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微纳塑料的制备、标记及在复杂基质中的定量检测研究
Alternative TitlePreparation, labeling and quantitative detection of micro-nanoplastics in complex matrices
高琦
Subtype硕士
Thesis Advisor王运庆 ; 陈令新
2022-05-13
Training institution中国科学院烟台海岸带研究所
Degree Grantor中国科学院大学
Degree Name工学硕士
Degree Discipline环境科学
Keyword微纳塑料 标记 体内分布 沉降行为 超疏水富集
Abstract微纳塑料(微塑料及纳米塑料的统称)作为一类新型污染物被广泛关注,目前已成为生态环境领域的研究热点。近年来,国内外科学家在微纳塑料的环境分布、来源调查、传输行为和生态毒理等方向开展了大量研究,取得了一定的进展,但仍存在两个关键问题还未解决。一是效应评价的微纳塑料模型环境相关性较差,许多研究使用聚苯乙烯(PS)球型颗粒进行微纳塑料的研究,但自然环境中的微纳塑料表面形状不规则,且表面性质、与生物分子的作用方式和毒理效应均因塑料材质不同而有所差异,因此通过PS球形颗粒得到的结论不应被认为是适用于所有塑料材质的普遍结论;二是缺乏针对微纳塑料的高灵敏度示踪方法,目前许多工作从成像分析、光谱鉴定及质谱检测等方面进行微纳塑料的研究,但这些方法对微纳塑料的检测存在灵敏度低、抗环境背景干扰差、不适合纳米级塑料的分析等问题,发展高灵敏的纳米塑料示踪方法仍处于瓶颈状态。本研究针对上述问题,发展了基于机械破碎的“真实”微纳塑料的制备方法及基于溶胀法标记微纳塑料的方法,将铱(Ir)标记的微纳塑料应用于小鼠体内分布及沉积物再悬浮体系中,得到了微纳塑料在小鼠体内的分布具有粒径依赖性及自然环境中的微纳塑料存在吸附沉降行为等结论;使用尼罗红标记的微纳塑料开展微量微纳塑料的超疏水富集成像研究,初步验证了微量微纳塑料快速成像检测的可行性,提高了微纳塑料检测的灵敏度。具体内容如下: (1)发展了一种基于机械破碎的“真实”微纳塑料的制备及标记方法 通过模拟自然环境中微纳塑料的形成过程,以市售的 PET 材质矿泉水瓶为原料,制备了 200 nm、900 nm 和 2500 nm 的微纳塑料(简称 PET200、PET900 和PET2500)。基于聚合物的溶胀特性,以铂族元素 Ir 的稠环配位化合物为标记物对微纳塑料进行标记,得到了 3 种标记后的微纳塑料 PET200-Ir、PET900-Ir 和PET2500-Ir。优化了标记及消解方法,以标记物浓度为 0.5 mg/mL,标记比例为微纳塑料与标记物的体积比 6:1,标记时间为 12 h 进行标记,以消解时间为 10 h,消解温度为 100℃进行消解。通过设置环境和生物介质中的模拟泄露实验,发现除胎牛血清基质中 PET-Ir 的泄露量达到 5%以外,其他基质中的 PET-Ir 的泄露量均小于 3%,表明该标记方法具有体内标记潜质。本研究中的制备方法做到了生成过程真实(物理磨损)、材料理化性质真实(源自真实的日用塑料),而标记方法具有抗背景干扰的优势,是微纳塑料模型制备的重要进展,为后续生物及环境分布的应用研究奠定了基础。 (2) 纳米塑料在小鼠体内的分布研究 通过静脉注射和灌胃两种方式,以Ir的水分散液作为对照,探究了PET200-Ir和PET900-Ir两种粒径的纳米塑料在小鼠体内的分布情况。发现了静脉注射条件下,肝脏为纳米塑料蓄积的主要器官且纳米塑料的分布行为具有粒径依赖性,PET200的蓄积速度和清除速度均快于PET900;灌胃条件下,在肠胃以外的其他组织中均未检测到两种粒径的纳米塑料及Ir的水分散液的存在,该现象可能是由于胃肠道屏障的影响。本研究探究了“真实”微纳塑料应用于生物体内的可行性,表明了生物体内纳米塑料行为的粒径依赖性,可为“真实”纳米塑料的毒理研究提供参考。 (3) 微纳塑料在海岸带沉积物再悬浮体系中的分布研究 沉积物再悬浮现象是海岸带区域一个比较普遍的物理现象,本研究以沉积物再悬浮体系为背景,探究了振荡时间对微纳塑料垂直分布的影响以及微纳塑料的垂直分布检测。结果表明,随振荡时间的增加,上清液中的微纳塑料浓度降低,沉积物中的微纳塑料浓度升高,符合吸附驱动的沉降行为。45.3%的微纳塑料集中在深度为1.4~1.8 cm范围内的沉积层,是相邻水体层的3.6倍,相邻沉积物层的5.5倍,说明了微纳塑料易于分布在沉积物顶层及水体底层。本研究阐明了“真实”微纳塑料应用于环境行为研究的可行性,探究了沉积物扰动以及同自然物质的相互作用影响微纳塑料的沉降,揭示了存在沉积物再悬浮现象的海岸带区域中,生活在沉积物表层的底栖生物可能更容易受到微纳塑料污染的影响。 (4)真实样品中微纳塑料的超疏水富集荧光检测 基于目前成像检测视野小,而载物基底上样品分布面积大、粒子稀疏,难以准确快速定位到粒子所处位置导致的分析效率低的现状,急需创新成像分析方法,扩大浓度检测范围。超疏水富集是一种适用于成像检测的有效富集方法,可适用于少体积、小粒径、低浓度颗粒样品的检测。本研究发展了超疏水富集法,可以将玻片上10 μL样品直径缩小到0.1 mm以下,促进了低浓度样品的检测。同时,将超疏水富集与荧光标记法相结合,初步解决了荧光背景干扰问题,开展了低浓度无标记微纳塑料样品的富集检测研究,实现海水及纯水样品中低浓度PET900样品(6.7×10-5 mg/mL)、纯水样品低浓度PET200样品(3.4×10-5 mg/mL)及PET2500(5.7×10-5 mg/mL)样品的高灵敏度成像检测。本研究降低了荧光成像纳米塑料的检测限,显著提高了检出效率及检测灵敏度,为真实环境中微纳塑料的检测提供方法学参考。
Other AbstractMicro-nanoplastics (the general name of microplastics and nano-plastics) have been widely concerned as a new type of pollutants, and have become a research hotspot in the field of ecological and environmental. In recent years, scientists at home and abroad have carried out a lot of research and made some progress on the environmental distribution, source investigation, behavior transmission and ecotoxicology of micro-nanoplastics, but there are still two key problems that have not been solved. First, the micro-nanoplastic model for effect evaluation is lack of authenticity. Many studies use polystyrene (PS) spherical particles to substitute micro-nanoplastics, but the surface shape of micro-nanoplastics in the natural environment is irregular, and the surface properties, interaction with biomolecules and toxicological effects are different due to different plastic materials. Therefore, the experimental results obtained from PS spherical particles should not be regarded as a general conclusion applicable to all plastic materials. Second, there is a lack of high sensitivity tracing methods for micro-nanoplastics. At present, a lot of work has been done on micro-nanoplastics from the aspects of imaging analysis, spectral identification and mass spectrometry detection. However, there are some problems in the detection of micro-nanoplastics by these methods, such as low sensitivity, poor resistance to environmental background interference, unsuitable for the analysis of nano plastics, and so on. The development of highly sensitive nano-plastic tracing methods is still in a bottleneck. In order to solve the above problems, the preparation method of "realistic" micro-nanoplastics based on mechanical fragmentation and the method of labeling micro-nanoplastics based on swelling were developed. The iridium (Ir) labeled micro-nanoplastics were applied to the distribution in mice and the sediment resuspension model. It was concluded that the distribution of micro-nanoplastics in mice was particle size dependent and the micro-nanoplastics in the natural environment had adsorption and sedimentation behavior. The ultra-hydrophobic enrichment imaging study of micro-nanoplastics marked with Nile red was carried out, which preliminarily verified the feasibility of rapid imaging detection of micro-nanoplastics and improved the sensitivity of micro-nanoplastics detection. The details are as follows: (1) A method for the preparation and labeling of "realistic" micro-nanoplastics based on mechanical crushing has been developed. By simulating the formation process of micro-nanoplastics in natural environment, 200 nm, 900 nm and 2500 nm micro-nanoplastics (PET200, PET900 and PET2500) were prepared from PET mineral water bottles sold in the market. The dense ring coordination compounds of platinum group element Ir were used as markers to label micro-nanoplastics based on the swelling characteristics of polymers, and three kinds of labeled micro-nanoplastics, including PET200-Ir, PET900-Ir and PET2500-Ir, were obtained. The labeling and digestion methods were optimized. The labeling concentration was 0.5 mg/mL, the labeling ratio was 6:1, the labeling time was 12 h, and the digestion time was 10 h and the digestion temperature was 100 ℃. Through the simulated leakage experiments in environment and biological media, it was found that the leakage of PET-Ir in other matrices was less than 3% except 5% in fetal bovine serum matrix, indicating that this labeling method has the potential of labeling in vivo. The preparation method in this study achieves the truth of the formation process (physical wear) and the physical and chemical properties of the material (derived from real daily plastics), and the labeling method has the advantage of anti-background interference, which is an important progress in the preparation of micro-nanoplastic models. It lays a foundation for the follow-up application research of biological and environmental distribution. (2) Study on the distribution of nanoplastics in mice. The distribution of PET200-Ir and PET900-Ir nanoplastics in mice was studied by intravenous injection and intragastric administration, with the aqueous dispersion of Ir marker as control. It was found that under the condition of intravenous injection, the liver was the main organ of nano-plastic accumulation and the distribution behavior of nano-plastic was particle size-dependent. The accumulation rate and clearance rate of PET200 were faster than that of PET900. The aqueous dispersions of Ir and nanoplastic with two kinds of particle size were not detected in other tissues except gastrointestinal tract, which may be due to the influence of gastrointestinal barrier. This study explores the feasibility of the application of "realistic" micro-nanoplastics in organisms, and shows the particle size dependence of the behavior of nano-plastics in organisms, which can provide a reference for the toxicological study of "realistic" nano-plastics. (3) Study on the distribution of micro-nanoplastics in the resuspension model of coastal sediments. Sediment resuspension is a common physical phenomenon in the intertidal zone. Based on the sediment resuspension model, this study explored the effect of oscillation time on the vertical distribution of micro-nanoplastics and the detection of the vertical distribution of micro-nanoplastics. The results show that with the increase of oscillation time, the concentration of micro-nanoplastics in the supernatant decreases and the concentration of micro-nanoplastics in sediment increases, which accords with the sedimentation behavior driven by adsorption. 45.3% of the micro-nanoplastics are concentrated in the sediment layer with a depth of 1.4~1.8 cm, which is 3.6 times of the adjacent water layer and 5.5 times of the adjacent sediment layer, indicating that the micro-nanoplastics are easy to be distributed in the top layer of the sediment and the bottom of the water body. This study illustrates the feasibility of applying "realistic" micro-nanoplastics to environmental behavior research, and explores the effects of sediment disturbance and interaction with natural materials on the settlement of micro-nanoplastics. It is revealed that the benthos living on the sediment surface may be more susceptible to micro-nanoplastic pollution in the coastal environment where sediment resuspension exists. (4) Fluorescence detection of micro-nanoplastics in real samples by super hydrophobic enrichment. Based on the current situation that the field of vision of imaging detection is small, the distribution area of samples on the carrier substrate is large, and the particles are sparse, it is difficult to locate the particles accurately and quickly, which leads to the low efficiency of analysis. There is an urgent need to expand the concentration detection range of imaging analysis. Superhydrophobic enrichment is an effective enrichment method for imaging detection, which is suitable for the detection of small volume, small particle size and low concentration particle samples. In this study, a super-hydrophobic enrichment method was developed, which can reduce the diameter of 10 mL samples on glass slides to 0.1 mm, which promotes the detection of low concentration samples. At the same time, the superhydrophobic enrichment combined with fluorescence labeling method tried to solve the problem of fluorescence background interference, and carried out the enrichment detection of low concentration unlabeled micro-nanoplastic samples. The lowest detectable concentration of PET900 in seawater and pure water samples was as low as 6.7× 10-5 mg/ml, and the lowest detectable concentrations of PET200 and PET2500 in pure water samples were 3.4×10-5 mg/mL and 5.7×10-5 mg/mL, respectively. This study reduces the detection limit of fluorescence imaging nanoplastics, significantly improves the detection efficiency and detection sensitivity, thus providing a methodological reference for the detection of micro-nanoplastics in real environment.
Table of Contents第 1 章 引言 1 1.1 微纳塑料概述 1 1.2 微纳塑料的行为及分布 1 1.2.1 微纳塑料的环境分布情况 1 1.2.2 影响微纳塑料分布的因素 2 1.3 微纳塑料对生态环境和人类健康影响 4 1.3.1 微纳塑料对生态环境的影响 4 1.3.2 微纳塑料对水生生物的影响 4 1.3.3 微纳塑料对人类健康的潜在影响 4 1.4 微纳塑料的分析方法 6 1.4.1 微纳塑料的样品前处理方法 6 1.4.2 微纳塑料的检测方法 7 1.5 研究意义、内容及技术路线 8 1.5.1 研究意义及内容 8 1.5.2 技术路线 9 第 2 章 微纳塑料的制备、标记与定量检测研究 11 2.1 引言 11 2.2 实验材料 12 2.2.1 实验试剂 12 2.2.2 其他材料 12 2.2.3 实验仪器 12 2.3 实验流程 13 2.3.1 微纳塑料的制备及表征 13 2.3.2 微纳塑料的标记及表征 15 2.3.3 标记稳定性检测 15 2.3.4 标记物浓度优化 16 2.3.5 消解时间优化 16 2.3.6 方法检出限与定量限 17 2.3.7 Ir 浓度和 PET 质量换算 17 2.4 实验结果与讨论 17 2.4.1 微纳塑料的产生 17 2.4.2 微纳塑料的表征 18 2.4.3 微纳塑料标记方法的原理 20 2.4.4 标记后微纳塑料的表征 21 2.4.5 标记稳定性 23 2.4.6 标记物浓度优化 24 2.4.7 消解时间优化 25 2.4.8 检出限与定量限 26 2.4.9 Ir 浓度与 PET 质量转换 27 2.5 小结 27 第 3 章 纳米塑料在小鼠体内的分布研究 29 3.1 引言 29 3.2 实验材料 29 3.3 实验流程 29 3.3.1 空白及生物基质回收率 29 3.3.2 静脉注射 PET 的小鼠体内分布 30 3.3.3 灌胃暴露途径下 PET 在小鼠体内的分布 30 3.3.4 数据统计方法 30 3.4 实验结果与讨论 30 3.4.1 空白及生物基质回收率 30 3.4.2 静脉注射纳米塑料的小鼠体内分布 31 3.4.3 灌胃暴露途径下 PET 在小鼠体内的分布特征 33 3.5 小结 34 第 4 章 微纳塑料在沉积物再悬浮体系中的分布研究 37 4.1 引言 37 4.2 实验材料 37 4.3 实验流程 38 4.3.1 空白及环境基质回收率 38 4.3.2 沉积物再悬浮体系的构建与表征 38 4.3.3 振荡时间对微纳塑料分布的影响 38 4.3.4 微纳塑料的空间分布检测 39 4.4 实验结果与讨论 39 4.4.1 空白及环境基质回收率 39 4.4.2 沉积物再悬浮体系的构建与表征 40 4.4.3 振荡时间对微纳塑料分布的影响 41 4.4.4 微纳塑料的空间分布 42 4.5 小结 43 第 5 章 真实样品中微纳塑料的超疏水富集联合荧光检测 45 5.1 引言 45 5.2 实验准备 46 5.3 实验流程 46 5.3.1 超疏水基底的制备与表征. 46 5.3.2 基于离心法去除荧光背景 47 5.3.3 基于萃取法消除荧光背景 47 5.3.4 基于冲洗法消除荧光背景 47 5.4 实验结果与讨论 48 5.4.1 超疏水基底的制备与表征 48 5.4.2 基于离心法去除荧光背景 49 5.4.3 基于萃取法去除荧光背景 52 5.4.4 基于冲洗法去除荧光背景 53 5.5 小结 54 第 6 章 总结与展望 55 6.1 本研究的主要结论 55 6.2 本研究的创新点 55 6.3 展望 56 参考文献 57 致谢 67 作者简历及攻读学位期间发表的学术论文与研究成果 69
Pages89
Language中文
Document Type学位论文
Identifierhttp://ir.yic.ac.cn/handle/133337/30984
Collection中国科学院烟台海岸带研究所知识产出_学位论文
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高琦. 微纳塑料的制备、标记及在复杂基质中的定量检测研究[D]. 中国科学院大学,2022.
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