去除微量元素的氧化铜纳米粉体制备铀<em>原位</em>恢复泌水及其对细胞活力的影响
Summary
Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.
Abstract
在原位恢复（ISR）是提取铀在美国的主要方法。期间的ISR，铀是从矿体浸出，并通过离子交换萃取。所得的生产泌水（PBW）含有污染物，如砷等重金属。 PBW从激活ISR铀设施处理样品与氧化铜纳米粒子（氧化铜纳米粒）。的CuO-NP处理PBW降低优先污染物，包括砷，硒，铀，和钒。未处理和CuO-NP处理通过MTT（3-（4,5-二甲基吡啶-2-基）-2,5-二苯基溴化）测定PBW用作细胞生长介质和变化生存能力的液体成分分析在人胚肾（HEK 293）和人肝细胞癌（Hep G2细胞）细胞。的CuO-NP处理用改进的HEK和HEP细胞活力有关。这种方法的限制包括稀释PBW由生长培养基成分和渗透压克分子期间先进而精湛调整以及必要pH调节。这种方法是在其更广泛的范围，由于稀释作用以及变化PBW的pH是传统微酸但是限制;这种方法可以有更广泛的用途评估的CuO-NP处理的较为中性的水域。
Introduction
大约是美国电力供应的20％是由核能，部分基于国家鼓励提高能源独立性提供，预计美国的核能力，以增加1。核能的全球增长预计也将继续，与许多美国以外的2出现的增长。截至2013年，美国铀的83％是进口的，但储量952544吨，在美国3,4存在。在2013年有7个新设施的应用和怀俄明州，新墨西哥州，内布拉斯加州和5之间14重启/扩展应用。在美国，铀为主通过原位恢复（ISR）处理6萃取。 ISR导致更少的土地破坏和避免造成尾矿堆，可以释放环境污染物7。 ISR使用水基氧化解决方案，以从地下矿石体，在这之后的铀从浸出液通过提取浸出铀离子交换过程8。维持负面水平衡在矿体，浸出液的一部分，被称为生产流血水（PBW）中，放血关闭。所述PBW的一部分被利用反渗透（RO）和净化重新引入开采过程中，但PBW也可以有利于工业或农业用途，如果有毒的污染物可以减少到由状态管理机构对于表面确定的可接受水平和地下水9。目前，大多数ISR铀的设施使用RO从PBW去除污染物。然而，反渗透处理是能源密集型，并产生有毒废物盐水，这需要监管的处置。
许多水净化方法都存在，包括吸附剂，膜和离子交换。在这些中，吸附是最常用的，并且最近的发展纳米颗粒合成具有增强的吸附剂基水净化处理10的功能。铜OXI德纳米颗粒（氧化铜纳米粒）以前没有被广泛研究对铀的ISR PBW，但在最近的污染物去除从地下水的研究，氧化铜纳米粒被发现有独特的性能，包括无需预处理或后处理的水处理工序（ 例如 ，调节pH值或氧化还原电位），并在不同的水成分表现良好（ 例如 ，在不同的pH值，盐的浓度，或竞争离子）11。另外，氧化铜纳米粒容易通过用氢氧化钠（NaOH），之后，将再生的CuO纳米粒可重复使用浸出再生。从天然水域的CuO-NP微量金属过滤功能细节已先前公布11-14。
虽然对水处理是有用的，金属氧化物纳米颗粒可能是有毒的活生物体，但毒性的程度取决于，部分地基于纳米粒子的特性和成分10,15,16。因此，研究SIMULT是很重要现场应用之前aneous污染物的去除和纳米粒子毒性。目前的研究确定的CuO纳米粒的能力以去除PBW优先污染物（包括砷，硒，钒和铀），和评估的CuO-NP处理对PBW细胞毒性的影响。
PBW从有源ISR铀设施收集并用于确定在优先的污染物去除的CuO-NP处理的功效。之前和之后的CuO-NP处理PBW细胞毒性也进行了评估。 PBW是一个复杂的地质（工业/环境）的混合物和环境都和健康科学（NIEHS）国家研究所和机构的有毒物质及疾病登记（ASTDR）是把重点放在学习环境相关的混合物，包括混合物的毒性因为它们在自然界或工业设置，以及在体外测试促进存在优先的化学品进行进一步的体内测试17-19。是慢性，低剂量暴露的混合物具有挑战性的研究，因为长期接触低剂量的混合物，不会产生明显的影响，至少在大多数实验室研究的很短的时间框架。同样，大多数在化学混合物的体外研究细胞暴露于两种或更多种金属20,21限定的实验室制造的混合物。这些研究提供基线信息，但简化的混合物不复制，可能会出现一个天然的，环境样品，其中所述全方位混合物组分都存在于该复合拮抗和协同相互作用。
本研究的目标是检查供PBW备用污染物去除工艺和评估（的CuO-NP）的治疗上PBW细胞毒性使用培养的人类细胞的效果。该结果可能通过更有效的或环保的方法来去除污染物的发展中受益的铀业。这项研究提供第一个证据是PBW减少污染物优先由氧化铜纳米粒在哺乳动物细胞中22毒性降低。
Protocol
Representative Results
Discussion
以前的研究报告说，氧化铜，纳米粒子去除砷地下水11,13,30,31。这项研究支持，这些以往的调查结果也报告说，氧化铜纳米粒删除PBW更多的污染物。本研究还证实了以前的报告的CuO纳米粒是在除砷有效，尽管其他污染物和可能的竞争离子11的存在。形态建模预测，钒物种PBW的97％的负电荷，从而允许吸附在CuO纳米粒和批量处理除去钒的92％。
这是第一次研究，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Dr. Roger Hopper and the Wyoming Department of Agriculture, Analytical Services Lab for the mass spectroscopy analysis of our samples. We would like to express our gratitude to the University of Wyoming, School of Pharmacy for allowing us to video this protocol in their laboratories. We would also like to thank the Theodore O. and Dorothy S. King Endowed Professorship Agreement for their graduate assistantship (SC), the University of Wyoming for the Graduate Assistantship support (JRS), and the Science Posse (NSF GK-12 Project # 084129) for the teaching fellowship (JRS). We would also like to thank Uranium One for allowing us to obtain samples and assisting us with questions. This work was supported by the School of Energy Resources, University of Wyoming.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| CuCl2 | Sigma | 203149 | |
| Borosilicate glass balls | VWR | 26396-639 | 6 mm |
| Nitric Acid | Fisher | A509-P500 | Trace metal grade |
| 0.45 mm syringe filter | Fisher | SLHA 033S S | |
| 10X EMEM | Fisher | BW12-684F | |
| Fetal Bovine Serum | ATCC | 30-2020 | |
| L-glutamine | Fisher | BP379-100 | |
| NaHCO3 | Sigma | S5761 | |
| Penicillin/Streptomycin | ATCC | 30-2300 | |
| 0.22 mm vacuum filter unit | Fisher | 09-740-28C | |
| HEK293 | ATCC | CRL-1573 | |
| HEPG2 | ATCC | HB-8065 | |
| Trypsin | Sigma | SV3003101 | |
| MTT | Sigma | M2128 | |
| D-penicillamine | Fisher | ICN15180680 | |
| 96-well plates | Fisher | 07-200-92 | |
| DMSO | Fisher | D12814 | |
| Spectra Max 190 | Molecular Devices | ||
| Visual MINTEQ version 3.0 | KTH Royal Institute of Technology | ||
| ICP-MS | Agilent | Details of instruments, models and detection limits were published in Reddy et al., 2013. | |
| IC DIONEX DX 500 | Dionex | Details of instruments, models and detection limits were published in Reddy et al., 2013. | |
| VWR Incubator | VWR |
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