阅读说明：本技术 一种抗菌多肽固定的导电聚合物电极及其制备方法与应用 (Antibacterial polypeptide-immobilized conductive polymer electrode and preparation method and application thereof ) 是由 宁成云 邢君 于鹏 王珍高 张珂嘉 代聪 于 2019-07-22 设计创作，主要内容包括：本发明公开了一种抗菌多肽固定的导电聚合物电极及其制备方法与应用,所述制备方法包括以下步骤：(1)沉积导电聚合物预成核层；(2)沉积聚多巴胺功能化的导电聚合物涂层；(3)将抗菌多肽溶解于磷酸盐缓冲液中,得抗菌多肽溶液；将步骤(2)制备的沉积有聚多巴胺功能化的导电聚合物涂层的导电电极浸入到抗菌多肽溶液中,得抗菌多肽固定的导电聚合物电极。聚多巴胺功能化的导电聚合物电极表面富含邻苯酚羟基,易于进一步固定抗菌多肽,从而提高电极表面的电化学性能；抗菌多肽固定的导电聚合物电极具有消除细菌粘附,在细菌环境中高电化学稳定性,以及长期抗菌效果的能力,实现了电极活性损失的最小化,从而拓宽了电化学传感器的应用。(The invention discloses a conductive polymer electrode fixed by antibacterial polypeptide and a preparation method and application thereof, wherein the preparation method comprises the following steps: (1) depositing a conductive polymer pre-nucleation layer; (2) depositing a poly-dopamine functionalized conductive polymer coating; (3) dissolving the antibacterial polypeptide in a phosphate buffer solution to obtain an antibacterial polypeptide solution; and (3) immersing the conductive electrode deposited with the poly-dopamine functionalized conductive polymer coating prepared in the step (2) into an antibacterial polypeptide solution to obtain the conductive polymer electrode fixed by antibacterial polypeptide. The poly-dopamine functionalized conductive polymer electrode surface is rich in o-phenolic hydroxyl groups, so that the antibacterial polypeptide can be further easily fixed, and the electrochemical performance of the electrode surface is improved; the conductive polymer electrode fixed by the antibacterial polypeptide has the capabilities of eliminating bacterial adhesion, realizing high electrochemical stability in a bacterial environment and long-term antibacterial effect, and realizing the minimization of the activity loss of the electrode, thereby widening the application of an electrochemical sensor.)

1. a preparation method of a conductive polymer electrode fixed by antibacterial polypeptide is characterized by comprising the following steps:

(1) Depositing a conductive polymer pre-nucleation layer:

depositing a conductive polymer pre-nucleation layer on the surface of a conductive electrode by an electrochemical method in a three-electrode mode, wherein the three electrodes comprise a counter electrode, a working electrode and a reference electrode, the working electrode is a conductive electrode, the electrolyte solution is a phosphate buffer solution containing conductive polymer monomers and chloride ions, and the electrochemical reaction is controlled by a timing current method to obtain the conductive electrode with the conductive polymer pre-nucleation layer deposited on the surface;

(2) Deposition of a poly-dopamine functionalized conductive polymer coating:

Electrochemically depositing a dopamine-doped conductive polymer coating on the surface of the conductive polymer pre-nucleation layer deposited in the step (1) in a three-electrode mode, wherein the three electrodes comprise a counter electrode, a working electrode and a reference electrode, the working electrode is the conductive electrode with the conductive polymer pre-nucleation layer deposited on the surface and prepared in the step (1), the electrolyte solution is phosphate buffer solution containing conductive polymer monomers and dopamine, and the electrochemical reaction is controlled by adopting a time potential method to obtain the conductive electrode with the deposited dopamine-functionalized conductive polymer coating;

(3) Grafted antimicrobial polypeptides

Dissolving the antibacterial polypeptide in a phosphate buffer solution to obtain an antibacterial polypeptide solution; and (3) immersing the conductive electrode deposited with the poly-dopamine functionalized conductive polymer coating prepared in the step (2) into an antibacterial polypeptide solution to obtain the conductive polymer electrode fixed by antibacterial polypeptide.

2. the method for preparing an antibacterial polypeptide-immobilized conductive polymer electrode according to claim 1, wherein the source of chloride ions in step (1) is hydrogen chloride, potassium chloride or sodium chloride;

the concentration of chloride ions in the electrolyte solution is 0.1-0.5 mol/L, and the concentration of the conductive polymer monomer is 0.01-0.5 mol/L; the time of the electrochemical reaction is 5-60 seconds, and the voltage of the electrochemical reaction is 0.6-1.2V.

3. The method for preparing the conductive polymer electrode with the immobilized antimicrobial polypeptide according to claim 1, wherein the concentration of the conductive polymer monomer in the electrolyte solution of the step (2) is 0.01-0.5 mol/L, the concentration of dopamine is 0.01-0.5 mol/L, the time of the electrochemical reaction is 5-50 minutes, and the current density of the electrochemical reaction is 0.4-2.0 m A/cm ².

4. the method for preparing the conductive polymer electrode with the immobilized antimicrobial polypeptide according to claim 1, wherein the conductive polymer monomer in the steps (1) and (2) is any one of thiophene, pyrrole or aniline; the counter electrode is made of conductive metal; the conductive metal is platinum or copper; the conductive electrode is any one of a titanium electrode, a platinum electrode or a gold electrode; the reference electrode is a saturated calomel electrode; the pH value of the phosphate buffer solution is 5.3-7.4.

5. the method for preparing a conductive polymer electrode having an antimicrobial polypeptide immobilized thereon according to claim 1, wherein the antimicrobial polypeptide of step (3) is marguerin 2; the concentration of the antibacterial polypeptide in the antibacterial polypeptide solution is 0.1-5 g/L; the pH value of the phosphate buffer solution is 6.8-8.5; the immersion condition is that the antibacterial polypeptide is immersed in the antibacterial polypeptide solution at the temperature of 20-37 ℃ for 24-72 hours.

6. The method for preparing an antibacterial polypeptide-immobilized conductive polymer electrode according to claim 1, wherein the source of chloride ions in step (1) is hydrogen chloride.

7. the method for preparing a conductive polymer electrode having an immobilized antimicrobial polypeptide according to claim 1, wherein the concentration of the conductive polymer monomer in the electrolyte solution in the step (2) is 0.2mol/L, the concentration of dopamine is 0.1mol/L, the time for the electrochemical reaction is 40 minutes, and the current density is 1.5mA/cm ².

8. the method for preparing a conductive polymer electrode having an antibacterial polypeptide immobilized thereon according to claim 1, wherein the conductive polymer monomer in the steps (1) and (2) is pyrrole; the pH of the phosphate buffer was 6.8; the counter electrode is a copper electrode; the conductive electrode is a titanium electrode.

9. an antibacterial polypeptide-immobilized conductive polymer electrode prepared by the preparation method of any one of claims 1 to 8.

10. Use of the antimicrobial polypeptide immobilized conducting polymer electrode of claim 9 in environmental, food or medical testing.

Technical Field

the invention relates to the technical field of electrode equipment, in particular to a conductive polymer electrode fixed by antibacterial polypeptide and a preparation method and application thereof.

background

Electrically conductive polymer electrodes are widely used for rapid signal detection and monitoring in the fields of clinical, food, environmental and bioprocessing due to their unique mechanical, redox and impedance properties, and are easily functionalized, however, in various environments, bacteria readily adhere to the electrode surface { V. Villalobos, A. Leiva, H.E. Rios, J. Pavez, C. P.Silva, M. Ahmar, Y. Queneau, J.M. Amay, F.18P. Chavez, M.D. Urzua, ACS Appl. Mater. Interfaces 2018, 10, 28147 } although the level of bacteria found in hospital environments is reduced by the use of sterile techniques, microorganisms can still be observed in most electrodes.

however, the ease of leaching and short duration of antimicrobial agents, the risk of developing resistant bacteria and the impaired electrochemical stability of the electrode material limit their application { R. ZHao, W. Kong, M. Sun, Y. Yang, W. Liu, M. Lv, S. Song, L. Wang, H. Song, R. Hao, ACS Appl. Mater. Interfaces 2018, 10, 17}

Studies have shown that antimicrobial polypeptides have rapid and broad-spectrum bactericidal properties, reducing the risk of inducing bacterial resistance. In addition, the antibacterial polypeptide is easy to be fixed on the surface of a target substrate through covalent grafting due to abundant functional groups (amino, carboxyl, sulfydryl and the like), and a large steric hindrance effect cannot be generated due to a small-molecule short-chain linear structure. Thus, antimicrobial polypeptides are ideal candidates for the functionalization of conductive electrodes, which can achieve both the elimination of bacteria and the maintenance of electrochemically stable properties in a bacterial environment.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the invention aims to provide a conductive polymer electrode with immobilized antibacterial polypeptide, and a preparation method and application thereof. According to the invention, a layer of dopamine-doped conductive polymer molecular layer is constructed on the surface of the electrode by an electrochemical deposition method, and the dopamine is further used as a molecular anchor to graft antibacterial polypeptide, so that the long-term effectiveness of the electrochemical stability and antibacterial performance of the electrode is realized.

The object of the invention is achieved by at least one of the following solutions.

The invention provides a preparation method of a conductive polymer electrode fixed by antibacterial polypeptide, which comprises the following steps:

(1) Depositing a conductive polymer pre-nucleation layer:

Depositing a conductive polymer pre-nucleation layer on the surface of a conductive electrode by an electrochemical method in a three-electrode mode, wherein the three electrodes comprise a counter electrode, a working electrode and a reference electrode, the working electrode is a conductive electrode, the electrolyte solution is a phosphate buffer solution containing conductive polymer monomers and chloride ions, and the electrochemical reaction is controlled by a timing current method to obtain the conductive electrode with the conductive polymer pre-nucleation layer deposited on the surface;

(2) deposition of a poly-dopamine functionalized conductive polymer coating:

Electrochemically depositing a dopamine-doped conductive polymer coating on the surface of the conductive polymer pre-nucleation layer deposited in the step (1) in a three-electrode mode, wherein the three electrodes comprise a counter electrode, a working electrode and a reference electrode, the working electrode is the conductive electrode with the conductive polymer pre-nucleation layer deposited on the surface and prepared in the step (1), the electrolyte solution is phosphate buffer solution containing conductive polymer monomers and dopamine, and the electrochemical reaction is controlled by adopting a time potential method to obtain the conductive electrode with the deposited dopamine-functionalized conductive polymer coating;

(3) Grafted antimicrobial polypeptides

Dissolving the antibacterial polypeptide in a phosphate buffer solution to obtain an antibacterial polypeptide solution; and (3) immersing the conductive electrode deposited with the poly-dopamine functionalized conductive polymer coating prepared in the step (2) into an antibacterial polypeptide solution to obtain the conductive polymer electrode fixed by antibacterial polypeptide.

preferably, the source of chloride ions in step (1) is hydrogen chloride, potassium chloride or sodium chloride;

The concentration of chloride ions in the electrolyte solution is 0.1-0.5 mol/L, and the concentration of the conductive polymer monomer is 0.01-0.5 mol/L;

The time of the electrochemical reaction is 5-60 seconds, and the voltage of the electrochemical reaction is 0.6-1.2V.

preferably, the concentration of the conductive polymer monomer in the electrolyte solution in the step (2) is 0.01-0.5 mol/L, and the concentration of dopamine is 0.01-0.5 mol/L;

the time of the electrochemical reaction is 5-50 minutes, and the current density of the electrochemical reaction is 0.4-2.0 m A/cm ².

Preferably, the conductive polymer monomer in steps (1) and (2) is any one of thiophene, pyrrole or aniline; the counter electrode is made of conductive metal; the conductive metal is platinum or copper; the conductive electrode is any one of a titanium electrode, a platinum electrode or a gold electrode; the reference electrode is a saturated calomel electrode; the pH value of the phosphate buffer solution is 5.3-7.4.

Preferably, the antibacterial polypeptide in step (3) is marenin 2; the concentration of the antibacterial polypeptide in the antibacterial polypeptide solution is 0.1-5 g/L; the pH value of the phosphate buffer solution is 6.8-8.5; the immersion condition is that the antibacterial polypeptide is immersed in the antibacterial polypeptide solution at the temperature of 20-37 ℃ for 24-72 hours.

Preferably, the source of chloride ions in step (1) is hydrogen chloride.

preferably, the concentration of the conductive polymer monomer in the electrolyte solution in the step (2) is 0.2mol/L, the concentration of dopamine is 0.1mol/L, the time of the electrochemical reaction is 40 minutes, and the current density is 1.5mA/cm ².

preferably, the conductive polymer monomer in steps (1) and (2) is pyrrole; the pH of the phosphate buffer was 6.8; the counter electrode is a copper electrode; the conductive electrode is a titanium electrode.

The invention also provides the conductive polymer electrode fixed by the antibacterial polypeptide prepared by the preparation method.

The invention also provides application of the conductive polymer electrode fixed with the antibacterial polypeptide in environmental, food or medical detection.

Compared with the prior art, the invention has the following beneficial effects and advantages:

(1) constructing a poly-dopamine functionalized conductive polymer coating on the surface of an electrode by adopting a pollution-free, rapid and controllable electrochemical method; the preparation method provided by the invention is simple, environment-friendly and easy to realize;

(2) The poly-dopamine functionalized conductive polymer electrode surface is rich in o-phenolic hydroxyl groups, so that the antibacterial polypeptide can be further easily fixed, and the electrochemical performance of the electrode surface is improved;

(3) The conductive polymer electrode fixed by the antibacterial polypeptide has the capabilities of eliminating bacterial adhesion, high electrochemical stability in a bacterial environment and long-term antibacterial effect, and realizes the minimization of the activity loss of the electrode, thereby widening the application of the electrochemical sensor.

drawings

FIG. 1 is a bar graph of the bacterial survival rates of the antimicrobial polypeptides immobilized conductive polymer electrodes prepared in examples 1-6;

FIG. 2 is an AC impedance spectrum of the antibacterial polypeptide-immobilized conductive polymer electrode prepared in example 2 in a bacterial environment.

Detailed Description

The present invention and the antibacterial effect achieved by the present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto, and the process parameters not particularly specified may be performed with reference to the conventional techniques.