CN112972769A - Method for preparing nano-silver-containing antibacterial peptide functional coating on metal surface - Google Patents
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Abstract
本发明公开了一种金属表面制备含纳米银抗菌肽功能涂层的方法，该方法包括：一、将硝酸银粉末和抗菌肽粉末加入到丝素蛋白溶液中混合得到混合溶液A；二、将混合溶液A进行紫外照射还原得到混合溶液B；三、将Tris溶液标定后加入盐酸多巴胺并浸泡金属，得到表面具有聚多巴胺辅助层的金属；四、将表面具有聚多巴胺辅助层的金属浸入混合溶液B中浸泡，在金属表面形成含纳米银抗菌肽功能涂层。本发明采用紫外照射法利用丝素蛋白将银离子还原成纳米银，有效控制了纳米银粒子的粒径大小和形貌，且抗菌肽与纳米银之间形成配位键，促进抗菌肽在纳米银颗粒表面富集，并在含纳米银抗菌肽功能涂层内呈现纳米级分散态，实现低抗菌剂用量下长期高效杀菌。
The invention discloses a method for preparing a functional coating containing nano-silver antibacterial peptides on a metal surface. The method includes: first, adding silver nitrate powder and antibacterial peptide powder into a silk fibroin solution and mixing to obtain a mixed solution A; second, mixing The mixed solution A is reduced by ultraviolet irradiation to obtain the mixed solution B; 3. After the Tris solution is calibrated, dopamine hydrochloride is added and the metal is soaked to obtain a metal with a polydopamine auxiliary layer on the surface; 4. The metal with a polydopamine auxiliary layer on the surface is immersed in the mixed solution Soak in B to form a functional coating containing nano-silver antimicrobial peptides on the metal surface. The invention adopts the ultraviolet irradiation method to reduce silver ions into nanometer silver by using silk fibroin, which effectively controls the particle size and shape of nanometer silver particles, and forms coordination bonds between antibacterial peptides and nanometer silver, so as to promote the effect of antibacterial peptides on nanometer silver particles. The silver particles are enriched on the surface and present in a nano-scale dispersion state in the functional coating containing nano-silver antimicrobial peptides, so as to achieve long-term and efficient sterilization at a low dosage of antimicrobial agents.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a method for preparing a nano-silver-containing antibacterial peptide functional coating on a metal surface.
Background
Pure titanium for medical use has been widely used in the field of clinical medicine due to its excellent biocompatibility. As early as 50 years in the 20 th century, pure titanium was processed into bone plates, bone nails, femoral heads and other implanted medical devices for orthopedic repair operations. However, as a common postoperative complication, bacterial infection caused by medical instruments has become one of the important problems to be solved urgently in the medical field of the 21 st century. Annual incidence of orthopedic implant-related infections in the united states is reported to reach around 4.3%. According to the data in the handbook of infection control and prevention in hospitals issued by the World Health Organization (WHO), more than 1400 million people are suffering from nosocomial infections every day around the world, of which 60% of bacterial infections are related to the medical devices used. Postoperative infection of orthopedics and the like can directly cause the wound of a patient to be not healed for a long time, often can cause operation failure, even can cause complications such as chronic osteomyelitis and the like, not only brings great physical and mental pain and heavy economic burden to the patient, but also can cause negative effects of different degrees to hospitals, society and the like. Therefore, the construction of the functional coating carrying the antibacterial agent (antibiotics, antibacterial peptide, nano silver and the like) on the surface of the medical titanium alloy has great social and economic significance for eliminating or reducing bacterial infectious diseases caused by related medical instruments.
At present, antibiotics are still the most widely used traditional antibacterial agents thanks to efficient and reliable sterilization capability, but the antibiotics easily cause bacterial drug resistance, and cause more serious drug-resistant bacterial infection problems. In contrast, nano-silver and antibacterial peptide are not easy to cause drug resistance of bacteria, and can be used for broad-spectrum antibiosis and effectively inhibiting a biological membrane for a long time, so that the nano-silver has great potential to replace antibiotics. Compared with other existing forms, the nano silver has higher antibacterial activity due to the combined action of the elution effect of metal ions and the bactericidal property of nano particles. At present, nano silver is generally considered to have a complex antibacterial mechanism: it can adhere to the surface of bacteria, thereby changing the permeability of bacterial cell membranes; it can be sterilized by interaction with thiol-containing proteins; it can bind to bacterial DNA and disrupt its function; in particular, the nano silver can release silver ions with bactericidal ability. Due to various biological activities, the antibacterial peptide becomes a research hotspot of biological molecules and biological materials. When the antibacterial peptide is directly used for treating infection caused by bacteria and fungi, the antibacterial efficiency of the antibacterial peptide is higher. Currently, antimicrobial peptides are receiving increasing attention in biomedical materials, mainly as surface-modified antimicrobial components or as delivered antimicrobial drugs for local delivery.
Silk fibroin is the main body of silk and is a natural biological macromolecule without physiological activity. Compared with other natural polymers, the fibroin protein has obvious superiority, and researches show that the fibroin protein has good biocompatibility, no toxicity, no pollution, no irritation and biodegradability. In recent years, silk fibroin is gradually applied to the fields of biosensing, biomedical materials, soft tissue compatible materials, tissue engineering and the like due to its good biocompatibility. In particular, the silk fibroin has reducibility, can be reduced in situ to obtain nano silver and antibacterial peptide particles, and avoids the use of toxic reducing agents.
At present, common antibiotic-carrying coatings easily cause bacterial drug resistance and cannot cope with bacterial infection environments with higher bacterial concentrations or stronger bacterial activities, and high-concentration antibacterial agents generate potential biotoxicity, so that the challenge is how to achieve long-term and high-efficiency antibacterial effects at low-concentration dosage.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for preparing a nano-silver-containing antibacterial peptide functional coating on a metal surface, aiming at the defects of the prior art. The method adopts an ultraviolet irradiation method to reduce silver ions into nano silver by utilizing silk fibroin, effectively controls the particle size and the morphology of nano silver particles, forms coordinate bonds between the antibacterial peptide and the nano silver, promotes the antibacterial peptide to be enriched on the surface of the nano silver particles, enables the nano silver with the antibacterial peptide enriched on the surface to present a nano-scale dispersion state in the nano-silver-containing antibacterial peptide functional coating, and is beneficial to realizing long-term and efficient sterilization under the low dosage of the antibacterial agent.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a method for preparing a nano-silver-containing antibacterial peptide functional coating on a metal surface is characterized by comprising the following steps:
adding silver nitrate powder and antibacterial peptide powder into a silk fibroin solution, and mixing to obtain a mixed solution A containing silk fibroin, silver ions and antibacterial peptide; the mass concentration of the silk fibroin in the mixed solution A is 2-5%, the concentration of silver ions is 1-20 mg/mL, and the concentration of the antibacterial peptide is 0.4-1 mg/mL;
step two, placing the mixed solution A obtained in the step one under an ultraviolet lamp for irradiation reduction, so that the color of the mixed solution A is gradually changed from milky white to dark brown, and obtaining a mixed solution B containing silk fibroin, nano silver and antibacterial peptide; the time for irradiation reduction is 0.5 h-2 h;
step three, calibrating a 10mM Tris solution to the pH value of 8.5 by adopting a standard NaOH solution, then adding dopamine hydrochloride to obtain a working solution, and then soaking the metal in the working solution for 12-24 hours in a dark condition to obtain the metal with the polydopamine auxiliary layer on the surface;
and step four, soaking the metal with the polydopamine auxiliary layer on the surface obtained in the step three into the mixed solution B obtained in the step two, and forming the nano-silver-containing antibacterial peptide functional coating on the surface of the metal.
According to the invention, firstly, a reduction method of ultraviolet irradiation is adopted, so that silver ions are reduced by silk fibroin, key process parameters including silk fibroin concentration, silver ion concentration and irradiation reduction time are controlled, the particle size and the morphology of nano silver particles are effectively controlled, nano silver particles with well dispersed and uniform particle size are obtained, a coordination bond is formed between antibacterial peptide and nano silver, and the enrichment of antibacterial peptide on the surface of the nano silver particles is promoted, so that the nano silver with antibacterial peptide enriched on the surface is in a nano-scale dispersion state in the silk fibroin-based nano silver antibacterial peptide-containing functional coating, the sterilization efficiency is improved, the preparation time is greatly shortened, and long-term and efficient sterilization with low antibacterial peptide dosage and low nano silver dosage is realized; then introducing a polydopamine auxiliary layer on the surface of the metal substrate, and combining the nano-silver-containing antibacterial peptide functional coating on the fibroin protein base carrying nano silver (with the antibacterial peptide enriched on the surface) on the surface of the metal substrate by adopting a soaking method, so as to form the nano-silver-containing antibacterial peptide functional coating on the metal surface, solve the problem of poor binding property between the nano-silver-containing antibacterial peptide functional coating and the metal substrate, and improve the binding effect between the nano-silver-containing antibacterial peptide functional coating and the metal surface. Meanwhile, as the nano silver and the antibacterial peptide in the nano silver-containing antibacterial peptide functional coating both have high-efficiency sterilization capability, especially the antibacterial peptide has a unique sterilization mechanism, the integrity of microbial cell membranes can be usually destroyed, invading microbes can be rapidly killed under the condition of not activating adaptive immunity, and the genetic adaptation mechanism of bacteria is effectively avoided, so that the drug resistance is not caused, the nano silver and the antibacterial peptide have a synergistic effect and can be controllably released for a long time at an implantation position, the long-term high-efficiency sterilization capability of the nano silver-containing peptide functional coating is endowed, the problem of bacterial infection related to an implant is effectively solved, the drug resistance of bacteria is not caused, and the concentration of the antibacterial peptide is reduced; in addition, since the silk fibroin has good human body affinity, biocompatibility, no toxicity and is beneficial to cell adhesion, and the osteoinduction of the low-concentration nano silver particles provides excellent osteogenesis induction capability, the nano silver-containing antibacterial peptide functional coating prepared on the surface of metal (particularly titanium metal) has good biocompatibility and osteoinduction, good applicability and high application value.
The method for preparing the nano-silver-containing antibacterial peptide functional coating on the metal surface is characterized in that the particle size of nano-silver in the mixed solution B in the step two is 20-500 nm, and the mass content of the silk fibroin with a beta-folded structure in the mixed solution B is 15-30%. The optimal antibacterial effect of the nano-silver is ensured by the optimal particle size; the optimized mass content of the silk fibroin with the beta-folded structure improves the stability of the mixed solution B, thereby ensuring the stability of the antibacterial peptide functional coating containing nano-silver formed on the metal surface.
The method for preparing the antibacterial peptide functional coating containing nano-silver on the metal surface is characterized in that the amino acid sequence of the antibacterial peptide in the mixed solution A in the step two is (NH)2)-NGIVKAGPAIAVLGEAAL-CONH2The quality purity of the antibacterial peptide is more than 99%. The preferred antibacterial peptide has specific and efficient bactericidal performance, and is not easy to cause drug resistance of bacteria.
The method for preparing the nano-silver-containing antibacterial peptide functional coating on the metal surface is characterized in that the metal in the third step is titanium, titanium alloy or stainless steel. The preparation method has wide application range and is suitable for various common medical metals.
The method for preparing the nano-silver-containing antibacterial peptide functional coating on the metal surface is characterized in that the concentration of dopamine hydrochloride in the working solution in the third step is 1-4 mg/mL.
The method for preparing the nano-silver-containing antibacterial peptide functional coating on the metal surface is characterized in that in the third step, the metal is cleaned and pretreated before being immersed into the working solution: and sequentially immersing the metal into acetone, ethanol and deionized water for ultrasonic cleaning for 15 min. The optimized cleaning process removes impurities and pollutants on the metal surface, and is beneficial to polymerization of dopamine on the metal surface to form a polydopamine auxiliary layer.
The method for preparing the nano-silver-containing antibacterial peptide functional coating on the metal surface is characterized in that the thickness of the polydopamine auxiliary layer in the third step is 200-500 nm. The polydopamine auxiliary layer with the optimal thickness ensures the combination effect between the metal and the nano-silver-containing antibacterial peptide functional coating.
The method for preparing the nano-silver-containing antibacterial peptide functional coating on the metal surface is characterized in that the metal with the surface provided with the polydopamine auxiliary layer in the fourth step is soaked in the mixed solution B obtained in the second step, then the metal is taken out and placed in a constant-temperature drying oven, and the metal is dried for 10-30 min at the temperature of 60 ℃. The drying process promotes the silk fibroin to form a stable structure, and is beneficial to improving the binding performance of the metal and the functional coating containing the nano-silver antibacterial peptide.
The method for preparing the nano-silver-containing antibacterial peptide functional coating on the metal surface is characterized in that the thickness of the nano-silver-containing antibacterial peptide functional coating in the step four is 1-20 microns.
Compared with the prior art, the invention has the following advantages:
1. the invention adopts an ultraviolet irradiation method to reduce silver ions into nano-silver by utilizing silk fibroin, effectively controls the particle size and the morphology of nano-silver particles, and forms coordinate bonds between the antibacterial peptide and the nano-silver to promote the enrichment of the antibacterial peptide on the surface of the nano-silver particles, so that the nano-silver with the antibacterial peptide enriched on the surface presents a nano-scale dispersion state in the nano-silver-containing antibacterial peptide functional coating, and the long-term efficient sterilization under the low antibacterial agent consumption is favorably realized.
2. According to the invention, the polydopamine auxiliary layer is introduced on the surface of the metal substrate, so that the combination effect of the nano-silver antibacterial peptide functional coating and the metal surface is improved, the problem of poor combination between the nano-silver antibacterial peptide functional coating and the metal substrate is solved, and the long-term and efficient synergistic sterilization effect of the nano-silver antibacterial peptide functional coating as a metal implant is favorably exerted.
3. The nano silver and the antibacterial peptide in the nano silver-containing antibacterial peptide functional coating formed on the metal surface have high-efficiency bactericidal capability, have synergistic effect and are controllably released at an implantation position for a long time, so that the aims of reducing the dosage of the antibacterial peptide (far lower than the biotoxicity critical concentration) and sterilizing efficiently for a long time are fulfilled, and the problem of bacterial drug resistance is solved.
4. The functional coating containing the nano-silver antibacterial peptide prepared on the metal surface has good biocompatibility and osteoinductivity, inhibits the absorption process of the original bone and promotes the generation of new bone after being implanted into organisms, realizes the purpose of fast and good integration of an orthopedic implant device (prosthesis) and surrounding bone tissues, effectively prevents implantation infection caused by bacteria, prolongs the service period of the orthopedic implant device, is particularly suitable for treating patients with pathologically induced bone absorption and fracture, and has good applicability and high application value.
The technical solution of the present invention is further described in detail by the accompanying drawings and examples.
Drawings
FIG. 1 is a transmission electron microscope image of nano-silver in a mixed solution B of example 1 of the present invention.
FIG. 2 is a graph showing the comparison of the antibacterial effects of the coating layers and the titanium sheet formed in example 1 of the present invention and comparative examples 1 to 2.
Detailed Description
Example 1
The embodiment comprises the following steps:
step one, silk is placed in 0.02M Na2CO3Boiling the solution for 40min, taking out, washing with deionized water, and adding into 9.3M LiBr solution at 60 deg.C for dissolving for 4h to obtain fibroin protein stock solution;
diluting the silk fibroin stock solution into a silk fibroin solution with the mass concentration of 2% by using deionized water, then adding 17mg of silver nitrate powder and 4mg of antibacterial peptide powder into 10mL of silk fibroin solution with the mass concentration of 2% and mixing to obtain a mixed solution A which contains silk fibroin, silver ions and antibacterial peptide and has the pH value of 8.5;
step two, placing the mixed solution A obtained in the step one under a 40W ultraviolet lamp for irradiation reduction for 0.5h, so that the color of the mixed solution A is gradually changed from milky white to dark brown, and obtaining a mixed solution B containing silk fibroin, nano silver and antibacterial peptide; the particle size of the nano silver in the mixed solution B is 20nm, and the mass content of the silk fibroin with a beta-folded structure in the mixed solution B is 15%; the amino acid sequence of the antibacterial peptide in the mixed solution A is (NH)2)-NGIVKAGPAIAVLGEAAL-CONH2The mass purity of the antibacterial peptide is 99.99 percent;
step three, calibrating a 10mM Tris (Tris (hydroxymethyl aminomethane) solution by using a standard NaOH solution until the pH value is 8.5, then adding dopamine hydrochloride to obtain a working solution, sequentially immersing the titanium sheet into acetone, ethanol and deionized water for ultrasonic cleaning for 15min, and immersing the titanium sheet into the working solution to soak for 12h under a dark condition to obtain the titanium sheet with the polydopamine auxiliary layer on the surface; the concentration of the dopamine hydrochloride in the working solution is 2 mg/mL; the thickness of the polydopamine auxiliary layer is 200 nm;
soaking the titanium sheet with the polydopamine auxiliary layer on the surface obtained in the third step into the mixed solution B obtained in the second step, taking out the titanium sheet, placing the titanium sheet in a constant-temperature drying box, drying the titanium sheet for 10min at the temperature of 60 ℃, and forming a nano-silver-containing antibacterial peptide functional coating on the surface of the titanium sheet; the thickness of the nano-silver-containing antibacterial peptide functional coating is 1 mu m.
Fig. 1 is a transmission electron microscope image of the nano silver in the mixed solution B of this embodiment, in which the black circular particles are nano silver particles, the diameter is about 20nm, the shaded portion around the black circular particles is the antimicrobial peptide, and it can be seen from fig. 1 that the antimicrobial peptide is enriched on the surface of the nano silver particles.
Comparative example 1
This comparative example comprises the following steps:
step one, silk is placed in 0.02M Na2CO3Boiling the solution for 40min, taking out, washing with deionized water, and adding into 9.3M LiBr solution at 60 deg.C for dissolving for 4h to obtain fibroin protein stock solution;
diluting the silk fibroin stock solution into a silk fibroin solution with the mass concentration of 2% by using deionized water, and then adding 17mg of silver nitrate powder into 10mL of the silk fibroin solution with the mass concentration of 2% for mixing to obtain a mixed solution A which contains silk fibroin and silver ions and has the pH value of 8.5;
step two, placing the mixed solution A obtained in the step one under a 40W ultraviolet lamp for irradiation reduction for 0.5h, so that the color of the mixed solution A is gradually changed from milky white to dark brown, and a mixed solution B containing silk fibroin and nano silver is obtained; the particle size of the nano silver in the mixed solution B is 20nm, and the mass content of the silk fibroin with a beta-folded structure in the mixed solution B is 15%; the amino acid sequence of the antibacterial peptide in the mixed solution A is (NH)2)-NGIVKAGPAIAVLGEAAL-CONH2；
Step three, calibrating a 10mM Tris (Tris (hydroxymethyl aminomethane) solution by using a standard NaOH solution until the pH value is 8.5, then adding dopamine hydrochloride to obtain a working solution, sequentially immersing the titanium sheet into acetone, ethanol and deionized water for ultrasonic cleaning for 15min, and immersing the titanium sheet into the working solution to soak for 12h under a dark condition to obtain the titanium sheet with the polydopamine auxiliary layer on the surface; the concentration of the dopamine hydrochloride in the working solution is 2 mg/mL; the thickness of the polydopamine auxiliary layer is 200 nm;
soaking the titanium sheet with the polydopamine auxiliary layer on the surface obtained in the third step into the mixed solution B obtained in the second step, taking out the titanium sheet, placing the titanium sheet in a constant-temperature drying box, drying the titanium sheet for 30min at 60 ℃, and forming a nano-silver-containing functional coating on the surface of the titanium sheet; the thickness of the functional coating containing the nano-silver is 1 mu m.
Comparative example 2
This comparative example comprises the following steps:
step one, silk is placed in 0.02M Na2CO3Boiling the solution for 40min, taking out, washing with deionized water, and adding into 9.3M LiBr solution at 60 deg.C for dissolving for 4h to obtain fibroin protein stock solution;
diluting the silk fibroin stock solution into a silk fibroin solution with the mass concentration of 2% by using deionized water, and then adding 4mg of antibacterial peptide powder into 10mL of silk fibroin solution with the mass concentration of 2% for mixing to obtain a mixed solution A which contains silk fibroin and antibacterial peptide and has the pH value of 8.5;
step two, placing the mixed solution A obtained in the step one under a 40W ultraviolet lamp for irradiation for 0.5h to obtain a mixed solution B containing silk fibroin and antibacterial peptide; the mass content of the silk fibroin with a beta-sheet structure in the mixed solution B is 15%, and the amino acid sequence of the antibacterial peptide in the mixed solution A is (NH)2)-NGIVKAGPAIAVLGEAAL-CONH2The mass purity of the antibacterial peptide is 99.99 percent;
step three, calibrating a 10mM Tris (Tris (hydroxymethyl aminomethane) solution by using a standard NaOH solution until the pH value is 8.5, then adding dopamine hydrochloride to obtain a working solution, sequentially immersing the titanium sheet into acetone, ethanol and deionized water for ultrasonic cleaning for 15min, and immersing the titanium sheet into the working solution to soak for 12h under a dark condition to obtain the titanium sheet with the polydopamine auxiliary layer on the surface; the concentration of the dopamine hydrochloride in the working solution is 2 mg/mL; the thickness of the polydopamine auxiliary layer is 200 nm;
soaking the titanium sheet with the polydopamine auxiliary layer on the surface obtained in the third step into the mixed solution B obtained in the second step, taking out the titanium sheet, placing the titanium sheet in a constant-temperature drying box, drying the titanium sheet for 30min at 60 ℃, and forming a functional coating containing antibacterial peptide on the surface of the titanium sheet; the thickness of the antibacterial peptide functional coating is 1 mu m.
The titanium sheet, the nano-silver-containing antibacterial peptide functional coating formed on the surface of the titanium sheet in example 1, the nano-silver-containing functional coating formed on the surface of the titanium sheet in comparative example 1 and the antibacterial peptide-containing functional coating formed on the surface of the titanium sheet in comparative example 2 are respectively mixed with staphylococcus aureus (1 multiplied by 10)8CFU) is co-cultured in a constant temperature box at 37 ℃ for 24h, then a plate coating method is adopted for counting, the number of the staphylococcus aureus after co-culture is respectively counted, and two different parts, namely a part 1 and a part 2, are respectively and correspondingly selected for observing the antibacterial effect of the titanium sheet and each group of coatings.
Fig. 2 is a comparison graph of antibacterial effects of different parts of the coatings and titanium sheets formed in example 1 and comparative examples 1 to 2, wherein green dots represent live bacteria and red dots represent dead bacteria, and as can be seen from fig. 2, compared with the coatings and titanium sheets formed in comparative examples 1 to 2, the functional coating containing nano-silver antibacterial peptide formed on the surface of the titanium sheet in example 1 of the present invention has the least number of staphylococcus aureus after co-culture, which indicates that the functional coating containing nano-silver antibacterial peptide has excellent synergistic antibacterial effect and is superior to the functional coating containing nano-silver and the functional coating containing antibacterial peptide.
Example 2
The embodiment comprises the following steps:
step one, silk is placed in 0.02M Na2CO3Boiling the solution for 40min, taking out, washing with deionized water, and adding into 9.3M LiBr solution at 60 deg.C for dissolving for 4h to obtain fibroin protein stock solution;
diluting the silk fibroin stock solution into a silk fibroin solution with the mass concentration of 3% by using deionized water, then adding 10mg of silver nitrate powder and 8mg of antibacterial peptide powder into 10mL of the silk fibroin solution with the mass concentration of 3% and mixing to obtain a mixed solution A which contains silk fibroin, silver ions and antibacterial peptide and has the pH value of 8;
step two, placing the mixed solution A obtained in the step one under a 40W ultraviolet lamp for irradiation reduction for 1h, so that the color of the mixed solution A is gradually changed from milky white to dark brown, and obtaining a mixed solution B containing silk fibroin, nano silver and antibacterial peptide; the particle size of the nano silver in the mixed solution B is 300nm, and the mass content of the silk fibroin with a beta-folded structure in the mixed solution A is 20%; the amino acid sequence of the antibacterial peptide in the mixed solution B is (NH)2)-NGIVKAGPAIAVLGEAAL-CONH2The mass purity of the antibacterial peptide is 99.99 percent;
step three, calibrating a 10mM Tris solution to the pH value of 8.5 by using a standard NaOH solution, then adding dopamine hydrochloride to obtain a working solution, sequentially immersing the titanium alloy into acetone, ethanol and deionized water for ultrasonic cleaning for 15min, and immersing the titanium alloy into the working solution for 18h under the condition of keeping out of the sun to obtain the titanium alloy with the polydopamine auxiliary layer on the surface; the concentration of the dopamine hydrochloride in the working solution is 2 mg/mL; the thickness of the polydopamine auxiliary layer is 300 nm;
soaking the titanium alloy with the polydopamine auxiliary layer on the surface obtained in the third step into the mixed solution B obtained in the second step, taking out the titanium alloy, placing the titanium alloy in a constant-temperature drying box, drying the titanium alloy for 30min at 60 ℃, and forming a nano-silver-containing antibacterial peptide functional coating on the surface of the titanium alloy; the thickness of the nano-silver-containing antibacterial peptide functional coating is 15 mu m.
Example 3
The embodiment comprises the following steps:
step one, silk is placed in 0.02M Na2CO3Boiling the solution for 40min, taking out, washing with deionized water, and adding into 9.3M LiBr solution at 60 deg.C for dissolving for 4h to obtain fibroin protein stock solution;
diluting the silk fibroin stock solution into a silk fibroin solution with the mass concentration of 5% by using deionized water, then adding 200mg of silver nitrate powder and 10mg of antibacterial peptide powder into 10mL of silk fibroin solution with the mass concentration of 5% for mixing to obtain a mixed solution A which contains silk fibroin, silver ions and antibacterial peptide and has the pH value of 10;
step two, placing the mixed solution A obtained in the step one under a 40W ultraviolet lamp for irradiation reduction for 2 hours, so that the color of the mixed solution A is gradually changed from milky white to dark brown, and obtaining a mixed solution B containing silk fibroin, nano silver and antibacterial peptide; the particle size of the nano silver in the mixed solution B is 500nm, and the mass content of the silk fibroin with a beta-folded structure in the mixed solution B is 30%; the amino acid sequence of the antibacterial peptide in the mixed solution A is (NH)2)-NGIVKAGPAIAVLGEAAL-CONH2The mass purity of the antibacterial peptide is 99.99 percent;
step three, calibrating a 10mM Tris solution to the pH value of 8.5 by adopting a standard NaOH solution, then adding dopamine hydrochloride to obtain a working solution, sequentially immersing the stainless steel into acetone, ethanol and deionized water for ultrasonic cleaning for 15min, and then immersing the stainless steel into the working solution for 24h under the condition of keeping out of the sun to obtain the stainless steel with the polydopamine auxiliary layer on the surface; the concentration of the dopamine hydrochloride in the working solution is 2 mg/mL; the thickness of the polydopamine auxiliary layer is 500 nm;
soaking the stainless steel with the polydopamine auxiliary layer on the surface obtained in the third step into the mixed solution B obtained in the second step, taking out the stainless steel, placing the stainless steel in a constant-temperature drying box, drying the stainless steel for 30min at 60 ℃, and forming a nano-silver-containing antibacterial peptide functional coating on the surface of the stainless steel; the thickness of the nano-silver-containing antibacterial peptide functional coating is 20 micrometers.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Any simple modification, change and equivalent changes of the above embodiments according to the technical essence of the invention are still within the protection scope of the technical solution of the invention.
Claims (9)
1. A method for preparing a nano-silver-containing antibacterial peptide functional coating on a metal surface is characterized by comprising the following steps:
adding silver nitrate powder and antibacterial peptide powder into a silk fibroin solution, and mixing to obtain a mixed solution A containing silk fibroin, silver ions and antibacterial peptide; the mass concentration of the silk fibroin in the mixed solution A is 2-5%, the concentration of silver ions is 1-20 mg/mL, and the concentration of the antibacterial peptide is 0.4-1 mg/mL;
step two, placing the mixed solution A obtained in the step one under an ultraviolet lamp for irradiation reduction, so that the color of the mixed solution A is gradually changed from milky white to dark brown, and obtaining a mixed solution B containing silk fibroin, nano silver and antibacterial peptide; the time for irradiation reduction is 0.5 h-2 h;
step three, calibrating a 10mM Tris solution to the pH value of 8.5 by adopting a standard NaOH solution, then adding dopamine hydrochloride to obtain a working solution, and then soaking the metal in the working solution for 12-24 hours in a dark condition to obtain the metal with the polydopamine auxiliary layer on the surface;
and step four, soaking the metal with the polydopamine auxiliary layer on the surface obtained in the step three into the mixed solution B obtained in the step two, and forming the nano-silver-containing antibacterial peptide functional coating on the surface of the metal.
2. The method for preparing the nano-silver-containing antibacterial peptide functional coating on the metal surface according to claim 1, wherein the particle size of nano-silver in the mixed solution B in the step two is 20nm to 500nm, and the mass content of the silk fibroin with a beta-sheet structure in the mixed solution B is 15% to 30%.
3. The method for preparing the functional coating containing the nano-silver antibacterial peptide on the metal surface according to claim 1, wherein the amino acid sequence of the antibacterial peptide in the mixed solution A in the second step is (NH)2)-NGIVKAGPAIAVLGEAAL-CONH2The quality purity of the antibacterial peptide is more than 99%.
4. The method for preparing the functional coating containing the nano-silver antibacterial peptide on the metal surface according to the claim 1, wherein the metal in the step three is titanium, titanium alloy or stainless steel.
5. The method for preparing the nano-silver-containing antibacterial peptide functional coating on the metal surface according to claim 1, wherein the concentration of dopamine hydrochloride in the working solution in the third step is 1 mg/mL-4 mg/mL.
6. The method for preparing the nano-silver-containing antibacterial peptide functional coating on the metal surface according to the claim 1, characterized in that the metal is subjected to cleaning pretreatment before being immersed in the working solution in the third step: and sequentially immersing the metal into acetone, ethanol and deionized water for ultrasonic cleaning for 15 min.
7. The method for preparing the nano-silver-containing antibacterial peptide functional coating on the metal surface according to claim 1, wherein the thickness of the polydopamine auxiliary layer in the third step is 200nm to 500 nm.
8. The method for preparing the nano-silver-containing antibacterial peptide functional coating on the metal surface according to claim 1, wherein the metal with the surface provided with the polydopamine auxiliary layer in the fourth step is soaked in the mixed solution B obtained in the second step, taken out and placed in a constant temperature drying oven, and dried at 60 ℃ for 10min to 30 min.
9. The method for preparing the functional coating containing the nano-silver antibacterial peptide on the metal surface according to the claim 1, wherein the thickness of the functional coating containing the nano-silver antibacterial peptide in the fourth step is 1 μm to 20 μm.
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