyme type sensors. The preparation method comprises the following steps: polishing and ultrasonically cleaning a carbon-based electrode to obtain a pretreated carbon-based electrode; the preparation method comprises the following steps: dissolving 4-(4-aminophenyl)-1, 2, 4-triazolidine-3, 5-diketone in a hydrochloric acid solution, and stirring to obtain a mixed solution A; dropwise adding a sodium nitrite solution into the mixed solution A, and stirring to obtain a mixed solution B; placing the pretreated carbon-based electrode in the mixed solution B for reduction modification and cleaning to obtain a pre-modified electrode; and placing the pre-modified electrode in an enzyme solution to carry out oxidation modification, thereby obtaining the electrode. The invention also discloses the stable enzyme type sensing electrode prepared by the method and application thereof. The stable enzyme type sensing electrode can maintain the structural stability and catalytic activity of enzyme molecules to a large extent under the low-voltage condition, the modification process is convenient and efficient, and the application prospect is wide.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme-based sensors, and particularly relates to a stable enzyme-based sensing electrode, a preparation method thereof, and an application thereof. Background Art

[0002] Enzyme bioelectrochemistry is an interdisciplinary subject that combines electrocatalysis and enzyme biocatalysis, giving rise to application fields such as electrochemical biosensors, biofuel cells, and bioelectrocatalysis. An electrochemical biosensor is an analytical detection platform that uses nucleic acids, enzymes, antibodies, protein receptors, organelles, or microorganisms as recognition elements. It integrates biorecognition molecules with electrochemical devices to generate measurable electrochemical signals related to changes in the analyte content. It shows great potential in medical diagnosis and some biotechnology industries, including food quality control, drug research and development, and environmental monitoring. In vivo electrochemical sensors are even more powerful tools for revealing changes in molecular mechanisms during life activities.

[0003] Among them, enzyme biosensors have developed into important detection tools in the field of bioelectroanalysis due to their advantages such as high activity, high selectivity, wide substrate range, and mild reaction conditions. However, enzyme molecules themselves have the characteristics of unstable structure and easy inactivation, and have poor resistance to environmental changes. Therefore, selecting a suitable immobilization method to efficiently and stably modify the recognition enzyme molecules on the electrode surface is the key issue in the design of enzyme-based electrochemical biosensors. Traditional enzyme protein immobilization strategies include non-covalent adsorption and deposition, covalent coupling, polymer embedding, cross-linking, and affinity interaction, etc. Among them, the non-covalent modification method has less impact on the structure of the enzyme itself and relatively more modification amounts, but at the same time, there are also problems such as weak connection force between the enzyme molecule and the electrode surface, desorption and leakage during detection, large diffusion barriers for substrates and catalytic products, uncontrollable modification process, and poor electrode reproducibility. The covalent modification method has become an important means for constructing enzyme electrodes due to its advantages such as stable bonding, fast response, site specificity, and no diffusion barrier. However, traditional covalent modification schemes are relatively single, mainly involving esterification and amidation reactions of free amino and carboxyl groups on the protein molecule surface, or pre-chemically modifying active groups on the enzyme molecule surface. Such a reaction process system is complex, requires the addition of a series of reaction aids and catalysts, and has a long reaction time, which is not conducive to the stable maintenance of enzyme activity. Therefore, there is a need to provide a new electrode modification strategy that can efficiently and stably modify enzyme molecules on the carbon electrode surface while ensuring the high specific recognition and catalytic ability of enzyme molecules to meet the stable analysis requirements of various catalytic substrates. Summary of the Invention

[0004] The problem to be solved by the present invention is to provide a stable enzyme-based sensing electrode, a preparation method thereof, and an application thereof to solve the problems of complex reaction system, low reaction efficiency, low enzyme activity, and uneven enzyme modification in the preparation of existing enzyme-based sensing electrodes.

[0005] The technical solution adopted to solve its technical problems is to provide a method for preparing a stable enzyme-type sensing electrode, comprising the following steps:

[0006] (1) Grinding and ultrasonically cleaning a carbon-based electrode to obtain a pretreated carbon-based electrode;

[0007] (2) Dissolving 4-(4-aminophenyl)-1,2,4-triazolidine-3,5-dione in a hydrochloric acid solution and stirring to obtain a mixed solution A;

[0008] (3) Dropping a sodium nitrite solution into the mixed solution A and stirring to obtain a mixed solution B;

[0009] (4) Placing the pretreated carbon-based electrode in the mixed solution B for reduction modification and cleaning to obtain a pre-modified electrode;

[0010] (5) Placing the pre-modified electrode in an enzyme solution for oxidation modification to obtain the product.

[0011] The beneficial effects of the present invention adopting the above technical solution are as follows: In the method for preparing a stable enzyme-type sensing electrode of the present invention, first, the carbon-based electrode is cleaned and polished to improve the electron transfer efficiency on the electrode surface; when a sodium nitrite solution is added to a mixed solution of 4-(4-aminophenyl)-1,2,4-triazolidine-3,5-dione and hydrochloric acid, the amino group in the 4-(4-aminophenyl)-1,2,4-triazolidine-3,5-dione molecule is diazotized by the nitrite to form a phenyl diazonium salt structure; during the reduction modification of the electrode, the diazotized 4-(4-aminophenyl)-1,2,4-triazolidine-3,5-dione molecule loses one molecule of N2 to generate a phenyl radical, which reacts with the surface of the carbon-based electrode to form a covalent bond of C-C. After the reduction modification is completed, the residual hydrochloric acid on the electrode surface and the unreacted 4-(4-aminophenyl)-1,2,4-triazolidine-3,5-dione molecules adsorbed on the surface are removed by washing, and then the oxidation modification of the enzyme is carried out. During the oxidation modification process, the -NH-NH- group in the 4-(4-aminophenyl)-1,2,4-triazolidine-3,5-dione molecule is oxidized to a highly reactive -N=N- group (TAD), and at the same time, a nucleophilic addition reaction occurs with the tyrosine residue on the surface of the enzyme molecule to form a stable C-N bond connection.

[0012] During the preparation of the stable enzyme-type sensing electrode of the present invention, by modifying a triazodione functional molecule on the surface of the carbon-based electrode and then covalently coupling it with the tyrosine residue on the surface of the enzyme molecule in an in-situ electrochemical manner, the two are stably connected through a C-N bond, so that the sensor prepared by the electrode has good stability under physiological conditions, can maintain the structural stability and catalytic activity of the enzyme molecule to a large extent under low voltage conditions, and the modification process is convenient and efficient. By modifying different types of enzyme molecules, the stable enzyme-type sensing electrode can be used for the detection of different substrates, and has a wide application prospect.

[0013] Preferably, step (1) includes the following steps: placing a carbon-based electrode with a smooth and stain-free surface on a flannel, and then successively placing it in alumina abrasive slurries of 2 - 3 μm and 40 - 60 nm for circular grinding, and then ultrasonically cleaning the abrasive with ethanol and ultrapure water alternately for 2 - 3 times.

[0014] More preferably, after the carbon-based electrode is ground and ultrasonically cleaned, it is placed in a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] electrolyte solution and cyclically scanned at a scanning rate of 100 mV / s within a potential range of -0.2 - 0.6 V. When the potential difference E P < 90 mV, a pretreated carbon-based electrode is obtained.

[0015] The beneficial effect of the present invention adopting the above technical solution is that when the potential difference E P < 90 mV, the electron transfer efficiency on the electrode surface is relatively high, and the effect is better for subsequent chemical modification.

[0016] More preferably, the carbon-based electrode is a glassy carbon electrode.

[0017] Preferably, in step (2), the material-liquid ratio of 4-(4-aminophenyl)-1,2,4-triazolidine-3,5-dione to the hydrochloric acid solution is 0.5 mg: 1.5 - 3 mL; the molar concentration of the hydrochloric acid solution is 0.4 - 0.6 M.

[0018] More preferably, in step (2), the material-liquid ratio of 4-(4-aminophenyl)-1,2,4-triazolidine-3,5-dione to the hydrochloric acid solution is 0.5 mg: 2 mL; the molar concentration of the hydrochloric acid solution is 0.5 M.

[0019] Preferably, the stirring time in both step (2) and step (3) is 2 - 4 min.

[0020] Preferably, in step (3), the molar concentration of the nitrite solution is 90 - 110 mM; the volume ratio of the nitrite solution to the mixed solution A is 0.04 - 0.06: 1.5 - 3.

[0021] Preferably, in step (4), the voltage for reduction modification is -0.2 - -0.4 V, and the time is 900 - 1100 s; the cleaning is successively carried out with water and acetonitrile.

[0022] More preferably, in step (4), the voltage for reduction modification is -0.3 V, and the time is 1000 s.

[0023] Preferably, in step (5), the concentration of the enzyme solution is 1.5 - 2.5 mg / mL; the enzyme in the enzyme solution is an enzyme containing tyrosine residues in its structure.

[0024] More preferably, the enzyme in the enzyme solution is glucose dehydrogenase, glucose oxidase or glutamate synthase.

[0025] More preferably, the concentration of the enzyme solution in step (5) is 2 mg / mL.

[0026] More preferably, the enzyme solution is prepared with 50 mM Tris buffer at pH 7.5.

[0027] Preferably, the voltage for oxidative modification in step (5) is +0.3 to +0.4 V, and the time is 4 to 6 min.

[0028] More preferably, the voltage for oxidative modification in step (5) is +0.36 V, and the time is 5 min.

[0029] The present invention also provides a stable enzyme-based sensing electrode prepared by the above preparation method.

[0030] The present invention also provides the application of the above stable enzyme-based sensing electrode in substrate detection.

[0031] The present invention has the following beneficial effects:

[0032] The stable enzyme-based sensing electrode of the present invention is connected by a -C-N- bond, with a relatively low bond energy and a relatively high bond strength, avoiding the hydrolysis and desorption phenomena of ester bonds or amide bonds formed during the covalent connection of traditional enzyme-based electrodes, having good connection stability, and no shedding and leaching of enzyme molecules will occur during the detection process; in the preparation method of the stable enzyme-based sensing electrode, the enzyme molecules are covalently modified on the surface of the carbon-based electrode gently and rapidly through electrochemical oxidation at a low potential, the process is easy to control and the electrode has high reproducibility; at the same time, this preparation method does not require pre-modification of the enzyme molecules, maintaining the catalytic activity of the enzyme to the greatest extent; in addition, this method has a simple modification process, does not require the participation of other auxiliaries and the reaction is rapid, and there is no restriction on the type of enzyme molecules, having a wide application prospect. Description of the Drawings

[0033] Figure 1 It is a flow chart for the preparation of a stable enzyme-based sensing electrode;

[0034] Figure 2 It is a graph of the glucose catalytic oxidation performance of the stable enzyme-based sensing electrode prepared in Example 1;

[0035] Figure 3 It is a graph of the concentration linear range of the electrochemical detection of the stable enzyme-based sensing electrode prepared in Example 1;

[0036] Figure 4 It is a graph of the catalytic selectivity of the stable enzyme-based sensing electrode prepared in Example 1 in the presence of interfering substances;

[0037] Figure 5Electrochemical signal stability diagram of the stable enzyme-based sensing electrode prepared in Example 1;

[0038] Figure 6 Glucose catalytic oxidation performance diagram of the stable enzyme-based sensing electrode prepared in Example 2;

[0039] Figure 7 Glutamate catalytic oxidation performance diagram of the stable enzyme-based sensing electrode prepared in Example 3. Detailed implementation manners

[0040] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention, that is, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0041] Therefore, the following detailed description of the provided embodiments of the present invention is not intended to limit the scope of the claimed present invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0042] The features and performance of the present invention will be further described in detail below in conjunction with embodiments. 4-(4-aminophenyl)-1,2,4-triazolidine-3,5-dione, CAS 140707-19-3; ferrocenylmethanol, CAS 1273-86-5.

[0043] Example 1

[0044] A preparation method of a stable enzyme-based sensing electrode includes the following steps:

[0045] (1) Vertically place a glassy carbon electrode with a smooth and stain-free surface on flannel, and successively place it in alumina abrasive slurries of 3 μm and 50 nm for circular polishing. Then, ultrasonically clean the abrasive with ethanol and ultrapure water alternately 3 times, and place it in a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] electrolyte solution, and perform cyclic scanning at a scanning rate of 100 mV / s within a potential range of -0.2 to 0.6 V. When the oxidation-reduction peak potential difference E P <90 mV, it is okay to obtain a pretreated glassy carbon electrode;

[0046] (2) Weigh 0.5 mg of 4-(4-aminophenyl)-1,2,4-triazolidine-3,5-dione and dissolve it in 2 mL of 0.5 M hydrochloric acid solution and stir for 2 min to obtain a mixed solution A;

[0047] (3) Prepare a 100 mM sodium nitrite solution. Use a pipette to measure 50 μL of the sodium nitrite solution and drop it into the mixed solution A, then stir for 2 min to obtain the mixed solution B;

[0048] (4) Place the pretreated glassy carbon electrode in the mixed solution B, perform reduction modification at a constant voltage of -0.3 V for 1000 s, and then rinse it successively with ultrapure water and ultrasonically clean it with acetonitrile for 3 min to obtain the pre-modified electrode;

[0049] (5) Prepare a 2 mg / mL glucose dehydrogenase (FAD-dependent glucose dehydrogenase) solution with 50 mM Tris buffer at pH 7.5. Place the pre-modified electrode in the glucose dehydrogenase solution and perform oxidation modification at a constant voltage of +0.36 V for 5 min to obtain the product.

[0050] The preparation process of the stable enzyme-type sensing electrode in this example is as Figure 1 shown.

[0051] Example 2

[0052] A method for preparing a stable enzyme-type sensing electrode includes the following steps:

[0053] (1) Vertically place the glassy carbon electrode with a smooth and stain-free surface on the flannelette, and successively place it in 3 μm and 50 nm alumina abrasive slurries for circular polishing. Then, ultrasonically clean it with ethanol and ultrapure water alternately for 3 times, and place it in a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] electrolyte solution. Perform cyclic scanning at a scanning rate of 100 mV / s within a potential range of -0.2 to 0.6 V. When the redox peak potential difference E P <90 mV, it is okay to obtain the pretreated glassy carbon electrode;

[0054] (2) Weigh 0.5 mg of 4-(4-aminophenyl)-1,2,4-triazolidine-3,5-dione and dissolve it in 2 mL of 0.5 M hydrochloric acid solution, and stir for 2 min to obtain the mixed solution A;

[0055] (3) Prepare a 100 mM sodium nitrite solution. Use a pipette to measure 50 μL of the sodium nitrite solution and drop it into the mixed solution A, then stir for 2 min to obtain the mixed solution B;

[0056] (4) Place the pretreated glassy carbon electrode in the mixed solution B, perform reduction modification at a constant voltage of -0.3 V for 1000 s, and then rinse it successively with ultrapure water and ultrasonically clean it with acetonitrile for 3 min to obtain the pre-modified electrode;

[0057] (5) Prepare a 2 mg / mL glucose oxidase solution with 50 mM Tris buffer at pH 7.5. Place the pre-modified electrode in the glucose dehydrogenase solution and perform oxidative modification at a constant voltage of +0.36 V for 5 min to obtain the product.

[0058] Example 3

[0059] A method for preparing a stable enzyme-based sensing electrode, comprising the following steps:

[0060] (1) Vertically place a glassy carbon electrode with a smooth and stain-free surface on flannel, polish it in 3 μm and 50 nm alumina abrasive slurries in a circular motion in turn, then ultrasonically clean the abrasive three times alternately with ethanol and ultrapure water. Place it in a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] electrolyte solution and perform cyclic scanning at a scanning rate of 100 mV / s within a potential range of -0.2 to 0.6 V. When the redox peak potential difference E P <90 mV, it is okay to obtain the pretreated glassy carbon electrode;

[0061] (2) Weigh 0.5 mg of 4-(4-aminophenyl)-1,2,4-triazolidine-3,5-dione and dissolve it in 2 mL of 0.5 M hydrochloric acid solution and stir for 2 min to obtain a mixed solution A;

[0062] (3) Prepare a 100 mM sodium nitrite solution, use a pipette to measure 50 μL of the sodium nitrite solution and drop it into the mixed solution A and stir for 2 min to obtain a mixed solution B;

[0063] (4) Place the pretreated glassy carbon electrode in the mixed solution B, perform reduction modification at a constant voltage of -0.3 V for 1000 s, and rinse it with ultrapure water and ultrasonically clean it with acetonitrile for 3 min in turn to obtain the pre-modified electrode;

[0064] (5) Prepare a 2 mg / mL glutamate synthase solution with 50 mM Tris buffer at pH 7.5. Place the pre-modified electrode in the glucose dehydrogenase solution and perform oxidative modification at a constant voltage of +0.36 V for 5 min to obtain the product.

[0065] Example 4

[0066] A method for preparing a stable enzyme-based sensing electrode, comprising the following steps:

[0067] (1) Vertically place a glassy carbon electrode with a smooth and stain - free surface on the flannelette, successively place it in alumina abrasive slurries of 2μm and 40nm and polish it by rotating in circles. Then, ultrasonically clean the abrasive twice alternately with ethanol and ultrapure water. After that, place it in a 5mM K3[Fe(CN)6] / K4[Fe(CN)6] electrolyte solution and cyclically scan in the potential range of - 0.2~0.6V at a scanning rate of 100mV / s. When the potential difference E of the oxidation - reduction peak P <90mV, it is okay, and a pretreated glassy carbon electrode is obtained;

[0068] (2) Weigh 0.5mg of 4-(4 - aminophenyl)-1,2,4 - triazolidine - 3,5 - dione and dissolve it in 1.5mL of 0.4M hydrochloric acid solution and stir for 3min to obtain a mixed solution A;

[0069] (3) Prepare a 90mM sodium nitrite solution, use a pipette to measure 40μL of the sodium nitrite solution and drop it into the mixed solution A and stir for 3min to obtain a mixed solution B;

[0070] (4) Place the pretreated glassy carbon electrode in the mixed solution B, perform reduction modification at a constant voltage of - 0.2V for 1100s, and successively rinse it with ultrapure water and ultrasonically clean it with acetonitrile for 3min to obtain a pre - modified electrode;

[0071] (5) Configure a 1.5mg / mL glucose dehydrogenase solution with a 50mM Tris buffer solution with a pH of 7.5. Place the pre - modified electrode in the glucose dehydrogenase solution and perform oxidation modification at a constant voltage of + 0.3V for 4min to obtain it.

[0072] Example 5

[0073] A preparation method of a stable enzyme - type sensing electrode, comprising the following steps:

[0074] (1) Vertically place a glassy carbon electrode with a smooth and stain - free surface on the flannelette, successively place it in alumina abrasive slurries of 3μm and 60nm and polish it by rotating in circles. Then, ultrasonically clean the abrasive three times alternately with ethanol and ultrapure water. After that, place it in a 5mM K3[Fe(CN)6] / K4[Fe(CN)6] electrolyte solution and cyclically scan in the potential range of - 0.2~0.6V at a scanning rate of 100mV / s. When the potential difference E of the oxidation - reduction peak P <90mV, it is okay, and a pretreated glassy carbon electrode is obtained;

[0075] (2) Weigh 0.5mg of 4-(4 - aminophenyl)-1,2,4 - triazolidine - 3,5 - dione and dissolve it in 2.5mL of 0.6M hydrochloric acid solution and stir for 4min to obtain a mixed solution A;

[0076] (3) Prepare a 110 mM sodium nitrite solution. Use a pipette to measure 60 μL of the sodium nitrite solution and drop it into the mixed solution A, then stir for 4 min to obtain the mixed solution B;

[0077] (4) Place the pretreated glassy carbon electrode in the mixed solution B, perform reduction modification at a constant voltage of -0.4 V for 900 s, and then rinse it with ultrapure water and ultrasonically clean it with acetonitrile for 3 min to obtain the pre-modified electrode;

[0078] (5) Prepare a 2.5 mg / mL glucose dehydrogenase solution with 50 mM Tris buffer solution at pH 7.5. Place the pre-modified electrode in the glucose dehydrogenase solution and perform oxidation modification at a constant voltage of +0.4 V for 6 min to obtain the product.

[0079] Comparative Example 1

[0080] A method for preparing a sensing electrode includes the following steps:

[0081] (1) Vertically place the glassy carbon electrode with a smooth and stain-free surface on the flannel, and successively place it in 3 μm and 50 nm alumina abrasive slurries for circular polishing. Then, ultrasonically clean the abrasive with ethanol and ultrapure water alternately for 3 times, and place it in a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] electrolyte solution. Cyclically scan within the potential range of -0.2 to 0.6 V at a scanning rate of 100 mV / s. When the oxidation-reduction peak potential difference E P < 90 mV, it is okay to obtain the pretreated glassy carbon electrode;

[0082] (2) Weigh 0.5 mg of 4-(4-aminophenyl)-1,2,4-triazolidine-3,5-dione and dissolve it in 2 mL of 0.5 M hydrochloric acid solution, and stir for 2 min to obtain the mixed solution A;

[0083] (3) Place the pretreated glassy carbon electrode in the mixed solution A, perform reduction modification at a constant voltage of -0.3 V for 1000 s, and then rinse it with ultrapure water and ultrasonically clean it with acetonitrile for 3 min to obtain the pre-modified electrode;

[0084] (4) Prepare a 2 mg / mL glucose dehydrogenase solution with 50 mM Tris buffer solution at pH 7.5. Place the pre-modified electrode in the glucose dehydrogenase solution and perform oxidation modification at a constant voltage of +0.36 V for 5 min to obtain the product.

[0085] Experimental Example

[0086] 1. Glucose catalytic oxidation performance of the glucose dehydrogenase (GDH) modified electrode

[0087] Electrochemical tests: Using a three - electrode system, the stable enzyme - type sensing electrode prepared in Example 1 was used as the working electrode, a Pt wire as the counter electrode, and Ag / AgCl as the reference electrode. All electrochemical experiments were completed using an electrochemical workstation 660E (Shanghai Chenhua). The test methods included cyclic voltammetry (CV), current - time (I - t) method, and coulometry.

[0088] 1.1 Catalytic oxidation performance test and results

[0089] Prepare a 0.2 mM ferrocene methanol mediator solution with 50 mM phosphate buffer (pH 7.8) and evacuate it with N2. Place the stable enzyme - type sensing electrode (GDH - GCE TAD ) prepared in Example 1 into the mediator solution, and obtain cyclic voltammograms at +0.2 V in systems with and without 10 mM D - glucose (D - Glu). The scanning rate is 100 mV / s. The results are as Figure 2 shown.

[0090] From Figure 2 it can be seen that in the presence of D - Glu, the peak current of the cyclic voltammogram curve increases significantly compared to the curve without D - Glu, indicating that the GDH - modified electrode has a catalytic oxidation effect on glucose. The stable enzyme - type sensing electrode prepared in Example 1 can be used for the qualitative detection of glucose.

[0091] 1.2 Concentration linear range of electrochemical detection

[0092] Prepare a 0.2 mM ferrocene methanol mediator solution with 50 mM phosphate buffer (pH 7.8) and evacuate it with N2. Place the glassy carbon electrode (GCE), the stable enzyme - type sensing electrode (GDH - GCE TAD ) prepared in Example 1, and the sensing electrode (GDH + GCE PU ) prepared in Comparative Example 1 into the mediator solution respectively, and obtain amperometric response curves of different concentrations of D - Glu (20, 100, 200, 400 μM and 1, 2, 4 mM) at 0.2 V (vs. Ag / AgCl). The results are as Figure 3 shown.

[0093] To evaluate the preservation of protein function after electrochemical coupling, glucose dehydrogenase (GDH) (an enzyme containing 22 tyrosine residues) was immobilized on GCE; GDH is a redox enzyme that catalyzes the conversion of glucose to gluconolactone and is widely used in blood glucose detection and biofuel cells. Monitor the catalytic activity of GDH through the ferrocene / ferrocenium (Fc / Fc + ) redox mediator. From Figure 3It can be seen that after adding D-glucose during the response process of the stable enzyme-type sensing electrode prepared by the present invention, the anodic current increases significantly. In the glucose concentration range of 20 μM to 4 mM, the sensor response is linear, and saturation is observed above 4 mM.

[0094] 1.3 Catalytic selectivity of the stable enzyme-type sensing electrode in the presence of interfering substances

[0095] The stable enzyme-type sensing electrode prepared in Example 1 was placed in a phosphate buffer solution containing 0.2 mM 1,4-benzoquinone (pH 6.5), and DA (dopamine), DOPAC (3,4-dihydroxyphenylacetic acid), L-DOPA (levodopa), UA (uric acid), E (epinephrine), and NE (norepinephrine) were continuously added. Amperometric response curves were obtained at 0.1 V (vs. Ag / AgCl), and the results are as Figure 4 shown.

[0096] As can be seen from Figure 4 it, the stable enzyme-type sensing electrode prepared in Example 1 has good compatibility with other redox mediators, such as the 1,4-hydroquinone / benzoquinone (H2Q / Q) pair, whose redox potential is relatively low, which can improve the sensor selectivity in biologically relevant samples; using H2Q / Q as the electron mediator, the sensing electrode shows minimal interference with physiologically relevant substances, including DA, DOPAC, L-DOPA, UA, NE, and E, while maintaining high selectivity for glucose detection, demonstrating its potential in precise biosensing applications.

[0097] 1.4 Electrochemical signal stability

[0098] The stable enzyme-type sensing electrode prepared in Example 1 was continuously detected for 5000 s by adding 50 mM D-Glu to verify the electrochemical signal stability during continuous long-term detection, and the results are as Figure 5 shown.

[0099] During the extended monitoring (>80 min) of adding 50 mM glucose at a constant potential of 0.2 V, as can be seen from Figure 5 it, the stable enzyme-type sensing electrode of the present invention shows a stable catalytic current.

[0100] 2. Glucose catalytic oxidation performance of the glucose oxidase modified electrode

[0101] The electrochemical test method is the same as that in 1 above. A 0.2 mM ferrocenemethanol mediator solution was prepared with 50 mM phosphate buffer (pH 7.8) and purged with N2. The stable enzyme-type sensing electrode (GluO X -GCE TAD)It was placed in a mediator solution to obtain amperometric response curves under a system containing 10 mM D-glucose (D-Glu) and a system without 10 mM D-Glu and at a voltage of +0.2 V. The results are as Figure 6 shown; the black curve in the figure represents the system without 10 mM D-glucose, and the red curve represents the system containing 10 mM D-glucose.

[0102] 3. Glutamate catalytic oxidation performance of the glutamate synthase (GltS) modified electrode

[0103] The electrochemical test method was the same as that in 1 above. A 0.2 mM ferrocene methanol mediator solution was prepared with 50 mM phosphate buffer (pH 7.8) and purged with N2. The stable enzyme-type sensing electrode (GltS-GCE TAD ) prepared in Example 3 was placed in the mediator solution, and amperometric response curves were obtained under a system without adding glutamate and a system with continuous addition of glutamate and at a voltage of +0.2 V. The results are as Figure 7 shown; curve (1) in the figure represents the system without adding glutamate, and curve (2) represents the system with continuous addition of glutamate.

[0104] It can be seen from Figures 6 - 7 that after the electrocoupling of GluO X and GltS on the GCE, GluO X -GCE TAD and GltS-GCE TAD showed good electrochemical reactions to glucose and glutamate respectively, indicating that the stable enzyme-type sensing electrode of the present invention has a wide application prospect.

[0105] The present invention has been described according to the above embodiments. It should be understood that the above embodiments do not limit the present invention in any form. Any technical solutions obtained by using equivalent replacements or equivalent transformations fall within the scope of the present invention.

Claims

1. A method for preparing a stable enzyme-type sensing electrode, characterized in that, It includes the following steps: (1) Polish and ultrasonically clean the carbon-based electrode to obtain a pretreated carbon-based electrode; (2) Dissolve 4-(4-aminophenyl)-1,2,4-triazolidine-3,5-dione in a hydrochloric acid solution and stir to obtain a mixed solution A; (3) Drop a sodium nitrite solution into the mixed solution A and stir to obtain a mixed solution B; (4) Place the pretreated carbon-based electrode in the mixed solution B for reduction modification and cleaning to obtain a pre-modified electrode; (5) Place the pre-modified electrode in an enzyme solution for oxidation modification to obtain the product.

2. The preparation method of the stable enzyme-based sensing electrode according to claim 1, wherein, The step (1) includes the following steps: Place the carbon-based electrode with a smooth and stain-free surface on a flannel, and then successively place it in alumina abrasive slurries of 2 - 3 μm and 40 - 60 nm for circular polishing, and then ultrasonically clean the abrasive with ethanol and ultrapure water alternately for 2 - 3 times.

3. The preparation method of the stable enzyme-type sensing electrode according to claim 1, wherein, In the step (2), the material ratio of 4-(4-aminophenyl)-1,2,4-triazolidine-3,5-dione to the hydrochloric acid solution is 0.5 mg:1.5 - 3 mL; the molar concentration of the hydrochloric acid solution is 0.4 - 0.6 M.

4. The preparation method of the stable enzyme-type sensing electrode according to claim 1, wherein The stirring time in both the step (2) and the step (3) is 2 - 4 min.

5. The preparation method of the stable enzyme-type sensing electrode according to claim 1, characterized in that, In the step (3), the molar concentration of the nitrite solution is 90 - 110 mM; the volume ratio of the nitrite solution to the mixed solution A is 0.04 - 0.06:1.5 - 3.

6. The preparation method of the stable enzyme-type sensing electrode according to claim 1, characterized in that, In the step (4), the voltage for reduction modification is -0.2 - -0.4 V, and the time is 900 - 1100 s; the cleaning is successively carried out with water and acetonitrile.

7. The preparation method of the stable enzyme-type sensing electrode according to claim 1, characterized in that, In the step (5), the concentration of the enzyme solution is 1.5 - 2.5 mg / mL; the enzyme in the enzyme solution is an enzyme containing tyrosine residues in its structure.

8. The preparation method of the stable enzyme-based sensing electrode according to claim 1, characterized in that, In the step (5), the voltage for oxidation modification is +0.3 - +0.4 V, and the time is 4 - 6 min.

9. A stable enzyme-based sensing electrode prepared by the preparation method according to any one of claims 1 - 8.

10. Application of the stable enzyme-based sensing electrode according to claim 9 in substrate detection.

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