CN115887793A

CN115887793A - Preparation and amination method of polyphenol oxidase catalyzed polyphenol coating material

Info

Publication number: CN115887793A
Application number: CN202211222823.XA
Authority: CN
Inventors: 洪枫; 胡高铨; 陈琳
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2022-10-08
Filing date: 2022-10-08
Publication date: 2023-04-04

Abstract

The invention relates to a method for preparing and aminating a polyphenol coating material catalyzed by polyphenol oxidase, which comprises the following steps: putting a substrate material to be subjected to surface modification into a buffer solution containing polyphenol oxidase, adding polyphenol, fully and uniformly stirring, carrying out oscillation reaction, and carrying out ultrasonic washing to obtain a polyphenol oxidase catalyzed polyphenol coating material; or amination of the polyphenol coating material. The method disclosed by the invention is environment-friendly, high in compounding efficiency, suitable for the surfaces of various hydrophilic and hydrophobic materials, capable of preparing uniform and stable poly-polyphenol coatings and aminated coatings on the surfaces of various materials, high in coating speed, high in active group content, universal in application and the like, and wide in application prospect in the fields of biomedicine, environmental pollution resistance and the like.

Description

Preparation and amination method of polyphenol oxidase catalyzed polyphenol coating material

Technical Field

The invention belongs to the field of surface modification, and particularly relates to a method for preparing and aminating a polyphenol coating material catalyzed by polyphenol oxidase.

Background

The polyphenol compound can be adhered and polymerized on the surface of almost all materials under the alkaline condition in the presence of oxygen to form a polymer coating, and is independent of the types and shapes of the materials. A large number of phenolic hydroxyl groups and quinone groups exist on the surface of the film, and the film can be used as a reaction platform to combine proteins, polypeptides and other macromolecules. Therefore, the polyphenol coating is a modification technology with application prospect.

However, in addition to dopamine, most polyphenol compounds such as catechol, tannic acid, gallic acid and the like have poor adhesion effect of polyphenol formed under alkaline conditions in the presence of oxygen, cannot form a firm coating, and are not stable enough. Moreover, alkaline conditions are destructive to some biomedical materials, e.g., resulting in degradation and mechanical loss of polyurethanes and the like. Thus, these polyphenolic compounds often require complexing metal ions to form a stable coating. For example, cu is introduced into tea polyphenol solution ²⁺ A stable copper-catechol coating can be obtained, or metal ions are added in the tannic acid solution, however, the introduction of the metal ions can form competitive coordination on one hand, so that the number of active phenolic hydroxyl groups is reduced, and the activity reaction of the coating is reduced; on the other hand, due to the complex physiochemical reaction of the human body, the metal ion mediated polyphenol medical material has the risk of unstable coating in the human body, so that a large amount of metal ions are released, and the threat to the human body is caused. Therefore, a novel polyphenol coating preparation technology is urgently needed at present to make up the defects of the metal ion mediated coating, reduce the safety risk and obtain a uniform and stable polyphenol coating.

In recent years, biological enzyme catalysis has attracted extensive attention in the field of catalytic synthesis of polymers due to the characteristics of mild reaction conditions, small environmental pollution and the like. Polyphenol oxidase such as laccase and the like can catalyze polyphenol substrates to polymerize under the acidic condition of oxygen, and a polyphenol coating is formed on the surface of the material. Compared with the traditional alkaline oxygen self-polymerization and metal ion-mediated polyphenol coating preparation technology, the preparation technology has the characteristics of mild reaction conditions and stable polymer coating, and simultaneously avoids the problems of mechanical loss and the like caused by excessive toxicity of metal ions and degradation of polyester materials under alkaline conditions.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for preparing and aminating a polyphenol coating material catalyzed by polyphenol oxidase, which solves the problems of poor coating adhesion effect and unstable coating and simultaneously avoids the toxicity of metal ions.

The invention provides a method for preparing a polyphenol coating material catalyzed by polyphenol oxidase and amination thereof, which comprises the following steps:

(1) Dissolving polyphenol oxidase in a buffer solution, fully and uniformly stirring, and measuring enzyme activity;

(2) Putting a substrate material to be subjected to surface modification into the solution obtained in the step (1), adding polyphenol, fully and uniformly stirring, carrying out oscillation reaction, and carrying out ultrasonic washing to obtain a polyphenol coating material catalyzed by polyphenol oxidase;

or (3) an amination method of the polyphenol coating material: adding polyphenol into the solution obtained in the step (1), adding a substrate material, stirring, carrying out oscillation reaction, carrying out ultrasonic washing to obtain a polyphenol coating material, and then placing the polyphenol coating material in an amino compound or imino compound aqueous solution for reaction to obtain a polyphenol-amino/imino compound coating material; or adding polyphenol and amino compound or imino compound into the solution obtained in the step (1), adding a substrate material, stirring, and carrying out oscillation reaction to obtain the polyphenol-amino/imino compound coating material.

The polyphenol oxidase in the step (1) is one or more of laccase, catechol oxidase, tyrosinase, bisphenol oxidase, phenol enzyme, cresol enzyme and catechol oxidoreductase, and the enzyme activity is 5U/mL-100U/mL; the mass-to-volume ratio of the polyphenol oxidase to the buffer solution is 1-5mg. The polyphenol oxidases are all purchased from Shanghai leaf Biotech limited.

The buffer solution in the step (1) is phosphate buffer solution or acetate buffer solution, the ionic strength is 20-100mM, and the pH value is 2.2-7.0. The enzyme activity is measured by an ABTS method and an ultraviolet spectrophotometer.

The substrate material in the step (2) is one or more of cellulose, polyvinyl alcohol, polyurethane, polyester, polytetrafluoroethylene, polymethyl methacrylate, medical polysiloxane, stainless steel, glass, titanium and alloy thereof, magnesium and alloy thereof.

The cellulose is plant-derived cellulose or microbial-derived bacterial cellulose, and comprises nanocellulose (nanofiber and nanocrystal).

The polyurethane is

AL polycarbonate-based ether-free polyurethane, liquid polyurethane->

AR、Carbothane ^TM 、Tecoflex ^TM 、/>

Tecothane ^TM One or more of thermoplastic polyurethane.

The remaining substrate materials are commercially available materials such as polyester available from Shanghai Testet medical science and technology; the polytetrafluoroethylene is commercially available from Boston science (BOSTON SCIENTIFIC EXXCEL), gole (GORE-TEX) and Shanghai Suokang medical materials, inc.

The polyphenol in the step (2) comprises dopamine (C) ₈ H ₁₁ NO ₂ Dopamine, DA), norepinephrine (C) ₈ H ₁₁ NO ₃ Norepinepherine, NE), epigallocatechin gallate (C) ₂₂ H ₁₈ O ₁₀ Epigallocatechin gallate (EGCG), epigallocatechin (C) ₁₅ H ₁₄ O ₇ Epigallocatachin, EGC), tannic acid (C) ₇₆ H ₅₂ O ₄₆ Tannic acid, TA), gallic acid (C) ₇ H ₆ O ₅ Gallic acid, GA), curcumin (C) ₂₁ H ₂₀ O ₆ Curcumin) is added.

The addition amount of the polyphenol in the step (2) is 0.1mg/mL-4mg/mL.

The oscillation reaction in the step (2) is oscillation reaction for 3-24h at 25-35 ℃, and the oscillation reaction frequency is 1-5.

And (3) carrying out ultrasonic washing in the step (2) by using 75% of ethanol and deionized water for 15 minutes, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using deionized water, and repeating for three times.

The polyphenol type, the polyphenol adding amount, the reaction temperature and the coating times in the step (3) are the same as those in the step (2).

Preferably, the concentration of the amino compound or imino compound in the aqueous solution in the step (3) is 3 to 5g/L.

Preferably, the concentration of the amino compound or imino compound in the solution after the amino compound or imino compound is added in said step (3) is 0.6-1.5mg/mL.

Preferably, the amino compound or imino compound in step (3) comprises polyethyleneimine, arginine or lysine.

Preferably, the reaction temperature of the amino compound or imino compound in the step (3) in the aqueous solution is 50-70 ℃ and the reaction time is 4-6h.

Preferably, the cleaning in the step (3) is performed by using ultrapure water.

Preferably, the ultrasonic washing in the step (3) is: and carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, and after 3 times of washing, continuing ultrasonic washing for 15 minutes by using the deionized water, and repeating the steps for three times.

The invention also provides a preparation method of the material with the heparin coating, which comprises the following steps:

placing polyphenol coating material catalyzed by polyphenol oxidase or polyphenol-amino/imino compound coating material in heparin aqueous solution containing EDC and NHS, reacting in dark place, cleaning, and drying to obtain material with heparin coating.

Preferably, the concentration of heparin in the heparin aqueous solution containing EDC and NHS is 0.5-1.5g/L, the concentration of NHS is 0.01-0.05M, and the molar ratio of EDC/NHS is 0.98-1.1.

Preferably, the reaction protected from light is: and reacting for 20-30h at 35-40 ℃ under the condition of pH =5.5 +/-0.5 and avoiding light.

Preferably, the cleaning uses ultrapure water.

Preferably, the obtained material with the heparin coating is used for activating and modifying the surface of the cardiovascular and cerebrovascular product.

Preferably, the obtained material with heparin coating is applied to the preparation of the anti-thrombosis material.

The invention overcomes the defects of unstable polymer coating, poor adhesion effect and uneven coating after oxygen self-polymerization of polyphenol in an alkaline environment; meanwhile, the preparation technology also avoids the use of metal ions, and compared with the preparation technology of the metal ion mediated polyphenol coating, the preparation technology avoids the toxicity caused by the excessive use and burst release of the metal ions, and avoids the potential safety risk. Besides the advantages, the technology has the characteristics of mild reaction conditions, adjustable and controllable parameters, small environmental pollution and the like. The substrate material cellulose used in the preparation technology is commercially available lignocellulose or bacterial cellulose prepared by fermentation, and the rest substrate materials are commercially available products; the polyphenol oxidase used in the preparation technology is all commercial polyphenol oxidase. And (2) placing the substrate material in a buffer solution in which polyphenol oxidase and polyphenol are dissolved at room temperature, catalyzing the polymerization of polyphenol on the surface of the substrate for several hours, and then carrying out ultrasonic washing to obtain the material coated with the polyphenol coating.

Advantageous effects

(1) Compared with the traditional polyphenol oxygen self-polymerization under the alkaline condition, the preparation method improves the adhesion and polymerization effects of polyphenol on the surface of the material, the coating speed is higher, the phenolic hydroxyl group of the obtained coating is higher, the polyphenol polymerized coating formed on the surface of the material is firmer, more uniform and stable, and more bioactive substances are grafted subsequently;

(2) Compared with the metal ion mediated polyphenol coating, the invention avoids the defects of competitive coordination caused by the introduction of metal ions, reduced number of active phenolic hydroxyl groups and reduced active reaction of the coating;

(3) Compared with a polyphenol coating mediated by metal ions, when the material coated with the polyphenol coating prepared by the invention is applied to medical materials and instruments and is contacted/implanted into a human body, the risks of instability of the coating and release of a large amount of metal ions are avoided, potential safety risks are avoided, and safety damage to the human body is avoided;

(4) According to the invention, most of hydrophilic and hydrophobic materials and organic and inorganic materials can be used as base materials, most of polyphenol compounds are used as catalytic substrates, and the catalyst has the advantages of wide application range and broad-spectrum applicability;

(5) The reaction pH is below neutral, so that the mechanical loss caused by degradation of polyester materials such as polyurethane under alkaline conditions in the traditional process can be avoided;

(6) The invention can change the polymerization conditions by adjusting parameters such as pH, reaction time, solution concentration, enzyme activity and the like, no additional reagent is required to be introduced in the reaction, the prepared polyphenol coating has more surface active groups, the preparation method is simple and easy to implement, the preparation conditions are mild and controllable, and the method is green and environment-friendly and has good market application prospect.

(7) Compared with the traditional process, the invention can graft more polyamino substances such as arginine, lysine and the like through a one-step method so as to improve the biocompatibility; can also be used as a secondary reaction platform to graft more anticoagulant substances (such as heparin and the like) compared with the traditional process so as to improve the activities of the blood compatibility and the like; short reaction flow, high efficiency and contribution to industrial application.

Drawings

FIG. 1 is an electron microscope image of 10 ten thousand times and an electron microscope image of 2 ten thousand times of the surface of the Lac-PU-PODA and Tris-PU-PODA materials of example 1;

FIG. 2 is a graph showing the content of phenolic hydroxyl groups contained in the various coatings obtained in examples 1 to 3;

FIG. 3 is a 10 ten thousand times electron micrograph of examples 4 to 6;

FIG. 4 is a graph showing the content of phenolic hydroxyl groups contained in the various coatings obtained in examples 4 to 6;

FIG. 5 is a 2 ten thousand fold electron micrograph of examples 7 to 8;

FIG. 6 is a 10 ten thousand fold electron micrograph of examples 9 and 6;

FIG. 7 is a graph showing the content of phenolic hydroxyl groups contained in the various coatings obtained in examples 7 to 9;

FIG. 8 shows the results of phenolic hydroxyl group contents after coating with tannic acid of different concentrations in example 10;

FIG. 9 shows the results of coating the phenolic hydroxyl content of TA with different enzyme activities in example 11;

FIG. 10 is the results of phenolic hydroxyl content using different catalytic times TA for example 12;

FIG. 11 is the results of the amount of amino grafting after coating of different polyphenol species in example 13 and example 14;

FIG. 12 is a flow chart of the preparation of grafted heparin after one-step amination catalyzed by laccase in example 18;

FIG. 13 is the cell viability data of human umbilical vein endothelial cells on PU membrane and argininated PU membrane of example 19;

FIG. 14 is the cell viability data of human umbilical vein endothelial cells on the PU membrane and the lysated PU membrane of example 20;

FIG. 15 is a schematic preparation scheme of the poly-polyphenol coating material and its amination of the present invention;

FIG. 16 shows the molecular structures of the polyphenols and the amino and imino compounds used.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

(1) The preparation method of the polyurethane film sheet comprises the following steps: dissolving the polyurethane particulate material with tetrahydrofuran

AL polycarbonate-based ether-free polyurethane available from HnG medical technology, inc, canada), preparing a PU solution in which the mass concentration of polyurethane is 15% or liquid polyurethane @, available from HnG medical technology, co, canada, is dissolved in DMF>

AR (solid content: 22%) was diluted to a mass concentration of 15% in polyurethane. Pouring the polyurethane solution into a Teflon mold with the length of 10mm, the width of 10mm and the depth of 2mm, slightly putting the Teflon mold filled with the polyurethane solution into water, immersing the mold on the liquid surface, removing the mold after 24 hours, repeatedly cleaning by using absolute ethyl alcohol and deionized water, and drying to obtain the polyurethane film sheet by the solvent solidification method.

(2) Preparation of phosphate buffer solution: a 0.2M disodium hydrogen phosphate solution and a 0.2M sodium dihydrogen phosphate solution were prepared in advance, and the above solutions were mixed to prepare a phosphate buffer solution having a pH =5.0.

(3) Preparation of phosphate buffer solution containing laccase: adding 10mg of laccase (with the activity of more than or equal to 0.5U/mg solid, purchased from Shanghai-sourced leaf Biotechnology Co., ltd., the same below) into the phosphate buffer solution in the step (2), measuring the enzyme activity of the laccase buffer solution by using an ABTS method, and regulating the enzyme activity to 40U/mL by using the phosphate buffer solution in the step (2).

(4) Preparation of a phosphate polyphenol solution containing laccase: and (3) adding dopamine monomer (dopamine hydrochloride, 98 percent and 25g, which is purchased from Shanghai Allantin Biotechnology GmbH, the same below) into the phosphate buffer solution containing laccase in the step (3), and controlling the concentration to be 2mg/mL.

(5) Enzymatic preparation of polyurethane materials with polydopamine coating: and (3) adding the polyurethane film obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, repeating for three times, and carrying out vacuum drying to obtain the polyurethane membrane with the enzymatic polydopamine coating, wherein the polyurethane membrane is marked as Lac-PU-PODA.

(6) Preparation of polyurethane material with polydopamine coating by oxygen autopolymerization: placing the polyurethane membrane sheet obtained in the step (1) in a Tris (Tris (hydroxymethyl aminomethane) buffer solution containing 2mg/mL of dopamine monomer and having a pH =8.5 +/-0.5, and carrying out a shaking reaction at 30 ℃ for 24 hours. And then, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, continuing to carry out ultrasonic washing for 15 minutes by using deionized water, repeating for three times, and carrying out vacuum drying to obtain the polyurethane membrane coated with the oxygen self-polymerization polydopamine, wherein the membrane is recorded as Tris-PU-PODA.

(7) And (3) measuring the content of phenolic hydroxyl on the surface of the material:

1. the sizes of the two diaphragms are measured by a vernier caliper, and the surface areas of the two diaphragms are calculated.

2. Since the BCA protein quantification kit can detect the concentration of a phenolic hydroxyl group, a BCA working solution was prepared according to the instructions of the BCA protein quantification kit (purchased from siemer feishel technologies), and a standard curve was drawn based on the protein standard used in the kit.

3. And (3) taking a new 24-pore plate, placing a polyurethane membrane in each pore, adding 1mL of BCA working solution, and placing in a shaking table at 37 ℃ to shake and incubate for 30min.

4. After completion of incubation, 200. Mu.L of BCA working solution per well was added to a 96-well plate, OD was measured at 550nm, and the concentration of phenolic hydroxyl groups on the material surface was calculated from the standard curve.

As can be seen from FIG. 1, the surfaces obtained by the two preparation methods are coated with granular substances, and the experimental results show that the two methods can form a polydopamine coating on the surface of PU.

Example 2

(1) The polyurethane film was prepared according to the procedure for preparing the polyurethane film in example 1, and dried for use.

(2) Preparation of phosphate buffer solution: a 0.2M disodium hydrogenphosphate solution and a 0.2M sodium dihydrogenphosphate solution were prepared in advance, and the above solutions were mixed to prepare a phosphate buffer solution having a pH =5.0.

(3) Preparation of phosphate buffer solution containing laccase: and (3) adding 10mg of laccase into the phosphate buffer solution in the step (2), determining the enzyme activity of the laccase buffer solution by using an ABTS method, and regulating the enzyme activity to 40U/mL by using the phosphate buffer solution in the step (2).

(4) Preparation of a phosphate polyphenol solution containing laccase: epigallocatechin gallate (EGCG, 25g, available from Merrill chemical technology, inc., shanghai, infra) was added to the laccase-containing phosphate buffer solution of step (3) at a concentration of 2mg/mL.

(5) Enzymatic preparation of polyurethane materials with EGCG coating: and (3) adding the polyurethane film obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using deionized water, repeating for three times, and carrying out vacuum drying to obtain the polyurethane film with the enzyme-catalyzed EGCG coating.

(6) Preparation of polyurethane material with EGCG coating by oxygen autopolymerization: placing the polyurethane membrane obtained in the step (1) in a Tris (Tris (hydroxymethyl aminomethane) buffer solution containing 2mg/mL of EGCG and having a pH of =8.5 +/-0.5, and carrying out a shaking reaction at 30 ℃ for 24h. And then, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, continuing to carry out ultrasonic washing for 15 minutes by using deionized water, repeating for three times, and carrying out vacuum drying to obtain the polyurethane film with the oxygen self-polymerization EGCG coating.

(7) The content of phenolic hydroxyl groups on the surface of the material was measured in accordance with the method for measuring phenolic hydroxyl groups in example 1.

Example 3

(4) Preparation of a phosphate polyphenol solution containing laccase: tannic acid (tannic acid, AR,100g, available from rayne reagent, shanghai Yi En chemical technology, ltd., same below) was added to the laccase-containing phosphate buffer solution described in step (3) at a concentration of 2mg/mL.

(5) Enzymatic preparation of polyurethane materials with tannic acid coating: and (3) adding the polyurethane film obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, repeating for three times, and carrying out vacuum drying to obtain the polyurethane film with the enzyme-catalyzed tannin coating.

(6) Oxygen autopolymerization of polyurethane materials with tannic acid coating preparation: placing the polyurethane film piece obtained in the step (1) in a Tris (Tris (hydroxymethyl) aminomethane) buffer solution containing 2mg/mL of tannic acid and having a pH of =8.5 +/-0.5, and carrying out a shaking reaction at 30 ℃ for 24 hours. And then, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, repeating the ultrasonic washing for three times, and carrying out vacuum drying to obtain the polyurethane film with the oxygen self-polymerized tannin coating.

(7) The content of phenolic hydroxyl groups on the surface of the material was measured according to the method for measuring phenolic hydroxyl groups in example 1.

FIG. 2 shows the content of phenolic hydroxyl groups contained in the various coatings obtained in examples 1 to 3, and the higher the content of phenolic hydroxyl groups, the better the coating effect of the coatings, and the more functional groups. As can be seen from FIG. 2, EGCG and TA can be better coated on the PU surface by the enzyme-catalyzed method to form a poly-polyphenol coating.

Example 4

(1) The preparation method of the polyvinyl alcohol (PVA) membrane comprises the following steps: 10g of PVA grains (PVA, molecular weight 1750, available from national pharmaceutical products chemical Co., ltd., the same below) were added to 100mL of deionized water, heated to 95 ℃, stirred and dissolved to obtain a 10wt% PVA solution, and allowed to stand overnight for deaeration. Subsequently, the above solution was poured into a teflon mold, left overnight at-80 ℃, and then immersed in absolute ethanol and left overnight at-20 ℃. After overnight, the mold was removed and repeatedly washed with deionized water at room temperature to obtain a frozen phase separated PVA film.

(4) Preparation of a phosphate polyphenol solution containing laccase: and (4) adding dopamine monomer into the phosphate buffer solution containing laccase in the step (3), and controlling the concentration to be 2mg/mL.

(5) Enzymatic preparation of PVA material with polydopamine coating: and (3) adding the PVA membrane obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, and repeating for three times to obtain the PVA membrane with the enzyme catalysis polydopamine coating, which is marked as Lac-PVA-PODA.

(6) Oxygen autopolymerization preparation of PVA material with polydopamine coating: placing the PVA film obtained in the step (1) in a Tris (Tris (hydroxymethyl) aminomethane) buffer solution containing 2mg/mL of dopamine monomer and having a pH of =8.5 +/-0.5, and carrying out a shaking reaction at 30 ℃ for 24h. And then, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, continuing to carry out ultrasonic washing for 15 minutes by using deionized water, and repeating for three times to obtain the PVA film with the oxygen self-polymerization polydopamine coating, wherein the PVA film is recorded as Tris-PVA-PODA.

Example 5

(1) The PVA film was prepared for use with reference to the step of preparing the PVA film in example 4.

(4) Preparation of a phosphate polyphenol solution containing laccase: EGCG is added into the phosphate buffer solution containing laccase in the step (3), and the concentration is controlled to be 2mg/mL.

(5) Enzymatic preparation of PVA material with EGCG coating: and (3) adding the PVA membrane obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, and repeating for three times to obtain the PVA membrane of the enzyme catalysis EGCG coating, which is marked as Lac-PVA-EGCG.

(6) Oxygen autopolymerization preparation of PVA material with EGCG coating: placing the PVA membrane obtained in the step (1) in a Tris (Tris (hydroxymethyl aminomethane) buffer solution containing 2mg/mL of EGCG and having the pH =8.5 +/-0.5, and carrying out a shaking reaction at the temperature of 30 ℃ for 24 hours. And then, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, and repeating for three times to obtain the PVA membrane with the oxygen self-polymerization EGCG coating, wherein the PVA membrane is marked as Tris-PVA-EGCG.

Example 6

(4) Preparation of a phosphate polyphenol solution containing laccase: and (4) adding tannic acid into the phosphate buffer solution containing laccase in the step (3), and controlling the concentration to be 2mg/mL.

(5) Enzymatic preparation of PVA material with tannic acid coating: and (3) adding the PVA film obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, and repeating for three times to obtain the PVA membrane with the enzyme catalysis tannic acid coating, which is marked as Lac-PVA-TA.

(6) Oxygen autopolymerization of PVA material with tannic acid coating preparation: placing the PVA membrane obtained in the step (1) in a Tris (trihydroxymethyl aminomethane) buffer solution containing 2mg/mL of tannic acid and having the pH =8.5 +/-0.5, and carrying out a shaking reaction at the temperature of 30 ℃ for 24 hours. And then, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, and repeating for three times to obtain the PVA membrane with the oxygen self-polymerized tannic acid coating, wherein the PVA membrane is marked as Tris-PVA-TA.

FIG. 3 is a 10 ten thousand times electron microscope image of examples 4 to 6, it can be seen that the surfaces obtained by the two preparation methods are coated with granular substances, and the experimental result shows that the two methods can form a polydopamine coating, an EGCG coating and a TA coating on the surface of PVA; however, compared with the Tris oxygen self-polymerization method, the enzyme-catalyzed polymerization method can enable EGCG and TA to form more granular coatings on the surface of PVA, and the effect is better.

FIG. 4 shows the content of phenolic hydroxyl groups contained in the various coatings obtained in examples 4 to 6, and the higher the content of phenolic hydroxyl groups, the better the coating effect of the coatings, and the more functional groups. As can be seen from FIG. 4, both the enzyme catalysis method and the Tris-oxygen self-polymerization method can form polydopamine and EGCG coatings on the PVA surface; however, TA can be better coated on the PVA surface by an enzyme-catalyzed method to form a polyphenol coating.

Example 7

(1) Preparation of Bacterial cellulose membrane (BNC): taking gluconacetobacter xylinus as a strain, performing static culture for 7 days at constant temperature by using a liquid culture medium, taking out, placing in a 1wt% sodium hydroxide solution, treating for 4 hours at 80 ℃, taking out, and rinsing with deionized water to be neutral to obtain the bacterial cellulose membrane.

(5) Enzymatic preparation of BNC membranes with polydopamine coating: and (3) adding the BNC membrane obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, and repeating for three times to obtain the BNC film with the enzyme-catalyzed polydopamine coating, which is marked as Lac-BNC-PODA.

(6) Oxygen autopolymerization of BNC films with polydopamine coatings: placing the BNC membrane obtained in the step (1) in a Tris (Tris (hydroxymethyl aminomethane) buffer solution containing 2mg/mL of dopamine monomer and having a pH =8.5 +/-0.5, and carrying out a shaking reaction at 30 ℃ for 24 hours. Subsequently, ultrasonic washing is carried out for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, the ultrasonic washing is carried out for 15 minutes by using the deionized water continuously, and the ultrasonic washing is repeated for three times, so as to obtain the BNC film coated with the oxygen self-polymerization polydopamine, and the film is recorded as Tris-BNC-PODA.

Example 8

(1) BNC films were prepared according to the BNC film preparation procedure in example 7.

(5) Enzymatic preparation of BNC membranes with EGCG coating: and (3) adding the BNC membrane obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, continuing to carry out ultrasonic washing for 15 minutes by using deionized water, repeating for three times, and thus obtaining a BNC film of the enzyme-catalyzed EGCG coating, which is marked as Lac-BNC-EGCG.

(6) Oxygen autopolymerization preparation of BNC films with EGCG coating: placing the BNC membrane obtained in the step (1) in a Tris (Tris (hydroxymethyl) aminomethane) buffer solution containing 2mg/mL of EGCG and having pH =8.5 +/-0.5, and carrying out a shaking reaction at 30 ℃ for 24h. And then, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, continuing to carry out ultrasonic washing for 15 minutes by using deionized water, and repeating for three times to obtain the BNC film coated with the oxygen self-polymerization EGCG, which is recorded as Tris-BNC-EGCG.

FIG. 5 is a 2 ten thousand electron micrograph of examples 7 to 8, from which it can be seen that two preparation methods can form polydopamine coating on the surface of BNC; compared with a Tris-oxygen self-polymerization method, the enzyme catalysis polymerization method can better enable EGCG to form more granular coatings on the surface of BNC, and the effect is better.

Example 9

(1) BNC films were prepared according to the BNC film preparation procedure in example 4.

(5) Enzymatic preparation of BNC membranes with tannic acid coating: and (3) adding the BNC membrane obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, repeating for three times to obtain a BNC film of the enzyme-catalyzed tannin coating, and marking the BNC film as Lac-BNC-TA.

(6) Oxygen autopolymerization of BNC films with tannic acid coating preparation: placing the BNC membrane obtained in the step (1) in a Tris (Tris-hydroxymethyl-aminomethane) buffer solution containing 2mg/mL of tannic acid and having a pH =8.5 +/-0.5, and carrying out a shaking reaction at 30 ℃ for 24h. Subsequently, ultrasonic washing is carried out for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, the ultrasonic washing is carried out for 15 minutes by using the deionized water continuously, and the ultrasonic washing is repeated for three times, so as to obtain the BNC film coated with the oxygen self-polymerized tannic acid, and the film is recorded as Tris-BNC-TA.

FIG. 6 is a 10 ten thousand fold electron micrograph of examples 9 and 6, which shows that more granular objects can be formed on the surface of BNC and PVA by the enzyme-catalyzed polymerization method, and the coating effect is better than that of the Tris-oxygen self-polymerization method.

Fig. 7 shows the content of phenolic hydroxyl groups contained in the different coatings obtained in examples 7 to 9, and the higher the content of phenolic hydroxyl groups, the better the coating effect of the coating, the more functional groups. As can be seen in FIG. 7, both the enzyme-catalyzed method and the Tris-oxygen autopolymerization method can form polydopamine coating on the surface of BNC; however, EGCG and TA can be better coated on the BNC surface using an enzymatic method to form a polyphenol coating.

Example 10

(4) Preparation of a phosphate polyphenol solution containing laccase: tannic acid is added into the phosphate buffer solution containing laccase in the step (3) to control the concentration to be 0.1mg/mL, 0.2mg/mL, 0.5mg/mL, 1mg/mL and 2mg/mL respectively.

(5) Enzymatic preparation of BNC membranes with tannic acid coating: and (3) adding the BNC membrane obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, continuing to carry out ultrasonic washing for 15 minutes by using deionized water, and repeating for three times to obtain the BNC film with the enzyme-catalyzed tannin coating.

(6) The content of phenolic hydroxyl groups on the surface of the material was measured according to the method for measuring phenolic hydroxyl groups in example 1.

FIG. 8 shows the results of phenolic hydroxyl group contents after coating with tannic acid of different concentrations in example 10, from which it can be seen that the phenolic hydroxyl group contents gradually increase with the increase of TA concentration; when the amount of TA is more than 0.5mg/mL, the phenolic hydroxyl group content tends to be flat.

Example 11

(3) Preparation of phosphate buffer solution containing laccase: and (3) adding 10mg of laccase into the phosphate buffer solution in the step (2), determining the enzyme activity of the laccase buffer solution by using an ABTS method, and regulating the enzyme activity to 5U/L, 10U/L, 20U/L, 40U/L and 60U/L by using the phosphate buffer solution in the step (2).

(4) Preparation of a phosphate polyphenol solution containing laccase: and (4) respectively adding tannic acid into the phosphate buffer solution containing laccase in the step (3), and controlling the concentration to be 0.5mg/mL.

(5) Enzymatic preparation of BNC membranes with tannic acid coating: and (3) adding the BNC membrane obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, and repeating for three times to obtain the BNC film with the enzyme-catalyzed tannin coating.

Fig. 9 shows the results of coating the phenolic hydroxyl group content of TA with different enzyme activities in example 11, and it can be seen from the results that the phenolic hydroxyl group content gradually increases with the increase of the enzyme activity.

Example 12

(4) Preparation of a phosphate polyphenol solution containing laccase: and (4) adding tannic acid into the phosphate buffer solution containing laccase in the step (3), and controlling the concentration to be 0.5mg/mL.

(5) Enzymatic preparation of BNC membranes with tannic acid coating: and (3) adding the BNC film obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 6h, 9h, 12h and 24h respectively. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, continuing to carry out ultrasonic washing for 15 minutes by using deionized water, and repeating for three times to obtain the BNC film with the enzyme-catalyzed tannin coating.

FIG. 10 shows the results of the phenolic hydroxyl group content in example 12 with different catalytic times TA, from which it can be seen that the phenolic hydroxyl group content increases slowly with increasing catalytic time; when the catalysis time is 24 hours, the content of phenolic hydroxyl groups is the highest, and the coating effect is better.

Example 13

(1) The polyurethane film (PU) was prepared according to the procedure for preparing the polyurethane film in example 1, and dried for use.

(4) Preparation of a phosphate polyphenol solution containing laccase: adding epigallocatechin gallate (EGCG) into the phosphate buffer solution containing laccase in the step (3), and controlling the concentration to be 0.5mg/mL.

(5) Enzyme-catalyzed preparation of PU membranes with EGCG coating: and (3) adding the PU membrane obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, repeating for three times, and carrying out vacuum drying to obtain the PU membrane of the enzyme-catalyzed EGCG coating, wherein the PU membrane is marked as L-EGCG.

(6) Oxygen autopolymerization preparation of PU membranes with EGCG coating: placing the PU membrane obtained in the step (1) in a Tris (Tris (hydroxymethyl aminomethane) buffer solution containing 0.5mg/mL of EGCG and having a pH =8.5 +/-0.5, and carrying out a shaking reaction at the temperature of 30 ℃ for 24 hours. And then, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, continuing to carry out ultrasonic washing for 15 minutes by using deionized water, repeating for three times, and carrying out vacuum drying to obtain the PU membrane with the oxygen self-polymerization EGCG coating, wherein the PU membrane is marked as Tris-EGCG.

(7) Surface amination of PU films with EGCG coating: preparing 4g/L aqueous solution of polyethyleneimine (M.W.10000, 99 percent and 25g, which is purchased from Shanghai Aladdin Biochemical technology Co., ltd., the same below), placing the PU membrane with the EGCG coating obtained in the step (5) and the step (6) in the 4g/L aqueous solution of polyethyleneimine, and reacting for 5h at the temperature of 60 ℃. After the reaction is finished, cleaning with a large amount of ultrapure water, and drying in vacuum to obtain the PU membrane grafted with the polyethyleneimine.

(8) And (3) measuring the amino content of the surface of the aminated PU membrane: and (4) measuring the amino content by using a golden orange II dyeing method. A0.2 mM golden orange II (golden orange II, 25g, hu test) stock solution was prepared using 0.1M hydrochloric acid pH = 3. The sample was placed in a centrifuge tube, 5mL of acid orange stock solution was added, and 120rpm was shaken at room temperature overnight. After the sample was taken out, it was washed with 0.1M hydrochloric acid having pH =3 for 2 times, and then the sample was transferred to a new centrifuge tube, 5mL of 0.1M NaOH solution was added, and shaken at room temperature for 4 hours, and the adsorbed aurantii ii was sufficiently eluted. And (3) measuring the absorbance at 485nm by using an ultraviolet spectrophotometer, and substituting the absorbance into a golden orange II standard curve to calculate the amino content on the surface of the aminated PU membrane.

Example 14

(4) Preparation of a phosphate polyphenol solution containing laccase: tannin (TA) is added into the phosphate buffer solution containing laccase in the step (3), and the concentration is controlled to be 0.5mg/mL.

(5) Enzyme-catalyzed preparation of PU membranes with TA coating: and (3) adding the PU membrane obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, repeating the ultrasonic washing for three times, and carrying out vacuum drying to obtain the PU membrane of the TA coating catalyzed by the enzyme.

(6) Oxygen autopolymerization preparation of PU films with TA coating: placing the PU membrane obtained in the step (1) in a Tris (Tris (hydroxymethyl aminomethane) buffer solution containing 0.5mg/mL of TA and having a pH of =8.5 +/-0.5, and carrying out a shaking reaction at 30 ℃ for 24h. And then, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, continuing to carry out ultrasonic washing for 15 minutes by using deionized water, repeating the ultrasonic washing for three times, and carrying out vacuum drying to obtain the PU membrane with the oxygen self-polymerization TA coating.

(7) Surface amination of PU films with TA coating: the surface amination of the polyurethane film sheet having a TA coating was completed in the manner of (7) in reference example 13.

(8) And (3) measuring the amino content of the surface of the aminated PU film: the same as in (8) in example 13.

FIG. 11 shows the results of the amount of amino groups grafted after the coating of different polyphenol species in example 13 and example 14. From the results, it can be seen that the enzyme-catalyzed polyphenol coating can graft more polyethyleneimine to increase the amino content on the surface of the polyurethane film compared with the conventional oxygen self-polymerization process.

Example 15

(5) Enzyme-catalyzed preparation of PU membranes with EGCG coating: and (3) adding the PU membrane obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, repeating for three times, and carrying out vacuum drying to obtain the PU membrane of the enzyme-catalyzed EGCG coating.

(6) Oxygen autopolymerization preparation of PU membranes with EGCG coating: placing the PU membrane obtained in the step (1) in a Tris (Tris (hydroxymethyl aminomethane) buffer solution containing 0.5mg/mL of EGCG and having a pH =8.5 +/-0.5, and carrying out a shaking reaction at the temperature of 30 ℃ for 24 hours. And then, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, continuing to carry out ultrasonic washing for 15 minutes by using deionized water, repeating for three times, and carrying out vacuum drying to obtain the PU membrane with the oxygen self-polymerization EGCG coating.

(7) Surface amination of PU films with EGCG coating: the procedure was as in (7) in example 13.

(8) Heparinizing the aminated PU membrane: MES solution with a molar concentration of 0.05M was prepared, pH = 5.5. + -. 0.5, and then heparin (heparin, 1g, hu test, BR grade), EDC and NHS were added to obtain heparin solution. Wherein the concentration of heparin is controlled to be 1g/L, the concentration of NHS is controlled to be 0.03M, and the molar ratio of EDC/NHS is controlled to be 1. And (4) placing the PU membrane grafted with the polyethyleneimine obtained in the step (7) in a heparin solution, and reacting for 24 hours at 37 ℃ in the dark. After the reaction is finished, the PU membrane is cleaned by a large amount of ultrapure water and then dried by a vacuum drying oven to obtain the PU membrane (HEP-PU) with the heparin coating.

(9) And (3) determining the heparin content on the surface of the heparinized PU membrane: and (3) determining the surface heparin grafting amount of the heparinized PU membrane by utilizing a toluidine blue method. And (3) placing the dried sample into a centrifuge tube, respectively adding 5mL of toluidine blue solution, shaking at 37 ℃ and 120rpm for 4h, adding 5mL of n-hexane, extracting for 4h, standing for 15 min, measuring the absorbance of a water phase at 625nm by using an ultraviolet spectrophotometer, and substituting into a standard curve to calculate the surface grafting amount of heparin.

Table 1 shows the heparin content on the surface of the EGCG-coated and polyethyleneimine-grafted PU membrane obtained by the different catalytic processes in example 15, which indicates that the heparin content on the surface of the PU membrane obtained by the enzymatic catalytic process is much higher (by more than 3 times) than that of the oxygen self-polymerization PU membrane, indicating that the enzymatic catalytic process can graft more heparin-reactive groups.

TABLE 1

Sample name	Heparinized EGCG (enzyme catalysis)	Heparinized EGCG (oxygen self-polymerization)
			Heparin content (μ g/cm) ² )	0.058±0.013	0.019±0.022

Example 16

(1) Cutting food-grade aluminum foil with the diameter of 10mm multiplied by 10mm, ultrasonically cleaning the aluminum foil by using 75% ethanol (v/v), and drying the aluminum foil in vacuum for later use.

(4) Preparation of a phosphate polyphenol solution containing laccase: epigallocatechin gallate (EGCG) is added into the phosphate buffer solution containing laccase in the step (3), and the concentration is controlled to be 0.5mg/mL.

(5) Enzyme-catalyzed preparation of aluminum foil with EGCG coating: and (3) adding the aluminum foil obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 6 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, repeating the ultrasonic washing for three times, and carrying out vacuum drying to obtain the aluminum foil with the enzyme-catalyzed EGCG coating.

(6) Oxygen self-polymerization preparation of aluminum foil with EGCG coating: placing the aluminum foil obtained in the step (1) in a Tris (Tris (hydroxymethyl aminomethane) buffer solution containing 0.5mg/mL of EGCG and having a pH =8.5 +/-0.5, and carrying out a shaking reaction at 30 ℃ for 6h. And then, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, continuing to carry out ultrasonic washing for 15 minutes by using deionized water, repeating the steps for three times, and carrying out vacuum drying to obtain the oxygen self-polymerized EGCG coated aluminum foil.

Table 2 shows the surface phenolic hydroxyl content of EGCG catalyzed in example 16 in different catalytic processes for 6 hours.

TABLE 2

Sample name	Enzyme-catalyzed EGCG	Oxygen self-polymerization EGCG
			Phenolic hydroxyl group content (mg/cm) ² )	0.172±0.041	0.109±0.042

From the results, it can be seen that when the polyphenol species is EGCG and the catalysis time is 6h, the enzyme-catalyzed aluminum foil has more phenolic hydroxyl groups than the oxygen self-polymerized aluminum foil.

Example 17

(2) Preparation of phosphate buffer solution: a 0.2M disodium hydrogenphosphate solution and a 0.2M sodium dihydrogenphosphate solution were prepared in advance, and the solutions were mixed at a certain ratio until the pH =5.0.

(5) Enzyme-catalyzed preparation of aluminum foil with TA coating: and (3) adding the aluminum foil obtained in the step (1) into the solution obtained in the step (4) for oscillation reaction at the reaction temperature of 30 ℃ for 3 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, repeating the ultrasonic washing for three times, and carrying out vacuum drying to obtain the aluminum foil with the TA coating catalyzed by the enzyme.

(6) Oxygen autopolymerization preparation of aluminum foil with TA coating: placing the aluminum foil obtained in the step (1) in a Tris (Tris (hydroxymethyl aminomethane) buffer solution containing 0.5mg/mL of TA and having a pH of =8.5 +/-0.5, and reacting at 30 ℃ for 3h with shaking. And then, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, repeating the ultrasonic washing for three times, and carrying out vacuum drying to obtain the aluminum foil with the oxygen self-polymerization TA coating.

(7) The content of phenolic hydroxyl groups on the surface of the material was measured according to the method for measuring phenolic hydroxyl groups in example 1, and the results of the experiment are shown in Table 3.

(8) After the reaction according to steps 7-8 of grafting heparin in example 15, the amount of heparin grafted was measured in step 9, and the results of the experiment are shown in Table 3. Table 3 shows the surface phenolic hydroxyl content of example 17 in which TA was catalyzed in a different catalytic process for a reaction time of 3 hours.

From the results, it can be seen that when the polyphenol species is TA and the catalysis time is 3h, the phenolic hydroxyl group content on the aluminum foil catalyzed by the enzyme is more than that of the aluminum foil polymerized by oxygen. The content of grafted heparin after enzyme catalysis is more than 3 times of that of the grafted heparin by an oxygen self-polymerization method.

TABLE 3

Sample name	Enzyme catalyzed TA	Oxygen self-polymerizing TA
			Phenolic hydroxyl group content (mg/cm) ² )	0.040±0.017	0.037±0.008
Heparin content (μ g/cm) ² )	0.029±0.011	0.0090±0.0024

Example 18

(4) The enzyme catalysis one-step method for preparing the aminated PU membrane comprises the following steps: and (4) adding gallic acid into the phosphate buffer solution containing laccase in the step (3), controlling the concentration to be 2mg/mL, then adding polyethyleneimine, controlling the concentration to be 1mg/mL, and controlling the pH of the reaction system to be 5.0. Subsequently, the PU membrane is added into the solution for oscillation reaction, the reaction temperature is 30 ℃, and the reaction time is 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, repeating for three times, and carrying out vacuum drying to obtain the PU membrane grafted with the polyethyleneimine by enzyme catalysis, wherein the PU membrane is marked as Lac-PEI-PU.

(5) The oxygen self-polymerization catalysis preparation of the aminated PU membrane comprises the following steps: and (2) placing the PU membrane obtained in the step (1) in a Tris (Tris-hydroxymethyl-aminomethane) buffer solution containing gallic acid and PEI (polyetherimide) and having the pH =8.5 +/-0.5, and carrying out oscillation reaction at the temperature of 30 ℃ for 24h, wherein the concentration of the gallic acid is controlled to be 2mg/mL, and the concentration of the PEI is controlled to be 1mg/mL. And then, ultrasonically washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuously ultrasonically washing for 15 minutes by using the deionized water, repeating for three times, and carrying out vacuum drying to obtain the PU membrane grafted with polyethyleneimine by oxygen self-polymerization, wherein the PU membrane is recorded as Tris-PEI-PU.

(6) And (3) measuring the amino content of the surface of the aminated PU membrane: the same as in (8) in example 13.

(7) Heparinizing an aminated PU membrane: MES buffer solution with a molar concentration of 0.05M was prepared, pH =5.5 ± 0.5 was controlled, and then heparin, EDC and NHS were added to give heparin solution. Wherein the concentration of heparin is controlled to be 1g/L, the concentration of NHS is controlled to be 0.03M, and the molar ratio of EDC/NHS is controlled to be 1. And (3) placing the PU membrane grafted with the polyethyleneimine obtained in the step (4) and the step (5) in a heparin solution, and reacting for 24 hours at 37 ℃ in the dark. After the reaction is finished, a large amount of ultrapure water is used for cleaning, and then a vacuum drying oven is used for drying to obtain the PU membrane with the heparin coating, which is respectively marked as Lac-HEP-PU and Tris-HEP-PU according to the process.

(8) And (3) measuring the heparin content on the surface of the heparinized PU membrane: the surface heparin contents of Lac-HEP-PU and Tris-HEP-PU were determined in the same manner as in (9) of example 15.

FIG. 12 is a flow chart of the preparation of grafted heparin after laccase catalyzed one-step amination in example 18, and Table 4 shows the results of the amount of amino grafting of PEI grafted by one-step method using different processes in example 18. From the results, it can be seen that compared to the conventional oxygen self-polymerization process, the enzyme-catalyzed one-step coating can graft more polyethyleneimine to increase the amino content on the surface of the polyurethane film, which is about 1.5 times higher than that of the conventional process.

TABLE 4

Sample name	Lac-PEI-PU	Tris-PEI-PU
			Amino group content (. Mu. Mol/cm) ² )	0.146±0.003	0.097±0.008

Table 5 shows the results of the heparin content of example 18 after grafting PEI and heparin by one-step method using different processes. The results show that compared with the conventional oxygen self-polymerization process, the enzyme catalysis one-step coating can further improve the grafting amount of subsequent heparin after grafting more polyethyleneimine, and the content of the heparin on the PU surface is about 1.3 times higher than that of the conventional process.

TABLE 5

Sample name	Lac-HEP-PU	Tris-HEP-PU
			Heparin contentAmount (. Mu.g/cm) ² )	1.045±0.017	0.821±0.013

Example 19

(2) Preparation of phosphate buffer solution: a 0.2M disodium hydrogen phosphate solution and a 0.2M sodium dihydrogen phosphate solution were prepared in advance, and the solutions were mixed at a certain ratio until pH =5.0.

(4) Enzyme catalysis preparation of the argininated PU membrane: EGCG was added to the phosphate buffer solution containing laccase in step (3) at a concentration of 2mg/mL, followed by L-arginine (Arg, 100g,98%, available from Shanghai Allantin Biotechnology Co., ltd., the same below) at a concentration of 1mg/mL, and the pH of the reaction system was controlled to 5.0. Subsequently, the PU membrane is added into the solution for oscillation reaction, the reaction temperature is 30 ℃, and the reaction time is 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, repeating for three times, and carrying out vacuum drying to obtain the PU membrane grafted with the L-arginine through enzyme catalysis, wherein the PU membrane is marked as Lac-Arg-PU.

(5) The preparation of the arginine PU membrane by oxygen self-polymerization catalysis comprises the following steps: and (2) putting the PU membrane obtained in the step (1) into a Tris (Tris (hydroxymethyl aminomethane) buffer solution containing EGCG and L-arginine and having the pH of =8.5 +/-0.5, and oscillating and reacting for 24h at the temperature of 30 ℃, wherein the concentration of the EGCG is controlled to be 2mg/mL, and the concentration of the L-arginine is controlled to be 1mg/mL. And then, ultrasonically washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuously ultrasonically washing for 15 minutes by using the deionized water, repeating for three times, and carrying out vacuum drying to obtain the PU membrane grafted with the L-arginine by oxygen self-polymerization, wherein the PU membrane is recorded as Tris-Arg-PU.

(6) XPS was used to measure the elemental content of the three elements C, N, O on the two film surfaces.

(7) Evaluation of cell compatibility of PU membrane: cleaning the membrane obtained in the steps (1), (4) and (5) by 75% (w/v) ethanol, placing the membrane in a 24-well plate, planting Human Umbilical Vein Endothelial Cells (HUVEC) on the surface of each material (10000 HUVEC per well), adding a proper amount of cell culture medium (containing penicillin-streptomycin mixed solution 1%, fetal bovine serum FBS 10% and high-sugar DMEM medium 89%), and testing the cell viability by using a cck-8 kit (Shanghai Biyunnan biotechnology Co., ltd., the same below) when culturing for 1, 3 and 5 days.

(8) The cck-8 cell viability experiment procedure at day 1 in step (7) was: the medium of each well was discarded, washed 3 times with PBS buffer, and DMEM (cck-8.

(9) The cck-8 cell viability experiment procedure on day 3 and day 5 is the same as that in step (8).

Table 6 shows the elemental analysis of the one-step grafting of L-arginine by the different procedures in example 19. From the results, the N element content of the coating of the enzyme catalysis one-step method is higher compared with the traditional oxygen self-polymerization process, and the process can graft more L-arginine.

TABLE 6

Sample name	C(％)	N(％)	O(％)
				Lac-Arg-PU	69.3	7.5	23.2
Tris-Arg-PU	70.1	5.1	24.8

FIG. 13 shows that: the higher the absorbance at 450nm, the better the cell proliferation, and as can be seen from the figure, after 5 days of culture, all the HUVECs on the PU surface are proliferated, and the activity of the HUVECs on Lac-Arg-PU is higher than that of Tris-Arg-PU due to PU contrast in the proliferation of argininated PU membrane cells, which indicates that more arginine is grafted by the enzyme catalysis one-step process, thereby promoting the proliferation of endothelial cells and having better biological activity.

Example 20

(4) Enzyme catalysis preparation of lysine acidification PU membrane: in step (3), dopamine was added to the laccase-containing phosphate buffer solution at a concentration of 2mg/mL, followed by lysine (Lys) (97%, 25g, available from Shanghai Aladdin Biochemical technology Co., ltd., the same applies hereinafter) at a concentration of 1mg/mL, and the pH of the reaction system was controlled to 5.0. Subsequently, the PU membrane is added into the solution for oscillation reaction, the reaction temperature is 30 ℃, and the reaction time is 24 hours. And after the reaction is finished, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using the deionized water, repeating for three times, and carrying out vacuum drying to obtain the PU membrane grafted with Lys through enzyme catalysis, wherein the PU membrane is marked as Lac-Lys-PU.

(5) Preparing the lysine-acidified PU membrane by oxygen self-polymerization catalysis: placing the PU membrane obtained in the step (1) in a Tris (Tris-hydroxymethyl-aminomethane) buffer solution containing dopamine and lysine with the pH =8.5 +/-0.5, and carrying out shaking reaction at the temperature of 30 ℃ for 24 hours, wherein the concentration of the dopamine is controlled to be 2mg/mL, and the concentration of the lysine is controlled to be 1mg/mL. And then, carrying out ultrasonic washing for 15 minutes by using 75% ethanol and deionized water, after 3 times of washing, continuing to carry out ultrasonic washing for 15 minutes by using deionized water, repeating for three times, and carrying out vacuum drying to obtain the PU membrane grafted with Lys by oxygen self-polymerization, wherein the PU membrane is marked as Tris-Lys-PU.

(6) The elemental contents of the three elements C, N, O were determined using XPS for both membrane surfaces.

(7) Evaluation of cell compatibility of PU membrane: after the membrane obtained in the steps (1), (4) and (5) is cleaned by 75% (w/v) ethanol, the membrane is placed in a 24-well plate, human Umbilical Vein Endothelial Cells (HUVEC) are planted on the surface of each material (10000 HUVEC per well), a proper amount of cell culture medium (containing 1% penicillin-streptomycin mixed solution, 10% fetal bovine serum FBS and 89% high-sugar DMEM medium) is added, and the cell viability is tested by using a cck-8 kit (Shanghai Biyunnan biotechnology Co., ltd.) when the membrane is cultured for 1, 3 and 5 days, wherein the testing steps are the same as the steps (8) and (9) in the example (19).

Table 7 shows the elemental analysis of the one-step lysine grafting carried out by the different procedures in example 20. From the results, the N element content of the coating of the enzyme catalysis one-step method is higher compared with the traditional oxygen self-polymerization process, and the process can graft more L-arginine.

TABLE 7

Sample name	C(％)	N(％)	O(％)
				Lac-Lys-PU	66.3	9.8	23.9
Tris-Lys-PU	70.2	6.4	23.4

FIG. 14 shows that: the higher the absorbance at 450nm, the better the cell proliferation, and as can be seen from the figure, after 5 days of culture, all HUVEC on the PU surface are proliferated, and the activity of HUVEC on Lac-Lys-PU is higher than that of Tris-Lys-PU due to PU contrast, which indicates that more lysine is grafted by the enzyme catalysis one-step process, the proliferation of endothelial cells is promoted, and the better biological activity is achieved.

In summary, the technical problem to be solved by the present invention is to provide a method for preparing and aminating a polyphenol coating material catalyzed by polyphenol oxidase. The method overcomes the defects of poor adhesion effect and uneven coating after the self-polymerization of polyphenol oxygen, avoids the toxicity caused by the excessive use and burst release of metal ions, and avoids potential safety risk. In addition, the technology has the advantages of mild reaction conditions, adjustable and controllable parameters and small pollution to the environment, and has universal applicability to various material substrates.

Claims

1. A preparation method and an amination method of polyphenol oxidase catalyzed polyphenol coating material comprise the following steps:

(2) Putting a substrate material to be subjected to surface modification into the solution obtained in the step (1), adding polyphenol, fully and uniformly stirring, carrying out oscillation reaction, and carrying out ultrasonic washing to obtain a polyphenol oxidase catalyzed polyphenol coating material;

2. The method of claim 1, wherein: the polyphenol oxidase in the step (1) is one or more of laccase, catechol oxidase, tyrosinase, bisphenol oxidase, phenol enzyme, cresol enzyme and catechol oxidoreductase, and the enzyme activity is 5U/mL-100U/mL; the mass-to-volume ratio of the polyphenol oxidase to the buffer solution is 1-5mg.

3. The method of claim 1, wherein: the buffer solution in the step (1) is phosphate buffer solution or acetate buffer solution, the ionic strength is 20-100mM, and the pH value is 2.2-7.0.

4. The method of claim 1, wherein: the substrate material in the step (2) is one or more of cellulose, polyvinyl alcohol, polyurethane, polyester, polytetrafluoroethylene, polymethyl methacrylate, medical polysiloxane, stainless steel, glass, titanium and alloy thereof, magnesium and alloy thereof.

5. The method of claim 1, wherein: the polyphenol in the step (2) is one or more of dopamine, norepinephrine, epigallocatechin gallate, epigallocatechin, tannic acid, gallic acid and curcumin.

6. The method of claim 1, wherein: the addition amount of the polyphenol in the step (2) is 0.1mg/mL-4mg/mL.

7. The method of claim 1, wherein: the oscillation reaction in the step (2) is oscillation reaction for 3-24h at 25-35 ℃, and the oscillation reaction frequency is 1-5.

8. The method of claim 1, wherein: and (3) carrying out ultrasonic washing in the step (2) by using 75% of ethanol and deionized water for 15 minutes, after washing for 3 times, continuing to carry out ultrasonic washing for 15 minutes by using deionized water, and repeating for three times.

9. The method according to claim 1, wherein the polyphenol species, polyphenol addition amount, shaking reaction temperature and shaking reaction times in step (3) are the same as those in step (2); the concentration of the amino compound or imino compound aqueous solution in the step (3) is 3-5g/L; after the amino compound or the imino compound is added, the concentration of the amino compound or the imino compound in the solution is 0.6-1.5mg/mL; the amino compound or imino compound includes polyethyleneimine, arginine or lysine.

10. The method according to claim 1, wherein the reaction in the aqueous solution of the amino compound or imino compound in the step (3) is carried out at a temperature of 50 to 70 ℃ for 4 to 6 hours.

11. A method of preparing a material having a heparin coating, comprising:

placing the polyphenol oxidase catalyzed polyphenol coating material or polyphenol-amino/imino compound coating material of claim 1 in heparin aqueous solution containing EDC and NHS, reacting away from light, cleaning, and drying to obtain material with heparin coating.

12. The method for producing according to claim 11, characterized in that: the obtained material with the heparin coating is used for the activation modification of the surface of the cardiovascular and cerebrovascular products.

13. The method of claim 11, wherein: the obtained material with the heparin coating is applied to the preparation of the anti-thrombosis forming material.