对水样的自动固相萃取小污染物的免疫学分析的简单方法
Summary
A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.
Abstract
对环境水样的固相萃取（SPE）的新方法。所开发的原型是成本效益和用户友好的，并能进行快速，自动化和简单的SPE。预浓缩的溶液与通过免疫测定分析兼容，具有低的有机溶剂含量。一种方法被描述为天然激素17β雌二醇在100ml水样提取和预浓缩。反相SPE执行与十八烷基二氧化硅吸附剂和洗脱用200μl甲醇50％体积/体积的完成。洗脱剂是通过将二 – 水，以降低甲醇的量稀释。手动制备SPE柱后，整体过程1小时内自动执行。在该过程结束时，雌二醇浓度通过使用市售酶联免疫吸附测定（ELISA）进行测定。 100倍预浓缩得以实现，在只有10％体积/体积的甲醇含量。完全恢复的分子用1ng得以实现/升掺入去离子和合成海水样品。
Introduction
样品制备是在任何分析过程中的重要一步。特别是，除去基体效应，干扰的缩减，且分析物的富集是必要的，以获得精确的结果，并达到检测的低限。内分泌干扰化合物（内分泌干扰物）是特别值得关注的，因为它们对活的生物体的动作，即使在环境中存在的非常低的水平。天然激素17β雌二醇存在于欧盟水污染的收藏和容易被加入到根据欧洲水框架指令调节优先物质清单。固相萃取（SPE）通常适用于小的污染物在水中的分析，与两个化学1-5（色谱，质谱）和免疫6-9检测方法。后者积累了环境监测领域的兴趣，如免疫测定法是在大量的各种格式提供，特定于たrget分析物，和达到低检测极限。6，7，10，11的各种酶联免疫吸附测定（ELISA）是可商购，并使得能够分析多个样品同时上的多孔板。该过程包括在连续的反应步骤，可能需要几个小时。反应的最终产物，可以检测光学来确定基于校准曲线对目标分子的浓度。
经典的SPE程序包括吸附剂预调节，样品萃取，清洗，洗脱，浓缩由洗脱液蒸发。用于稀释该提取物的溶剂选自根据检测方法。对于免疫学方法中，有机溶剂的影响强烈的方法的灵敏度的量。12
除了回收和预浓缩的表演中，该方法也需要简单和成本效益。在再修改的自动化Ë有助于减少人类相关的错误。在我们以前的工作13我们推出了样机的自动固相萃取，而我们的方法适用于天然激素17β雌二醇海水样品中的分析。在本视频中，我们想强调我们方法的技术优势相对于传统的离线和在线SPE，以及它与通过检测免疫反应的具体的兼容性。我们描述适用于水样品17β雌二醇的检测的协议。 SPE与十八烷基硅胶（C18）吸附剂相和洗脱用稀甲醇进行执行。
Protocol
Representative Results
Discussion
提出了水样然后分析使用免疫的制备新方法。该仪器能够以自动化和用户友好的方式进行固相萃取。
水样其注射之前到系统的过滤是非常关键的。溶液中存在的任何微粒仍然会潜在地引起流体网络的堵塞和阻碍SPE柱。另一个重要步骤是在SPE柱的制备。在列颗粒的量是至关重要的，以达到最佳的性能成为可能。制备吸附剂颗粒的悬浮液时，避免了粒子的附聚特必须小心。?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was funded by the European Union Seventh Framework Program FP7/2007-2013 under grant agreement no. 265721. The authors thank the RIKILT Institute for Food Safety (NL) for their support in this project.
Materials
| Filter membrane 0.2 μm pore size | Merck Millipore | GNWP04700 | For sample filtration |
| Nylon membrane 11 μm pore size | Merck Millipore | NY1104700 | For SPE column |
| Disposable biopsy punch 5 mm | Medical Budget | 39302439 | |
| Nucleodur C18 ec | Macherey Nagel | 713550.01 | 50 μm particle diameter |
| Synthetic sea water | Sigma Aldrich | SSWS500-500ML | |
| Methanol | VWR | ||
| 17beta-estradiol standard | Enzo Life Science | 300 ng/ml | |
| 17beta-estradiol ELISA kit | Enzo Life Science | ADI-900-008 | 96 wells, range 30 – 3000 ng/L |
Riferimenti
- Chang, H. S., Choo, K. H., Choi, S. J. The methods of identification, analysis and removal of endocrine disrupting compounds (EDCs) in water. J. Hazard. Matter. 172, 1-12 (2009).
- Azzouz, A., Souhail, B., Ballesteros, E. Continuous solid-phase extraction and gas chromatography-mass spectrometry determination of pharmaceuticals and hormones in water samples. J. Chromatogr. A. 1217, 2956-2963 (2010).
- Tomsikova, H., Aufartova, J., Solich, P., Novakova, L. High sensitivity analysis of female-steroid hormones in environ-mental samples. Trends Anal. Chem. 34, 35-58 (2012).
- Ciofi, L., Fibbi, D., Chiuminatto, U., Coppini, E., Checchini, L., Del Bubba, M. Fully automated on-line solid phase extraction coupled to high-performance liquid chromatography-tandem mass spectrometric analysis at sub-ng/L levels of selected estrogens in surface water and wastewater. J. Chromatogr. A. 1283, 53-61 (2013).
- Robles-Molina, J., Lara-Ortega, F. J., Gilbert-Lopez, B., Garcia-Reyes, J. F., Molina-Diaz, A. Multi-residue method for the determination of over 400 priority and emerging pollutants in water and wastewater by solid-phase extraction and liquid chromatography-time-of-flight mass spectrometry. J. Chromatogr. A. 1350, 30-43 (2014).
- Huang, C. H., Sedlak, D. L. Analysis of estrogenic hormones in municipal waste water effluent and surface water using enzyme-linked immunosorbent assay and gas chromatography/tandem mass spectrometry. Environ. Toxicol. Chem. 20, 133-139 (2001).
- Hintemann, T., Schneider, C., Schöler, H. F., Schneider, R. J. Field study using two immunoassays for the determination of estradiol and ethinylestradiol in the aquatic environment. Water Res. 40, 2287-2294 (2006).
- Farré, M., Kuster, M., Brix, R., Rubio, F., Lopez de Alda, M. J., Barcelo, D. Comparative study of an estradiol enzyme-linked immunosorbent assay kit, liquid chromatography-tandem mass spectrometry, and ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry for part-per-trillion analysis of estrogens in water samples. J. Chromatogr. A. 1160, 166-175 (2007).
- Pu, C., Wu, Y. F., Yang, H., Deng, A. P. Trace analysis of contraceptive drug levonorgestrel in waste water samples by a newly developed indirect competitive enzyme-linked immunosorbent assay (ELISA) coupled with solid phase extraction. Anal. Chim. Acta. 628, 73-79 (2008).
- Van Emon, J. M., Gerlach, C. L. A status report on field-portable immunoassay. Environ. Sci. Technol. 29 (7), 312A-317A (1995).
- Farré, M., Brix, R., Barcelò, D. Screening water for pollutants using biological techniques under European Union funding during the last 10 years. Trends Anal. Chem. 24 (6), 532-545 (2005).
- Schneider, C., Schöler, H. F., Schneider, R. J. A novel enzyme-linked immunosorbent assay for ethinylestradiol using a long-chain biotinylated EE2 derivative. Steroids. 69, 245-253 (2004).
- Heub, S., et al. Automated and portable solid phase extraction platform for immuno-detection of 17β-estradiol in water. J. Chrom. A. 1381, 22-28 (2015).
- Petrovic, M., Eljarrat, E., Lopez de Alda, M. J., Barcelo, D. Endocrine disrupting compounds and other merging contaminants in the environment: a new survey on new monitoring strategies and occurrence data. Anal. Bioanal. Chem. 378, 549-562 (2004).
- Richardson, S. D., Ternes, T. A. Water analysis: Emerging contaminants and current issues. Anal. Chem. 86, 2813-2848 (2014).
