Cyclic peptide-enzyme composite material-based modified electrode and application thereof in hydrogen peroxide detection
Through the technology of modifying electrodes based on cyclic peptide-enzyme composite materials, the problems of low sensitivity and high cost of hydrogen peroxide detection in the existing technology have been solved, and rapid, sensitive and highly selective detection has been achieved, which is suitable for on-site applications.
Patent Information
- Application Number
- CN202411467051.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-21
- Publication Date
- 2025-01-24
AI Technical Summary
The prior art used in hydrogen peroxide detection has problems such as low sensitivity, high cost and complex operation, and it is difficult to meet the needs of rapid on-site inspection.
The electrodes based on cyclopanide-enzyme composite materials are used to form a nanoparticle composite material by combining the ring dilateine and the peroxidase of the spicy root, and combined with Nafion to modify the glass carbon electrode to build a high-sensitivity electrical electrical electrical electrical electrode Chemical sensor.
It achieves rapid, sensitive and highly selective detection of hydrogen peroxide, has a lower detection limit and good biocompatibility, is low in cost and is suitable for on-site application.
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Figure CN119355082A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of electrochemical analysis, and in particular relates to a cyclic peptide-enzyme composite material modified electrode and application thereof in hydrogen peroxide detection. Background Art
[0002] Hydrogen peroxide, commonly known as hydrogen peroxide, is a common chemical substance with strong oxidizing, bleaching and bactericidal properties. It is widely used in daily life, food safety, medical and other industries. At the same time, in many enzymatic reactions, hydrogen peroxide is an intermediate metabolite and is associated with the metabolic processes in many organisms. Excessive levels of hydrogen peroxide can be toxic to cells. This shows that hydrogen peroxide has a significant impact on people's daily lives, environmental pollution, and health and safety.
[0003] The methods currently used for hydrogen peroxide detection mainly include electrochemical analysis, chemiluminescence, high performance liquid chromatography, surface Raman enhancement, colorimetry and fluorescence, but most of these detection methods have the problems of high cost, complicated operation and long detection time, and cannot meet the requirements of on-site rapid detection. Electrochemical analysis is widely used due to its advantages of high specificity, fast response speed, simplicity, convenient operation and on-site analysis. In particular, amperometric biosensors based on redox proteins or enzymes (such as hemoglobin, horseradish peroxidase or cytochrome C) are attractive tools for detecting H2O2 due to their significant specificity and high sensitivity. Summary of the invention
[0004] In view of the technical problems of low detection sensitivity and high development cost in the current traditional hydrogen peroxide detection, the present invention provides a cyclic peptide-enzyme composite modified electrode and its application in hydrogen peroxide detection. The cyclic peptide-enzyme composite modified electrode provided by the present invention has the characteristics of fast response, low cost, high sensitivity, low detection limit, good biocompatibility, etc. for detecting hydrogen peroxide.
[0005] In order to achieve the above object, the technical solution adopted by the present invention is: a cyclopeptide-enzyme composite material modified electrode, the preparation method comprises the following steps:
[0006] 1) dissolving cyclic ditryptophan (c-WW) in an aqueous solution at room temperature, heating in a water bath, and centrifuging to obtain a cyclic ditryptophan peptide nanoparticle solution (c-WW NPs solution);
[0007] 2) dissolving horseradish peroxidase (HRP) in a cyclic ditryptophan peptide nanoparticle solution (c-WW NPs solution) at room temperature, and vortexing and vortexing on a vortexer to fully mix to obtain an HRP-(c-WW NPs) mixed solution;
[0008] 3) Use a pipette to transfer the HRP-(c-WW NPs) mixed solution and drop it on the surface of the glassy carbon electrode. After freeze-drying, immediately add the Nafion aqueous solution on the surface and spread it evenly to obtain a cyclopeptide-enzyme composite modified electrode (Nafion / HRP-(c-WW NPs) / GCE modified electrode).
[0009] Furthermore, in step 1), the concentration of the obtained c-WW NPs solution is 4-5 mg / mL.
[0010] Furthermore, in step 1), the water bath heating temperature is 95-105° C., and the heating time is 2-3 hours.
[0011] Furthermore, in step 1), the centrifugation is performed at 10,000 rpm for 10 to 20 min.
[0012] Furthermore, in step 2), the concentration of HRP in the obtained HRP-(c-WW NPs) mixed solution is 0.5-1.0 mg / mL.
[0013] Furthermore, in step 2), the oscillation time is 5 to 10 minutes.
[0014] Furthermore, in step 3), the volume percentage concentration of the Nafion aqueous solution is 0.1-0.2%.
[0015] The invention provides an application of a cyclopeptide-enzyme composite material modified electrode as a working electrode in detecting hydrogen peroxide.
[0016] Furthermore, the method is as follows: a three-electrode system is formed by using a Nafion / HRP-(c-WW NPs) / GCE modified electrode as a working electrode, a platinum wire as a counter electrode, and a silver-silver chloride electrode as a reference electrode. The three-electrode system is assembled and connected to an electrochemical workstation to form an electrochemical sensor. PBS with a pH of 7 and a concentration of 0.1 M is used as an electrolyte, and electrochemical CV detection parameters are set to detect the hydrogen peroxide sample to be tested.
[0017] Furthermore, the electrochemical CV detection parameters were: starting potential 0.2 V, ending potential -0.8 V, standing time 2 s, and scan rate 200 mV / s.
[0018] Compared with the prior art, the present invention has the following advantages:
[0019] 1. The electrode modified based on the cyclopeptide-enzyme composite material provided by the present invention utilizes cycloditryptophan with good biocompatibility as a carrier for fixing the enzyme. Due to its good biocompatibility, direct electron transfer of the enzyme is achieved. The method for constructing the enzyme electrode is simple, non-toxic and low-cost.
[0020] 2. The cyclopeptide-enzyme composite modified electrode provided by the present invention realizes rapid, sensitive and highly selective detection of hydrogen peroxide, which is significantly superior to existing reported literature and has potential application prospects.
[0021] 3. The electrode modified based on the cyclopeptide-enzyme composite material provided by the present invention has a relatively wide linear range of 5-3355 μM when detecting hydrogen peroxide. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 Cyclic voltammograms of different modified electrodes (a: Nafion / HRP / GCE, b: Nafion / c-WW NPs / GCE, c: Nafion / HRP-(c-WW NPs) / GCE) prepared in Example 1 in 0.1 M PBS (pH=7) solution.
[0023] Figure 2 The cyclic voltammetry curves of the modified electrode Nafion / HRP-(c-WW NPs) / GCE prepared in Example 1 in different concentrations of hydrogen peroxide.
[0024] Figure 3 This is the linear relationship between the reduction peak current corresponding to the Nafion / HRP-(c-WW NPs) / GCE modified electrode at -0.4V and the hydrogen peroxide concentration. DETAILED DESCRIPTION
[0025] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.
[0026] In a first aspect of an embodiment of the present invention, there is provided an electrode modified based on a cyclopeptide-enzyme composite material, the preparation method of which comprises the following steps:
[0027] 1) Dissolving cyclic ditryptophan (c-WW) in an aqueous solution at room temperature, heating in a water bath, and centrifuging to obtain a cyclic ditryptophan peptide nanoparticle solution (c-WW NPs solution);
[0028] 2) at room temperature, dissolving horseradish peroxidase (HRP) in the c-WW NPs solution, and vortexing and vortexing on a vortexer to fully mix, to obtain an HRP-(c-WW NPs) mixed solution;
[0029] 3) Use a pipette to transfer the HRP-(c-WW NPs) mixed solution and drop it on the surface of the glassy carbon electrode. After freeze-drying, immediately add the Nafion aqueous solution on the surface and spread it evenly to obtain a cyclopeptide-enzyme composite modified electrode (Nafion / HRP-(c-WW NPs) / GCE modified electrode).
[0030] The chemical formula of cycloditryptophan (c-WW) is C 22 N4H 20 The structural formula of O2 is shown in (I).
[0031]
[0032] The invention utilizes c-WW dipeptide to self-assemble into spherical nanoparticles, and combines with horseradish peroxidase (HRP) to form a cyclic peptide-enzyme nanocomposite material. The prepared Nafion / HRP-(c-WW NPs) / GCE modified electrode has good biocompatibility, a large electrochemically active specific surface area, a faster mass transfer rate in the electrochemical process, and can improve the activity of hydrogen peroxide catalytic reaction.
[0033] In some feasible embodiments, further, in step 1), the concentration of the obtained c-WW NPs solution is 4-5 mg / mL. In the present invention, the particle size synthesized within this concentration range is relatively uniform, the sample does not dissolve when the concentration is too high, and the sample self-assembles unevenly when the concentration is too low.
[0034] In some feasible embodiments, further, in step 1), the water bath heating temperature is 95-105°C, and the heating time is 2-3 hours. The method provided by the present invention dissolves cyclic ditryptophan (c-WW) in an aqueous solution at room temperature, and heats the solution in a water bath, which can promote self-assembly.
[0035] In some feasible embodiments, further, in step 1), the centrifugation is performed at 10,000 rpm for 10 to 20 min.
[0036] In some feasible embodiments, further, in step 2), the concentration of HRP in the obtained HRP-(c-WW NPs) mixed solution is 0.5-1.0 mg / mL. In the present invention, if the concentration of HRP is too low, no redox signal will appear, and if the concentration is too high, the peak current signal will be reduced.
[0037] In some feasible embodiments, further, in step 2), the oscillation time is 5 to 10 minutes. Vortex oscillation can fully mix the two to make them more uniform.
[0038] In some feasible embodiments, further, in step 3), the volume percentage concentration of the Nafion aqueous solution is 0.1-0.2%. In the present invention, within this concentration range, the composite material can be better adsorbed on the electrode to avoid falling off from the electrode during the experiment.
[0039] According to a second aspect of the embodiments of the present invention, a cyclic peptide-enzyme composite modified electrode is provided as a working electrode for detecting hydrogen peroxide.
[0040] In some feasible embodiments, further, the method is as follows: a three-electrode system is formed using a Nafion / HRP-(c-WW NPs) / GCE modified electrode as a working electrode, a platinum wire as a counter electrode, and a silver-silver chloride electrode as a reference electrode. The three-electrode system is assembled and connected to an electrochemical workstation to form an electrochemical sensor. PBS with a pH of 7 and a concentration of 0.1 M is used as an electrolyte, and electrochemical CV detection parameters are set to detect the hydrogen peroxide sample to be tested.
[0041] The Nafion / HRP-(c-WW NPs) / GCE modified electrode provided by the present invention has good biocompatibility and a large electrochemically active specific surface area, has a faster mass transfer rate in the electrochemical process, and can improve the activity of the hydrogen peroxide catalytic reaction.
[0042] In some feasible embodiments, further, the electrochemical CV detection parameters are: starting potential 0.2V, ending potential -0.8V, standing time 2s, and scanning rate 200mV / s.
[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0044] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0045] Example 1
[0046] (I) Electrode modified based on cyclic peptide-enzyme composite material (Nafion / HRP-(c-WW NPs) / GCE modified electrode)
[0047] The preparation method is as follows:
[0048] 1. Preparation of c-WW NPs:
[0049] At room temperature, 46 mg of cyclic ditryptophan (c-WW) was dissolved in 10 mL of aqueous solution to obtain a mixed solution with a concentration of 4.6 mg / mL. The mixed solution was placed in a water bath and heated at 100°C for 2 h to promote self-assembly. The mixed solution was centrifuged at 10,000 rpm for 10 min in a high-speed rotating centrifuge to obtain a cyclic ditryptophan peptide nanoparticle solution with a concentration of 4.6 mg / mL, which was named c-WWNPs solution.
[0050] 2. Preparation of HRP-(c-WW NPs):
[0051] At room temperature, 7.5 mg of horseradish peroxidase (HRP) was dissolved in 10 mL of c-WW NPs solution and vortexed on a vortexer for 5 min to mix thoroughly to obtain an HRP-(c-WW NPs) mixed solution with an HRP concentration of 0.75 mg / mL.
[0052] 3. Preparation of electrodes modified by cyclopeptide-enzyme composite materials:
[0053] The bare glassy carbon electrode (GCE) was pretreated by polishing it with aluminum oxide powder and cleaning its surface with ethanol.
[0054] 5 μL of HRP-(c-WW NPs) mixed solution was pipetted onto the surface of the glassy carbon electrode and placed in a refrigerator at 4°C until it was completely dry. Then, 5 μL of 0.125% Nafion aqueous solution was immediately added onto the surface and spread evenly to obtain a modified electrode based on a cyclopeptide-enzyme composite material, named Nafion / HRP-(c-WW NPs) / GCE modified electrode. (II) Preparation of Nafion / HRP / GCE modified electrode:
[0055] The bare glassy carbon electrode (GCE) was pretreated by polishing it with aluminum oxide powder and cleaning its surface with ethanol.
[0056] Use a pipette to transfer 5 μL of 0.75 mg / mL HRP aqueous solution onto the surface of the glassy carbon electrode and place it in a refrigerator at 4°C until it is completely dry. Immediately after drying, add 5 μL of Nafion (0.125%) solution onto the surface and spread it evenly to obtain a Nafion / HRP / GCE modified electrode.
[0057] (III) Preparation of Nafion / c-WW NPs / GCE modified electrode:
[0058] The bare glassy carbon electrode (GCE) was pretreated by polishing it with aluminum oxide powder and cleaning its surface with ethanol.
[0059] Use a pipette to transfer 5 μL of 4.6 mg / mL c-WW NPs solution onto the surface of the glassy carbon electrode and place it in a refrigerator at 4°C until it is completely dry. Immediately after drying, add 5 μL of Nafion (0.125%) solution onto the surface and spread it evenly to obtain a Nafion / c-WW NPs / GCE modified electrode.
[0060] Example 2 Application
[0061] (I) Electrochemical test
[0062] The different modified electrodes prepared in Example 1 were used as working electrodes, platinum wire as counter electrode, and silver-silver chloride electrode as reference electrode to form a three-electrode system. The three-electrode system was assembled and connected to an electrochemical workstation to form an electrochemical sensor. 0.1M PBS (pH=7) was added to the electrolytic cell as an electrolyte. Cyclic voltammetry was selected with a starting potential of 0.2V, a termination potential of -0.8V, a standing time of 2s, and a scan rate of 200mV / s.
[0063] (II) Results
[0064] Figure 1 The cyclic voltammetry curves of different modified electrodes (a: Nafion / HRP / GCE, b: Nafion / c-WW NPs / GCE, c: Nafion / HRP-(c-WW NPs) / GCE) prepared in Example 1 in 0.1M PBS (pH=7) solution. Figure 1 It can be seen that only Nafion / HRP-(c-WW NPs) / GCE showed redox peaks, indicating that the composite electrode achieved good enzyme protein immobilization and direct electron transfer.
[0065] Figure 2 The cyclic voltammograms of the Nafion / HRP-(c-WW NPs) / GCE working electrode prepared in Example 1 in electrolytes containing different concentrations of hydrogen peroxide are shown in FIG. Figure 2 As can be seen from the graph, as the concentration of hydrogen peroxide increases from 1 μM to 1 mM, the reduction peak current increases and the oxidation peak current decreases. This indicates that HRP immobilized on c-WW NPs undergoes good electrocatalytic reduction of H2O2.
[0066] Figure 3 The linear relationship between the reduction peak current and the hydrogen peroxide concentration at -0.4V of the Nafion / HRP-(c-WW NPs) / GCE modified electrode is shown in Figure 2. Figure 3 It can be seen that the response current is linear with the hydrogen peroxide concentration, and the corresponding linear regression equations are I(μA)=10.26381x+0.98812(R2 =0.996) and I(μA)=7.70843x+4.11317(R 2 =0.998), the sensor has a sensitivity of 142.55 μA·mM -1 cm -2 , it can be seen that the electrode has a good detection effect on hydrogen peroxide.
[0067] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.
Claims
1. A modified electrode based on a cyclic peptide-enzyme composite material, characterized in that: The preparation method comprises the following steps: 1) Dissolving cyclic ditryptophan c-WW in an aqueous solution at room temperature, heating in a water bath, and centrifuging to obtain a cyclic ditryptophan peptide nanoparticle c-WW NPs solution; 2) at room temperature, dissolving horseradish peroxidase HRP in the cyclic ditryptophan peptide nanoparticle c-WW NPs solution, and vortexing and vortexing on a vortexer to fully mix, thereby obtaining a HRP-(c-WW NPs) mixed solution; 3) Use a pipette to transfer the HRP-(c-WW NPs) mixed solution and drop it on the surface of the glassy carbon electrode. After freeze-drying, immediately add the Nafion aqueous solution on the surface and spread it evenly to obtain the Nafion / HRP-(c-WW NPs) / GCE modified electrode based on the cyclopeptide-enzyme composite material.
2. The electrode modified by cyclopeptide-enzyme composite material according to claim 1, characterized in that: In step 1), the concentration of the obtained cyclic ditryptophan peptide nanoparticle c-WW NPs solution is 4-5 mg / mL.
3. The electrode modified by cyclic peptide-enzyme composite material according to claim 1, characterized in that: In step 1), the water bath heating temperature is 95-105° C., and the heating time is 2-3 hours.
4. The electrode modified by cyclic peptide-enzyme composite material according to claim 1, characterized in that: In step 1), the centrifugation is performed at 10,000 rpm for 10 to 20 minutes.
5. The electrode modified by cyclic peptide-enzyme composite material according to claim 1, characterized in that: In step 2), the concentration of HRP in the obtained HRP-(c-WW NPs) mixed solution is 0.5-1.0 mg / mL.
6. The electrode modified by cyclic peptide-enzyme composite material according to claim 1, characterized in that: In step 2), the shaking time is 5 to 10 minutes.
7. The electrode modified by cyclic peptide-enzyme composite material according to claim 1, characterized in that: In step 3), the volume percentage concentration of the Nafion aqueous solution is 0.1-0.2%.
8. Use of the cyclopeptide-enzyme composite material modified electrode according to any one of claims 1 to 7 as a working electrode in detecting hydrogen peroxide.
9. The use according to claim 8, characterized in that: The method is as follows: a three-electrode system is formed using a Nafion / HRP-(c-WW NPs) / GCE modified electrode as a working electrode, a platinum wire as a counter electrode, and a silver-silver chloride electrode as a reference electrode. The three-electrode system is assembled and connected to an electrochemical workstation to form an electrochemical sensor. PBS with a pH of 7 and a concentration of 0.1 M is used as the electrolyte. The electrochemical CV detection parameters are set to detect the hydrogen peroxide sample to be tested.
10. The use according to claim 9, characterized in that: The electrochemical CV detection parameters were as follows: starting potential 0.2 V, ending potential -0.8 V, standing time 2 s, and scan rate 200 mV / s.