水环境及生物基质中纳米银的分离与转化研究
文献类型：学位论文
| 作者 | 于素娟 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 刘景富 |
| 关键词 | 纳米银 溶解态有机质 光照 HepG2细胞 浊点萃取 Silver Nanoparticles Dissolved Organic Matter Sunlight HepG2 Cells Cloud Point Extraction |
| 其他题名 | Separation and transformation of silver nanoparticles in aquatic environments and biological matrices |
| 学位专业 | 环境科学 |
| 中文摘要 | 纳米银优良的杀菌消炎特性及物理化学性能使其在工业生产、生物医药及日常生活中得到了大量的应用，是目前商业化程度最高、应用最为广泛的纳米材料之一。但是，在为人们带来便利的同时，纳米银的负面效应也逐渐引起了公众的关注。纳米银本身性质活泼，在水环境中是高度动态的，一旦释放到环境里，在迁移过程中可能会发生不同的物理化学转变，而不同形态银的毒性、生物有效性相差很大。因此，发展不同形态的银的分离测定方法，研究纳米银在环境及生物体内的物理化学转化，有助于更加合理地评估纳米银的潜在环境风险。 本文针对水环境及生物基质中纳米银与银离子的分离和转化开展了相关的研究工作，将超滤技术和基于 Triton X-114浊点萃取技术与电感耦合等离子体质谱及其他多种表征技术相结合，发展了环境水体和细胞中纳米银与银离子的分离测定技术，并研究了它们在环境相关条件下及生物体内的相互转化。论文主要包括以下几个部分： 首先，我们从多种合成纳米银的方法中选择了两种快速简单的水相合成方法，并对合成及纯化的条件进行了优化，从而得到了两种粒径分布均一、能长时间稳定的纳米银，为后续的实验奠定了基础。 然后，我们选择 PVP包裹的纳米银（25 nm），考察了自然光照、天然有机质、Ca2+/Mg2+、Cl-和 S2-等重要的环境因素对环境水体中纳米银的形貌及化学转变的影响。研究表明，光照能够引发纳米银形貌变化，使原来分散良好的纳米银逐渐融合，自组装形成 2D的纳米结构，并进一步生长成大的团聚体。银离子的释放也非常明显，光照 48 h，大约有 80%的纳米银转化为银离子。但加入天然有机质后，光照条件下纳米银也能长时间分散，表明在有机质含量高的水体中纳米银能够长时间稳定存在，可能会发生长距离的迁移，对水体生物造成持久的危害；同时银离子的释放大大下降，大约只有 20%的纳米银被氧化，且呈现先升高又快速下降的趋势。TEM检测观察到有大量小的纳米银颗粒的生成，表明纳米银在环境中高度动态，存在着氧化释放出银离子和银离子再还原生成纳米银的循环。进一步的实验发现，在自然水体 pH范围内（pH 5 - 8）及 Ca2+/Mg2+、Cl-和 S2-存在下，该现象是普遍发生的。虽然纳米银毒性的机理并不非常明确，但普遍认为与银离子的释放密切相关。我们的研究发现在有机质丰富的水体中，经日光照射一段时间后银离子释放逐渐下降，表明纳米银的毒性可能会发生一定程度的下降。以往的研究结果也发现，纳米银的富集、生物有效性及毒性都与纳米颗粒的尺寸有关，我们实验中发现的在光照条件下有小颗粒的生成及大的团聚体的出现，表明传统的毒性实验只关注纳米银原始的颗粒并不科学，需要进一步关注在实际环境条件下纳米颗粒的变化及毒性。 在上述研究工作的基础上，我们分别制备了富同位素 107Ag的纳米银和富同位素 109Ag的银离子，用同位素示踪的方法，初步研究了在环境相关条件下，纳米银与银离子之间的相互转化。结果发现，在简单的纳米银水溶液中，没有还原性物质加入时，体系中以纳米银的氧化为主。避光条件下，纳米银的氧化符合近一级反应动力学过程，反应速率为 0.01976 h-1；光照条件下，氧化过程较复杂，不能用简单近一级反应来描述。当有腐殖酸存在时，纳米银水溶液中银离子进一步还原生成纳米银不容忽略，且光照条件下该还原反应更加明显，反应速率为0.00632 h-1，约是避光条件下的 3倍。但腐殖酸存在时纳米银的氧化更为复杂，需要进一步深入的研究。 论文的最后一部分主要针对细胞中纳米银的分离测定和纳米银的细胞摄入而展开。纳米银的毒性究竟是来源于纳米颗粒本身还是银离子，还存在很大的争议，这是因为二者往往共存，且目前没有好的方法能够将二者从生物基质中分离。因此，我们发展了基于表面活性剂 TX-114的浊点萃取的方法，分离 HepG2细胞中的纳米银和银离子。向破碎的细胞液中加入 Na2S2O3后，一方面它能够和银离子络合生成水溶性的复合物防止银离子被萃取到 TX-114相，另一方面盐效应能够促进相分离。将萃取溶液加热至浊点温度以上，出现浑浊以后，通过低速离心，纳米银被萃取至下层富表面活性剂相，而银离子保留在上层水相，从而实现它们的分离。然后分别收集上下层，经微波消解后用 ICP-MS定量。在最优的条件下，细胞裂解液中超过 67%的纳米银被萃取至 TX-114相，而 94%的银离子保留在上层水相。该方法可以定量测定纳米银暴露的 HepG2细胞中银离子和纳米银的含量。基于建立的分析方法，研究了纳米银在 HepG2细胞中摄入。细胞经过 10 mg/L纳米银暴露 24 h后，约有 67.8 ng/104的银进入细胞，其中约 10.3%为银离子。与最初纳米银溶液中银离子含量（约 5.2%）相比，明显升高的银离子的含量表明可能有部分纳米银在细胞体内转变为银离子，也可能由于细胞对银离子的吞噬速率明显高于纳米银。由于已报道的数据中，银离子的毒性明显高于纳米银，而暴露的细胞中大量银离子的存在表明在考察纳米银对生物体的毒性效应时，银离子的生物毒性不可忽略。同时先前的研究将生物体内银的总量作为纳米银的吞噬量也需要重新考虑。 |
| 英文摘要 | Silver nanoparticles (AgNPs) are well known for their excellent antibacterial ability and superior physical properties, and are widely used in a growing number of applications including as home disinfectants, medical devices and water purificants.However, with the accelerating production and introduction of AgNPs into commercial products, there is likelihood of release of AgNPs into the environment,which raises growing health and environmental concerns. AgNPs are highly dynamic in the aqueous system, and once released into the environment, they could undergo different chemical and morphology transformation during transport, which would in turn greatly affect their final fate, transport, and potential bioavailability and toxicity. In order to make a rational assessment of the environmental risks posed by AgNPs,further data about the transformation and speciation of AgNPs in the real environment are urgently required. This dissertation focuses on the separation and transformation of AgNPs in the environmental and biological matrices. Coupling ultrafiltration/Triton X-114-based cloud point extraction (CPE) with ICP-MS and other characterization techniques, we have developed methods for separation, characterization,quantification and monitoring the transformation of AgNPs in aquatic environments and HepG2 cells. Firstly, we developed two fast and simple methods for respective synthesis of PVP and citrate stabilized AgNPs in water. After optimizing the key reaction parameters, stable and monodispersed AgNPs was synthesized with high preparation success rates, which was used in the following experiments. Using PVP coated AgNPs (PVP-AgNPs, 25 nm) as the model, we examined how environmentally relevant factors like dissolved organic matter (DOM), sunlight,Ca2+/Mg2+, Cl-, and S2- individually or in combination affect the chemical transformation of AgNPs. It was demonstrated that simulated sunlight induced the aggregation of AgNPs, causing particle fusion or self-assembly to form larger structures and aggregates. Meanwhile, AgNPs were significantly stabilized by DOM, indicating that AgNPs may exist as single particles and be suspended in natural water for a long time or delivered far distances. Dissolution (ion release) kinetics of AgNPs in sunlit DOM-rich water showed that dissolved Ag concentration increased gradually first and then suddenly decreased with external light irradiation, along with the regeneration of new tiny AgNPs. pH variation and addition of Ca2+ and Mg2+ within environmental levels did not affect the tendency, showing that this phenomenon was general in real aquatic systems. Given that a great number of studies have proved the toxicity of dissolved Ag (commonly regarded as the source of AgNP toxicity) to many aquatic organisms, our finding that the effect of DOM and sunlight on AgNP dissolution can regulate AgNP toxicity under these conditions is important. The fact that the release of Ag+ and regeneration of AgNPs could both happen in sunlit DOM-rich water implies that previous results of toxicity studies gained by focusing on the original nature of AgNPs should be reconsidered and highlights the necessity to monitor the fate and toxicity of AgNPs under more environmentally relevant conditions. Based on the results above, we further synthesized isotopically enriched 107AgNPs and 109AgNO3 to track the transformation rate of AgNPs and Ag+ in aquatic environments. It was found that the oxidation of AgNPs dominated the reaction while the reduction of Ag+ was much lower without the addition of reducing agents. The oxidation of AgNPs in the dark followed a pseudo first-order rate law, and the rate coefficient was 0.01976 h-1; however, the oxidation of AgNPs under the light irradiation was much more complex. With the addition of DOM, the reduction of Ag+ was enhanced significantly under both conditions, especially after solar exposure. The reduction of Ag+ also followed a pseudo first-order rate law, and the rate coefficient under light irradiation was 0.00632 h-1, which was about three times larger than that in the dark. However, the oxidation of AgNPs was more complicated, and needs further investigation. In the last part, we focused on quantification and transformation of AgNPs in biological matrices. The toxic mechanism of AgNPs is still debating, partially because of the common co-occurrence and the lack of methods for separation of AgNPs and Ag+ in biological matrices. To overcome this bottleneck, Triton X-114-based CPE was proposed to separate AgNPs and Ag+ in the cell lysates of exposed HepG2 cells. Cell lysates were subjected to CPE after adding Na2S2O3, which facilitated the transfer of AgNPs into the nether Triton X-114-rich phase by salt effects and the preserve of Ag+ in the upper aqueous phase through the formation of hydrophilic complex. Then the AgNP and Ag+ contents in the exposed cells were determined by ICP-MS after microwave digestion of the two phases, respectively. Under the optimized conditions, over 67% of AgNPs in cell lysates were extracted into the Triton X-114-rich phase while 94% of Ag+ remained in the aqueous phase. This developed analytical method was applied to quantify the uptake of AgNPs to the HepG2 cells. After exposure to 10 mg/L AgNPs for 24 h, about 67.8 ng Ag were assimilated per 104 cells, in which about 10.3% silver existed as Ag+. Compared to the pristine AgNPs (with 5.2% Ag+) for exposure, the higher ratio of Ag+ to AgNPs in the exposed cells (10.3% Ag+) suggests the transformation of AgNPs into Ag+ in the cells and/or the higher uptake rate of Ag+ than that of AgNPs. Given that the toxicity of Ag+ is much higher than that of AgNPs, the substantial content of Ag+ in the exposed cells suggests that the contribution of Ag+ should be taken into account in evaluating the toxicity of AgNPs to organisms, and previous results obtained by regarding the total Ag content in organisms as AgNPs should be reconsidered. |
| 公开日期 | 2015-06-16 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/13472]  |
| 专题 | 生态环境研究中心_环境化学与生态毒理学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 于素娟. 水环境及生物基质中纳米银的分离与转化研究[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：生态环境研究中心
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