纳米材料对细菌的生物效应研究
摘要
纳米材料(Nanomaterials,简称NMs)的生物效应研究已有大量相关报道，主要是通过研究NMs对动物、植物、微生物的生物效应，探讨NMs对生态环境及人体的潜在危害。微生物中利用藻类测试NMs的毒性报道较多，而对于细菌毒性测试相对较少，因此不能全面地评价NMs对整个生态系统的毒性效应。本文通过研究NMs对细菌的毒性效应，探讨NMs对微生物系统的危害，旨在从微生物系统层面为NMs的生态毒理效应提供一定的科学依据。本文研究的主要内容及结论如下：
（1）采用平板计数法、光密度值法、MTT法三种不同方法测试NMs对常用指示菌大肠杆菌的生长抑制率。结果表明，三种测试方法测得的NMs对细菌的毒性效应趋势基本一致，但是操作繁琐程度、灵敏性、重复性各不相同。其中MTT法操作简单，重复性好，可以较好地运用于NMs对细菌的毒性测试中。
（2）主要选取微生物生态系统中三种常见的细菌（革兰氏阴性大肠杆菌、革兰氏阳性枯草芽孢杆菌及革兰氏阳性金黄色葡萄球菌）作为受试物测试NMs的微生物效应，探讨了MTT法测试枯草芽孢杆菌及金黄色葡萄球菌细胞活性的可行性及具体实施过程中的实验条件。通过MTT法考察了不同种类NMs及同种不同粒径NMs对大肠杆菌、枯草芽孢杆菌及金黄色葡萄球菌的生物毒性，利用半数效应浓度（EC50）值比较了不同NMs的毒性大小。结果表明：随着NMs浓度的增加，其对细菌的毒性逐渐增强；不同种NMs生物毒性大小不同，对于纳米氧化物，其毒性大小顺序为：nZnO > nTiO_2>nFe_3O_4> nSiO_2> nFe_2O_3；对于载银材料，有Ag-doped nZrP > Ag-doped nTiO_2；对于纳米银分散液，有SNP-1 > SNP-2；不同粒径的同种NMs毒性大小不同，粒径越小，NMs毒性越大；同种NMs对不同细菌的毒性大小不同，其中NMs对枯草芽孢杆菌的毒性最大，对金黄色葡萄球菌的毒性最小。
（3）通过考马斯亮蓝法测定了菌液蛋白质含量的变化，并通过电导率法测试了菌液电导率的变化，以此探讨了NMs对细菌细胞膜通透性的影响；通过细胞膜是否受损探讨了NMs的抑菌机理。结果表明，NMs对细菌细胞膜通透性有一定影响，而且不同NMs对细菌细胞膜通透性影响不同，同种NMs对不同细菌细胞膜影响程度不同。
总之，NMs对细菌具有一定的毒性效应，其毒性大小与NMs种类和粒径等因素有关，且对不同细菌的生物效应不同。通过研究NMs对细菌的生物效应能够为NMs的微生物效应提供充分的依据。
In recent years, much attention has been paid to the toxicity of nanomaterials (NMs) onanimals, plants and microorganism. For microorganism, alga is employed as model organisms.However, the study about toxicity of NMs on bacteria is limited. In this paper, bacteria is usedto test the toxicity of NMs. In order to provide base date for the toxicity on microbialecosystem, the results are listed as follows:
(1) Plate counting, optical density and MTT assays are applied to investigate the toxiceffects of nanomaterials on bacteria. Among these three different methods, MTT assay issimple, rapid, steady and more suitable for evaluating the toxicity of NMs.
(2) Gram-negative Escherichia coli, Gram-positive Bacillus subtilis and Staphylococcusaureus Gram-positive, common bacteria in microbial ecosystem, are selected to test thetoxicity of nanomaterials. The effects of concentration and particle size on the toxicity ofNMs to these three different kinds of bacteria are systematically examined with MTT assay.The different toxicities of nanomaterials are distinguished by 12 h EC_(50). The results show thatantibacterial activity increase with the increase of dose. Different NMs have different toxicity.As for nanoparticles of oxides, the most toxic is nZnO followed by nTiO_2, nFe_3O_4, nSiO_2andnFe2O3. For silver doped nanoparticles, Ag-doped nTiO_2is less toxic than Ag-doped nZrP.About silver nanoparticles, SNP-1 is more toxic than SNP-2. In addition, the toxicity of NMsincreases with decreasing particle size. The toxicity of the same NMs to different bacteria isdifferent, and these nanomaterials are more toxic to Bacillus subtilis than Escherichia coli andStaphylococcus aureus.
(3) Coomassie brilliant blue is used to test the content of protein in bacterial solution andconductimetric methods is used to test the conductivity of bacterial solution, discussing theeffect of nanomaterials on cell membrane permeability of different bacteria. The resultsindicate that nanomaterials destroy the cell membrane to a diferent degree. The effects ofnanomaterials on different bacteria are different.
In short, NMs present toxic effect on bacteria, the kind and particle size influence thetoxicity of NMs. The NMs present different toxic effect on different bacteria. This study canprovide support for the toxicity of nanomaterials on microorganism.
（1）采用平板计数法、光密度值法、MTT法三种不同方法测试NMs对常用指示菌大肠杆菌的生长抑制率。结果表明，三种测试方法测得的NMs对细菌的毒性效应趋势基本一致，但是操作繁琐程度、灵敏性、重复性各不相同。其中MTT法操作简单，重复性好，可以较好地运用于NMs对细菌的毒性测试中。
（2）主要选取微生物生态系统中三种常见的细菌（革兰氏阴性大肠杆菌、革兰氏阳性枯草芽孢杆菌及革兰氏阳性金黄色葡萄球菌）作为受试物测试NMs的微生物效应，探讨了MTT法测试枯草芽孢杆菌及金黄色葡萄球菌细胞活性的可行性及具体实施过程中的实验条件。通过MTT法考察了不同种类NMs及同种不同粒径NMs对大肠杆菌、枯草芽孢杆菌及金黄色葡萄球菌的生物毒性，利用半数效应浓度（EC50）值比较了不同NMs的毒性大小。结果表明：随着NMs浓度的增加，其对细菌的毒性逐渐增强；不同种NMs生物毒性大小不同，对于纳米氧化物，其毒性大小顺序为：nZnO > nTiO_2>nFe_3O_4> nSiO_2> nFe_2O_3；对于载银材料，有Ag-doped nZrP > Ag-doped nTiO_2；对于纳米银分散液，有SNP-1 > SNP-2；不同粒径的同种NMs毒性大小不同，粒径越小，NMs毒性越大；同种NMs对不同细菌的毒性大小不同，其中NMs对枯草芽孢杆菌的毒性最大，对金黄色葡萄球菌的毒性最小。
（3）通过考马斯亮蓝法测定了菌液蛋白质含量的变化，并通过电导率法测试了菌液电导率的变化，以此探讨了NMs对细菌细胞膜通透性的影响；通过细胞膜是否受损探讨了NMs的抑菌机理。结果表明，NMs对细菌细胞膜通透性有一定影响，而且不同NMs对细菌细胞膜通透性影响不同，同种NMs对不同细菌细胞膜影响程度不同。
总之，NMs对细菌具有一定的毒性效应，其毒性大小与NMs种类和粒径等因素有关，且对不同细菌的生物效应不同。通过研究NMs对细菌的生物效应能够为NMs的微生物效应提供充分的依据。
In recent years, much attention has been paid to the toxicity of nanomaterials (NMs) onanimals, plants and microorganism. For microorganism, alga is employed as model organisms.However, the study about toxicity of NMs on bacteria is limited. In this paper, bacteria is usedto test the toxicity of NMs. In order to provide base date for the toxicity on microbialecosystem, the results are listed as follows:
(1) Plate counting, optical density and MTT assays are applied to investigate the toxiceffects of nanomaterials on bacteria. Among these three different methods, MTT assay issimple, rapid, steady and more suitable for evaluating the toxicity of NMs.
(2) Gram-negative Escherichia coli, Gram-positive Bacillus subtilis and Staphylococcusaureus Gram-positive, common bacteria in microbial ecosystem, are selected to test thetoxicity of nanomaterials. The effects of concentration and particle size on the toxicity ofNMs to these three different kinds of bacteria are systematically examined with MTT assay.The different toxicities of nanomaterials are distinguished by 12 h EC_(50). The results show thatantibacterial activity increase with the increase of dose. Different NMs have different toxicity.As for nanoparticles of oxides, the most toxic is nZnO followed by nTiO_2, nFe_3O_4, nSiO_2andnFe2O3. For silver doped nanoparticles, Ag-doped nTiO_2is less toxic than Ag-doped nZrP.About silver nanoparticles, SNP-1 is more toxic than SNP-2. In addition, the toxicity of NMsincreases with decreasing particle size. The toxicity of the same NMs to different bacteria isdifferent, and these nanomaterials are more toxic to Bacillus subtilis than Escherichia coli andStaphylococcus aureus.
(3) Coomassie brilliant blue is used to test the content of protein in bacterial solution andconductimetric methods is used to test the conductivity of bacterial solution, discussing theeffect of nanomaterials on cell membrane permeability of different bacteria. The resultsindicate that nanomaterials destroy the cell membrane to a diferent degree. The effects ofnanomaterials on different bacteria are different.
In short, NMs present toxic effect on bacteria, the kind and particle size influence thetoxicity of NMs. The NMs present different toxic effect on different bacteria. This study canprovide support for the toxicity of nanomaterials on microorganism.
引文
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[3] Dhawan A, Sharma V. Toxicity assessment of nanomaterials: methods and challenges [J]. Anal BioanalChem, 2010, 398: 589-605.
[4]杨运梁,胡和平.纳米材料的环境毒理学研究进展[J].广东化工, 2010, 5 (37): 171-173.
[5] Borm P J, Robbins D, Haubold S, et al. 2006. The potential risks of nanomaterials: A reviewcarried out for ECETOC [J]. Particle and Fibre Toxicology, 3: 1- 35.
[6]曲秋莲,张英鸽.纳米技术和材料在医学上应用的现状与展望[J].东南大学学报,2011,30(1) :157-163.
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