纳米氢氧化镁的抗菌性研究
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摘要
为控制有害微生物的生长繁殖,避免细菌的传播和感染,研发高效无毒且效力持久的抗菌剂是一项与人类健康生活息息相关的重要课题。目前无机抗菌剂的研究多以重金属和半导体化合物为主,部分材料的抗菌机理不明,并且氢氧化物固体材料的抗菌性研究还是空白。针对以上问题,本论文研究了纳米Mg(OH)2的抗菌性能及抗菌机理,并加以应用。
首先考察了悬浊液中纳米Mg(OH)2对大肠杆菌、金黄色葡萄球菌、吩嗪伯克氏菌的抗菌性能,结果表明在纳米Mg(OH)2的作用下,细菌存活率随着时间的增长而降低,且细菌死亡不可修复；与其它纳米材料包括CuO、NiO以及粘土的抗菌效率相比,对革兰氏阳性菌,纳米Mg(OH)2的抗菌效率低于CuO,而对革兰氏阴性菌,与CuO类似,高于NiO,展示了纳米Mg(OH)2作为抗菌剂的良好应用前景。另外,实验建立了多次顶空抽提气相色谱技术(MHE-GC)对微生物生长情况进行检测,绘制了大肠杆菌生长曲线,避免了原顶空法实验工作量和误差较大的问题。与比浊法相比,MHE-GC法可以早1.2 h检测出大肠杆菌的生长速率,具有更高的灵敏度；其对大肠杆菌数目的检测结果与平板菌落计数法结果一致,表明MHE-GC法具有较高的精确度。
其次,以大肠杆菌为测试对象,对纳米Mg(OH)2的抗菌机理进行了讨论。实验结果表明Mg(OH)2纳米颗粒悬浊液中的OH和Mg2+对大肠杆菌的生长并无杀灭作用,但pH---10的碱性溶液对大肠杆菌的繁殖有一定的抑制作用；其杀菌过程在黑暗中也可进行,表明纳米Mg(OH)2杀菌并不依赖于光源。提出胞吞一离解机理,即Mg(OH)2纳米颗粒由胞吞作用进入细胞内部,在细胞体内的水性环境中释放大量OH-,破坏了细胞体内正常的中性环境,引起核酸和蛋白质的化学变性,从而导致细菌死亡。实验结果证实阻碍或促进细胞对Mg(OH)2的胞吞作用,比如选用微米级Mg(OH)2颗粒、加入能量抑制剂、引入长时间的紫外光照等,都严重影响了杀菌过程的进行。
最后,将纳米Mg(OH)2的抗菌性加以应用,制备了抗菌纸和PVDF/Mg(OH)2共混膜。采用湿部添加法制备了纳米Mg(OH)2抗菌纸,对纸张样品中Mg(OH)2的留着率和纸张抗菌性能进行考察,结果表明纳米Mg(OH)2的留着率高于75%,可以满足实际应用的要求；在实验操作条件下,添加3 wt%Mg(OH)2纳米颗粒的纸张样品对大肠杆菌在18 h内可以达到100%的杀菌率,具有良好的应用前景。采用相转化法制备了PVDF/Mg(OH)2共混膜,实验测定了共混膜的组成结构、机械稳定性、牛血清白蛋白(BSA)吸附量等,并考察了Mg(OH)2纳米颗粒对膜孔结构和渗透性能的影响。结果表明Mg(OH)2纳米颗粒的添加,改变了PVDF/Mg(OH)2共混膜的孔结构和亲水性,当PEG浓度为5wt%时,共混膜处理含菌溶液的滤饼阻力与PVDF原膜相比下降为原来的1/5,显著提高了膜的抗生物污染性。
The study on the antibacterial agents with high efficiency and no toxicity is of great importance for the healthy development of society to control the growth of microorganism and the spread and infection of bacteria. The existing researches on the inorganic antibacterial agents are mainly focused on the metals and metal oxides, as well as novel engineered nanomaterials. The major antibacterial mechanisms reported in the literature fall into three general views, including the release of metal ions, generation of reactive oxygen species of semiconductor materials, and contact of nanoparticles with microorganisms. In this thesis, the antibacterial activity of Mg(OH)2 has been explored, and a novel antibacterial mechanism has been proposed. The Mg(OH)2 nanoparticles have been mixed into the fibers and Poly(vinylidene fluoride) (PVDF) to prepare antibacterial paper and hybrid membrane respectively, which extends the applications of the Mg(OH)2 nanoparticles. In addition, the multiple headspace extraction (MHE) technique for monitoring the growth of bacteria has been established in order to improve the existing headspace gas chromatography (HS-GC) method which gives a larger data scattering and causes larger errors.
Firstly, the antibacterial activity of Mg(OH)2 nanoparticles was investigated and the Escherichia coli(E.coli), Staphyloccocus aureus, Burkholderia phenazinium were used as testing bacteria. The antibacterial efficiencies of different nanomaterials were also compared. Results indicate that Mg(OH)2 nanoparticles have a broad antibacterial spectrum, and the antibacterial efficiency of Mg(OH)2 against G- bacteria is almost the same as CuO, while a little lower for G+ bacteria, suggesting that Mg(OH)2 nanoparticles have an excellent potential application as antibacterial agents. Meanwhile, the MHE-GC technique for monitoring the growth of bacteria was established, and the growth curve of E.coli was obtained. The comparisons among the results from MHE-GC, turbidity measurement and viable cell counts indicate that the present method is very simple, sensitive and safe, which can easily perform an automatic measurement for bacteria growth at various desired incubation conditions.
Secondly, the factors affecting antibacterial efficiency of Mg(OH)2 nanoparticles were investigated, including the ions in the suspension, UV and visible irradiation, the contact mode of nanoparticles and cells, the size and shape of Mg(OH)2. Results indicate that OH-and Mg2+ in Mg(OH)2 suspension can not kill E.coli; the antibacterial activity of Mg(OH)2 depends on its size rather than the shape; Mg(OH)2 nanoparticles can kill E.coli in the dark, suggesting its potential wide application. A novel antibacterial mechanism has been proposed that Mg(OH)2 nanoparticles can penetrate into the cells by endocytosis and release large amount of OH-, which will increase the pH value of the inter cells and cause the protein denaturation, leading to the cell death of bacterial.
Finally, the Mg(OH)2 antibacterial paper and hybrid membrane were prepared, which extends the applications of the Mg(OH)2 nanoparticles. The retention rate of Mg(OH)2 and the antibacterial activity of the paper sheet were investigated. Results indicate that the retention rate of Mg(OH)2 in the paper sheet is above 75%, which reaches the standard of the industrial production. In our experiment, the paper sheet with 3% Mg(OH)2 nanoparticles performed a 100% antibacterial ratio in 18 h, suggesting an excellent potential application for Mg(OH)2 nanoparticles. In addition, the PVDF/Mg(OH)2 hybrid membrane was prepared by phase inversion method. Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) measurements were performed, and the ultrasonic stability, porosity, hydrophilicity, permeation and bovine serum albumin (BSA) and E. coli adsorption of the membrane were investigated. Results indicate that the addition of Mg(OH)2 alters the pore structures and hydrophilicity of the modified membrane, and the relative flux of membrane to pure water has been improved by more than 1.5 times with 10 wt% Mg(OH)2 added, suggesting it will be very effective in preventing flux losses caused by biofilm formation.
首先考察了悬浊液中纳米Mg(OH)2对大肠杆菌、金黄色葡萄球菌、吩嗪伯克氏菌的抗菌性能,结果表明在纳米Mg(OH)2的作用下,细菌存活率随着时间的增长而降低,且细菌死亡不可修复；与其它纳米材料包括CuO、NiO以及粘土的抗菌效率相比,对革兰氏阳性菌,纳米Mg(OH)2的抗菌效率低于CuO,而对革兰氏阴性菌,与CuO类似,高于NiO,展示了纳米Mg(OH)2作为抗菌剂的良好应用前景。另外,实验建立了多次顶空抽提气相色谱技术(MHE-GC)对微生物生长情况进行检测,绘制了大肠杆菌生长曲线,避免了原顶空法实验工作量和误差较大的问题。与比浊法相比,MHE-GC法可以早1.2 h检测出大肠杆菌的生长速率,具有更高的灵敏度；其对大肠杆菌数目的检测结果与平板菌落计数法结果一致,表明MHE-GC法具有较高的精确度。
其次,以大肠杆菌为测试对象,对纳米Mg(OH)2的抗菌机理进行了讨论。实验结果表明Mg(OH)2纳米颗粒悬浊液中的OH和Mg2+对大肠杆菌的生长并无杀灭作用,但pH---10的碱性溶液对大肠杆菌的繁殖有一定的抑制作用；其杀菌过程在黑暗中也可进行,表明纳米Mg(OH)2杀菌并不依赖于光源。提出胞吞一离解机理,即Mg(OH)2纳米颗粒由胞吞作用进入细胞内部,在细胞体内的水性环境中释放大量OH-,破坏了细胞体内正常的中性环境,引起核酸和蛋白质的化学变性,从而导致细菌死亡。实验结果证实阻碍或促进细胞对Mg(OH)2的胞吞作用,比如选用微米级Mg(OH)2颗粒、加入能量抑制剂、引入长时间的紫外光照等,都严重影响了杀菌过程的进行。
最后,将纳米Mg(OH)2的抗菌性加以应用,制备了抗菌纸和PVDF/Mg(OH)2共混膜。采用湿部添加法制备了纳米Mg(OH)2抗菌纸,对纸张样品中Mg(OH)2的留着率和纸张抗菌性能进行考察,结果表明纳米Mg(OH)2的留着率高于75%,可以满足实际应用的要求；在实验操作条件下,添加3 wt%Mg(OH)2纳米颗粒的纸张样品对大肠杆菌在18 h内可以达到100%的杀菌率,具有良好的应用前景。采用相转化法制备了PVDF/Mg(OH)2共混膜,实验测定了共混膜的组成结构、机械稳定性、牛血清白蛋白(BSA)吸附量等,并考察了Mg(OH)2纳米颗粒对膜孔结构和渗透性能的影响。结果表明Mg(OH)2纳米颗粒的添加,改变了PVDF/Mg(OH)2共混膜的孔结构和亲水性,当PEG浓度为5wt%时,共混膜处理含菌溶液的滤饼阻力与PVDF原膜相比下降为原来的1/5,显著提高了膜的抗生物污染性。
The study on the antibacterial agents with high efficiency and no toxicity is of great importance for the healthy development of society to control the growth of microorganism and the spread and infection of bacteria. The existing researches on the inorganic antibacterial agents are mainly focused on the metals and metal oxides, as well as novel engineered nanomaterials. The major antibacterial mechanisms reported in the literature fall into three general views, including the release of metal ions, generation of reactive oxygen species of semiconductor materials, and contact of nanoparticles with microorganisms. In this thesis, the antibacterial activity of Mg(OH)2 has been explored, and a novel antibacterial mechanism has been proposed. The Mg(OH)2 nanoparticles have been mixed into the fibers and Poly(vinylidene fluoride) (PVDF) to prepare antibacterial paper and hybrid membrane respectively, which extends the applications of the Mg(OH)2 nanoparticles. In addition, the multiple headspace extraction (MHE) technique for monitoring the growth of bacteria has been established in order to improve the existing headspace gas chromatography (HS-GC) method which gives a larger data scattering and causes larger errors.
Firstly, the antibacterial activity of Mg(OH)2 nanoparticles was investigated and the Escherichia coli(E.coli), Staphyloccocus aureus, Burkholderia phenazinium were used as testing bacteria. The antibacterial efficiencies of different nanomaterials were also compared. Results indicate that Mg(OH)2 nanoparticles have a broad antibacterial spectrum, and the antibacterial efficiency of Mg(OH)2 against G- bacteria is almost the same as CuO, while a little lower for G+ bacteria, suggesting that Mg(OH)2 nanoparticles have an excellent potential application as antibacterial agents. Meanwhile, the MHE-GC technique for monitoring the growth of bacteria was established, and the growth curve of E.coli was obtained. The comparisons among the results from MHE-GC, turbidity measurement and viable cell counts indicate that the present method is very simple, sensitive and safe, which can easily perform an automatic measurement for bacteria growth at various desired incubation conditions.
Secondly, the factors affecting antibacterial efficiency of Mg(OH)2 nanoparticles were investigated, including the ions in the suspension, UV and visible irradiation, the contact mode of nanoparticles and cells, the size and shape of Mg(OH)2. Results indicate that OH-and Mg2+ in Mg(OH)2 suspension can not kill E.coli; the antibacterial activity of Mg(OH)2 depends on its size rather than the shape; Mg(OH)2 nanoparticles can kill E.coli in the dark, suggesting its potential wide application. A novel antibacterial mechanism has been proposed that Mg(OH)2 nanoparticles can penetrate into the cells by endocytosis and release large amount of OH-, which will increase the pH value of the inter cells and cause the protein denaturation, leading to the cell death of bacterial.
Finally, the Mg(OH)2 antibacterial paper and hybrid membrane were prepared, which extends the applications of the Mg(OH)2 nanoparticles. The retention rate of Mg(OH)2 and the antibacterial activity of the paper sheet were investigated. Results indicate that the retention rate of Mg(OH)2 in the paper sheet is above 75%, which reaches the standard of the industrial production. In our experiment, the paper sheet with 3% Mg(OH)2 nanoparticles performed a 100% antibacterial ratio in 18 h, suggesting an excellent potential application for Mg(OH)2 nanoparticles. In addition, the PVDF/Mg(OH)2 hybrid membrane was prepared by phase inversion method. Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) measurements were performed, and the ultrasonic stability, porosity, hydrophilicity, permeation and bovine serum albumin (BSA) and E. coli adsorption of the membrane were investigated. Results indicate that the addition of Mg(OH)2 alters the pore structures and hydrophilicity of the modified membrane, and the relative flux of membrane to pure water has been improved by more than 1.5 times with 10 wt% Mg(OH)2 added, suggesting it will be very effective in preventing flux losses caused by biofilm formation.
引文
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